Life Sciences 80 (2007) 1921 – 1943
www.elsevier.com/locate/lifescie
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The magic glue hyaluronan and its eraser hyaluronidase:
A biological overview
K.S. Girish ⁎, K. Kemparaju
Department of Biochemistry, University of Mysore, Manasagangothri, Mysore, Karnataka State, 560007, India
Received 2 November 2006; accepted 19 February 2007
Abstract
Hyaluronan (HA) is a multifunctional high molecular weight polysaccharide found throughout the animal kingdom, especially in the
extracellular matrix (ECM) of soft connective tissues. HA is thought to participate in many biological processes, and its level is markedly elevated
during embryogenesis, cell migration, wound healing, malignant transformation, and tissue turnover. The enzymes that degrade HA,
hyaluronidases (HAases) are expressed both in prokaryotes and eukaryotes. These enzymes are known to be involved in physiological and
pathological processes ranging from fertilization to aging. Hyaluronidase-mediated degradation of HA increases the permeability of connective
tissues and decreases the viscosity of body fluids and is also involved in bacterial pathogenesis, the spread of toxins and venoms, acrosomal
reaction/ovum fertilization, and cancer progression. Furthermore, these enzymes may promote direct contact between pathogens and the host cell
surfaces. Depolymerization of HA also adversely affects the role of ECM and impairs its activity as a reservoir of growth factors, cytokines and
various enzymes involved in signal transduction. Inhibition of HA degradation therefore may be crucial in reducing disease progression and
spread of venom/toxins and bacterial pathogens. Hyaluronidase inhibitors are potent, ubiquitous regulating agents that are involved in maintaining
the balance between the anabolism and catabolism of HA. Hyaluronidase inhibitors could also serve as contraceptives and anti-tumor agents and
possibly have antibacterial and anti-venom/toxin activities. Additionally, these molecules can be used as pharmacological tools to study the
physiological and pathophysiological role of HA and hyaluronidases.
© 2007 Elsevier Inc. All rights reserved.
Contents
Introduction Hyaluronan . . . . . . . . . . . .
HA binding proteins and receptors . . . . . . .
Extracellular hyaladherins. . . . . . . . . . . .
HA receptors—cellular hyaladherins . . . . . .
Hyaluronan and cancer . . . . . . . . . . . . .
Biological activities of HA and HA oligomers .
Hyaluronidases . . . . . . . . . . . . . . . . .
Mammalian hyaluronidases . . . . . . . . . . .
Venom hyaluronidases . . . . . . . . . . . . .
Microbial hyaluronidases . . . . . . . . . . . .
Hyaluronidase inhibitors . . . . . . . . . . . .
Medical applications of hyaluronan. . . . . . .
Treatment of osteoarthritis . . . . . . . . . . .
Surgery and wound healing. . . . . . . . . . .
Embryo implantation . . . . . . . . . . . . . .
As a disease indicator. . . . . . . . . . . . . .
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⁎ Corresponding author. Department of Orthopedics, Cobb hall, BO57, P.O. Box # 800374, University of Virginia, Charlottesville, Virginia-22908, USA. Tel.: +1
434 924 1717 (o), +1 412 805 1951 ®; fax: +1 434 924 1691.
E-mail address: [email protected] (K.S. Girish).
0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2007.02.037
1922
K.S. Girish, K. Kemparaju / Life Sciences 80 (2007) 1921–1943
Hyaluronan in drug delivery. . . . .
Medical application of hyaluronidase
Concluding remarks . . . . . . . . .
Acknowledgements . . . . . . . . .
References . . . . . . . . . . . . . .
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Introduction Hyaluronan
Meyer and Palmer first biochemically purified hyaluronan
[HA] from bovine vitreous humour and solved its structure in
1954 (Meyer and Palmer, 1934; Weissman and Meyer, 1954).
Further studies have shown HA to be an acidic, negatively
charged, high molecular weight polysaccharide having an
uniformly repetitive, linear polysaccharide comprised of
disaccharide units of N-acetyl-D-glucosamine (GlcNac) and Dglucuronic acid (GlcA) (Laurent, 1970; Laurent and Fraser,
1992; Scott and Heatley, 2002). Though classified as a
glycosaminoglycan (GAG), it differs from other GAGs
(Table 1). HA is a megadalton molecule, with a typical molecular weight between ∼ 2 × 105 to ∼ 10 × 107 Da and an extended length of 2–25 μm. Whereas other GAGs are relatively
smaller in size (b50 kDa, commonly 15–20 kDa) with a short
chain length. Also, HA is synthesized at the inner face of the
plasma membrane as a free linear polymer without any protein
core, while other GAGs are synthesized by resident Golgi
enzymes and covalently attached to core proteins (Laurent and
Fraser, 1992; Lee and Spicer, 2000; Toole, 2004).
The biosynthesis of HA is regulated by three transmembrane
glycosyltransferase isoenzymes: HAS1, HAS2 and HAS3. The
active site of these enzymes protrudes from the inner face of the
plasma membrane. HA is extruded through the plasma membrane
onto the cell surface or into the ECM as it is being synthesized
(Weigel et al., 1997). The isoenzymes are unique in that they
possess two enzymatic components (i.e., Glycosyltransferases),
one to add on the GlcUA and another to add on the GlcNAc.
Although the amino acid sequences of these isozymes are 50 to
71% identical, the gene sequences are located on different
chromosomes (hCh19-HAS1, hCh8-HAS2, and hCh16-HAS3)
and encode three different proteins with distinct enzymatic
properties (Spicer and McDonald, 1998; Itano et al., 1999).
HAS3 synthesizes shorter forms of HA molecules (b 3 × 105 Da)
and is thought to be more active than HAS1 and HAS2, both of
which produce high molecular mass HA molecules
Table 1
Sugar composition of glycosaminoglycans
Name
Sugar composition
Sulfation Protein Linkage
core
Hyaluronan (HA)
Chondroitin sulfate (CS)
Dermatan sulfate (DS)
Keratan sulfate (KS)
Heparan sulfate (HS)
Heparin
GlcA/GlcNAc
GlcA/GalNAc
GlcA or IdoA/GalNAc
Gal/GlcNAc
GlcA or IdoA/GlcNAc
GlcA or IdoA/GlcNAc
−
+
+
+
+
+
+ = positive; − = negative.
−
+
+
+
+
+
β (1,3)
β (1,3)
β (1,3)
β (1,4)
α (1,4)
α (1,4)
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1935
1936
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1937
(3.9 × 106 Da). Mouse and human HA synthase genes are located
on different chromosomes, suggesting that gene duplication and
divergent evolution has resulted in differing gene regulation and
kinetic properties of the encoded synthases (Table 2). The
function of hyaluronan synthase (HAS) isoforms appears to be
cell and tissue specific, but their exact function and role in cell
signaling remains to be elucidated. Misregulation of HAS genes
results in abnormal production of HA and promotion of aberrant
biological processes such as transformation and metastasis
(Toole, 2004; Adamia et al., 2005).
HA is present in almost every tissue of all vertebrates but is
most abundant in the ECM of soft connective tissues (Fraser
et al., 1983). Depending on the tissue source, the polymer
usually consists of 2000–25,000 disaccharide units with
various functions. It is also found on the surface of certain
pathogenic Streptococcus and Pasteurella bacteria. In mammals, a high concentration of HA is found in connective tissues
such as umbilical cord, skin, synovial fluid and the vitreous
humour.
HA performs three basic molecular functions. First, it
interacts in an autocrine manner with cell surface HA receptors
on the same cell. Second, it interacts in a paracrine manner with
a variety of ECM molecules on neighboring cells. Due to its
giant physical structure, HA can interact with more than one
cell. Such interactions have been shown to be essential in the
structure and assembly of several tissues (Tammi et al., 2002;
Turley et al., 2002; Toole, 2004). A single HA polymer can bind
to hundreds of ECM proteins, which can in turn bind additional
matrix proteins. These large matrix complexes can also be
linked to the cell surface via HA receptors (Toole, 2004; Spicer
and Tien, 2004).
Third, newly synthesized HA may be secreted and
subsequently interact with several cell surface receptors,
including cluster determinant 44 (CD44), receptor for
hyaluronate-mediated motolity (RHAMM), lymphatic vessel
endothelial HA receptor (LYVE-1), hyaluronan receptor
for endocytosis (HARE), liver endothelial cell clearance
receptor (LEC receptor) and TLR-4. These interactions
mediate three important physiological processes: signal
transduction, the formation of pericellular coats and receptor-mediated internalization (Laurent et al., 1996; Vercruysse
et al., 1999; Toole et al., 2002; Turley et al., 2002; Toole,
2004; Spicer and Tien, 2004; Adamia et al., 2005; Taylor and
Gallo, 2006).
HA binding proteins and receptors
Hyaladherins are a heterogeneous group of proteins with the
ability to bind HA. These proteins can be grouped according to
K.S. Girish, K. Kemparaju / Life Sciences 80 (2007) 1921–1943
their location (extracellular/cellular) and by the sequence of HA
binding site (Table 3). Most of the known HA binding proteins
and receptors share a common 100 amino acid globular binding
domain called the “link module”, which was first described in
cartilage protein. The link module region is comprised of an
immunoglobulin domain and two adjacent link modules. The
immunoglobulin domains are most likely responsible for the
link protein–proteoglycan interaction, whereas the link modules
mediate binding to HA (Day and Prestwich, 2002; Spicer et al.,
2003). This molecular architecture is common in G1-domains
of aggrecan, versican, neurocan and brevican.
1923
Table 3
Hyaladherin family proteins
Cellular
CD44 family, RHAMM, cdc37, p68 (gelqR), HBP (hepatocyte
binding protein), IHABP4, TSG-6, LYVE-1, LEC,
Extracellular Versican, link protein, aggrecan, neurocan, brevican, fibrinogen,
Trypsin inhibitor (IαI)
matrix hyaladherins (Rauch et al., 1992; Blundell et al., 2004;
Seyfried et al., 2006).
HA receptors—cellular hyaladherins
Extracellular hyaladherins
Extracellular hyaladherins are a group of HA-binding
proteoglycans that include aggrecan, neurocan and brevican,
constituting a gene family collectively termed ‘hyalectins’.
These proteoglycans are components of the ECM and each
proteoglycan has a characteristic distribution, with versican
present in different soft tissues, aggrecan prominent in cartilage,
and neurocan and brevican prominent in the central nervous
system (CNS).
In cartilage, HA functions as the central filament of
proteoglycan aggregates, which are responsible for cartilage's
resistance to compression. Acting as a scaffold, HA stabilizes
the ECM structure through its interaction with several matrix
hyaladherins. The major cartilage proteoglycan, aggrecan, can
interact strongly with HA through the HA-binding domain (link
module) on the intact proteoglycan molecule. The binding of
proteoglycans to the HA chain is strengthened greatly by the
link-protein. The retention of proteoglycans within the ECM is
based on their interaction with HA, and thus they are the major
Table 2
Chromosomal location of hyaluronidase and hyaluronan synthase genes
Family
Species
Chromosomal
location
Gene
Protein
pH
optimum
HA synthase
Human
19q13.3–13.4
8q24.12
16q22.1
17
15
8
3p21.3
HAS1
HAS2
HAS3
Has1
Has2
Has3
HYAL1
HYAL2
HYAL3
HYAL4
SPAM1
HYALP1
Hyal1
Hyal2
Hyal3
Hyal4
Spam1
Hyalp1
Hyal5
HAS1
HAS2
HAS3
Has1
Has2
Has3
Hyal1
Hyal2
Hyal3
Hyal4
HPH-20
–
HYAL1
HYAL2
HYAL3
HYAL4
PH-20
–
HYAL5
–
–
–
–
–
–
3–4
4, 7.5
ND
ND
4, 7.5
ND
3–4
4, 7.5
ND
ND
4, 7.5
ND
4, 7.5
Mouse
Hyaluronidase
Human
7p31.3
Mouse
9F1–F2
6 A2
# Human and mouse hyaluronidase genes are located in equivalent locations of
respective chromosomes.
CD44, considered the principal receptor for HA, is a multifunctional single-pass transmembrane glycoprotein consisting
of four functional domains. CD44 is expressed in a number of
different isoforms, due to alternate splicing of multiple variant
exons, and variations in CD44 polypeptide sequence, glycosylation, and oligomerization influence its affinity for HA
binding (Ponta et al., 1998). The distal extracellular domain is
primarily responsible for the HA binding. Recent studies have
revealed that CD44 binds to HA via a single extracellular link
domain. Mutations in this region stall CD44 attachment to HA
and inhibit the affinity of anti-CD44 monoclonal antibodies that
block HA binding (Day and Prestwich, 2002). In addition to
HA, CD44 interacts with several other molecules, many of
which bind to carbohydrate side groups that are attached to
spliced-in regions. Other ligands of CD44 include fibroblast
growth factor, osteopontin (Knudson and Knudson, 2004),
matrix metalloproteases (Toole, 2004), SRC kinases (Thorne
et al., 2004), RHO GTPases (Ponta et al., 2003), VAV2, GAB1,
ezrin (Toole, 2004), and ankyrin (Zhu and Bourguignon, 2000).
CD44 is transcriptionally up-regulated by pro-inflammatory
cytokines such as IL-1 and growth factors such as epidermal
growth factor (EGF), transforming growth factor-beta (TGF-β)
and bone morphogenic protein (BMP-7) (Knudson and
Knudson, 2004; Heldin, 2003). IL-1 increases CD44 mRNA
and protein expression level in both chondrocytes and vascular
smooth muscle cells (Ohno et al., 2005; Iacob and Knudson,
2006). In chondrocytes, the increased CD44 expression results
in an increased capacity for HA binding, internalization and
degradation. IL-1-induced elevation of CD44 increases the
capacity of smooth muscle cells to bind HA. In atherosclerotic
lesions, the increased expression of CD44 and HA may
influence the proliferation and migration of smooth muscle
cells, which contributes to the development of pathological
lesions (Evanko et al., 1998; Wight and Merrilees, 2004).
Furthermore, EGF up-regulates CD44 expression in fibroblasts,
several tumor cell types, and epithelial cells (Toole, 2004;
Knudson and Knudson, 2004).
HA–CD44 interactions participate in a wide variety of
cellular functions, including cell–cell aggregation, retention of
pericellular matrix, matrix–cell and cell–matrix signaling,
receptor-mediated internalization/degradation of HA, and
regulating aspects of cell movement and cell–cell adhesion
(Li et al., 2000a; Knudson et al., 2002; Toole, 2002, 2004;
Knudson, 2003; Spicer and Tien, 2004). Studies of embryonic
1924
K.S. Girish, K. Kemparaju / Life Sciences 80 (2007) 1921–1943
development, regeneration and healing, cancer and vascular
disease have demonstrated that pericellular matrices surrounding proliferating and migrating cells are highly enriched in HA
(Toole, 2004; Knudson et al., 2002). Interactions of HA with
CD44 or hyaluronan synthase, versican, aggrecan, tumor
necrosis factor-stimulated gene-6 (TSG-6), inter-α-trypsin
inhibitor (IαI) and other hyaladherins in this matrix create a
complex, hydrated microenvironment that supports and promotes the cellular characteristics of dividing and migrating cells
(Toole, 2004; Spicer and Tien, 2004).
In recent investigations, three approaches were used to
manipulate endogenous HA–protein interaction. First, overexpression of soluble hyaladherins, which completely displace
HA from its endogenous cell surface receptors (Peterson et al.,
2000; Ahrens et al., 2001; Ward et al., 2003). Second,
administration of HA oligomers, compete for endogenous
polymeric HA–receptor interactions, thus resulting in lowvalency, low-affinity binding rather than polyvalent, high
affinity interactions with receptors (Toole, 2004). Overexpression of soluble hyaladherins and administration of HA oligomer
leads to inhibition of in vivo growth, local invasion and
metastasis of melanoma cells (Lesley et al., 2000; Ghatak et al.,
2002). The presence of HA oligomers in tissues is only
speculation, but the influence of HA oligomers may affect
different signals resulting from native HA interactions with
different receptors. However, recent study confirmed the
presence of HA fragments (b100 kDa) in sterile UVB-induced
inflammation in vivo (Averbeck et al., 2007). Third, treatment
with antibodies that block HA–CD44 interaction, inhibits tumor
growth and invasion (Lesley et al., 2000; Ghatak et al., 2002).
Among these approaches, overexpression of soluble receptors
and administration of HA oligomers, could have multiple
biochemical effects with differing downstream consequences.
RHAMM is a HA receptor that can be present at the cell
surface, within the cytoplasm, and in the nucleus depending on
alternative splicing of the transcript. RHAMM is expressed by
several cell types and, depending on which isoform is present,
contributes to HA mediate migration, rearrangement of the
cytoskeleton and intracellular signal transduction. Interactions
of HA with RHAMM can trigger a number of cellular signaling
pathways, including those that involve protein kinase C, focal
adhesion kinase (FAK), MAP kinases, nuclear factor-κB, RAS,
phosphatidylinositol kinase (PI3K), tyrosine kinases, and
cytoskeletal components (Hall et al., 1996; Fieber et al., 1999;
Politz et al., 2002; Ponta et al., 2003; Thorne et al., 2004; Toole,
2004).
RHAMM has also been implicated in cell motility. Several
studies have demonstrated its involvement in the locomotion of
transforming growth factor-β-stimulated fibroblasts, smooth
muscle cells, and macrophages, as well as in ras-transformed
fibroblasts (Savani et al., 2001). RHAMM dependent cell
migration appears to regulate the turnover of focal adhesions via
a protein tyrosine phosphorylation pathway. Of the two major
isoforms of RHAMM, cell surface RHAMM has been
implicated in promoting the motility and invasion of a number
of cell types by a HA-dependent mechanism (Zhang et al.,
1998). The intracellular variant of RHAMM (referred to as
intracellular HA binding protein [IHABP]) is localized in the
centrosome and modulates cell cycle control and mitotic spindle
formation and integrity through crosslinking and association
with dynein. It has been shown to associate with actin and
microtubule cytoskeletal elements (Zhang et al., 1998; Turley
et al., 2002).
The genes stabilin-1 and stabilin-2 encode the MS-1 antigen
and hepatic HA clearance receptor, respectively. These
functional HA receptors are present on the surface of
endothelial cells and activated macrophages present in the
liver, placenta, spleen and the lymph node. The hepatic HA
clearance receptor is thought to be important for removing of
HA from the blood during steady-state tissue remodeling (Politz
et al., 2002; Spicer and Tien, 2004).
LYVE-1 (lymphatic vascular endothelial hyaluronan receptor)
is a recently discovered HA binding protein expressed only in
lymph vessel endothelium. LYVE-1 is structurally related to
CD44 and other binding proteins that contain a consensus HAbinding domain (Link module). LYVE-1 cDNA codes for a 322residue integral membrane glycoprotein with a single link module
located at the N-terminal of the extracellular domain. The
membrane proximal domain is predicted to be heavily oglycosylated with an unpaired cysteine residue. Overall, LYVE1 is 43% similar to CD44 (Jackson et al., 2001; Jackson, 2003;
North et al., 2004). LYVE-1 is abundantly expressed in the
lymphatic endothelium of humans, mice, and rats. It is also
expressed in discrete populations of activated tissue macrophages
and in the sinusoidal endothelium of the liver and spleen, the sites
where uptake and degradation of high molecular weight HA is
known to occur (Jackson, 2004). Recent studies revealed that the
interaction between LYVE-1 and HA in lymphatics is tightly
regulated and that the mechanisms may be broadly similar to
those that regulate CD44–HA interactions in blood vasculature
(Jackson et al., 2001; Banerji et al., 1999). It is considered to be
HA specific since it has no affinity for any other GAGs tested
(Jackson, 2003).
Layilin is a recently cloned and characterized HA receptor.
Layilin exhibits a membrane-binding site for talin, a member of
the ERM superfamily of linkers between the actin cytoskeleton
and the cell membrane. The receptor has therefore been
proposed to contribute to cell migration and morphology
(Bono et al., 2001). Studies have revealed that layilin is a
functional HA receptor capable of mediating cell adhesion.
Layilin shares no sequence homology to the other known HA
receptors; it does not contain a link domain, a common HA
binding module found in many ECM proteins and cell surface
receptors. However, layilin contains a C-type lectin domain that
may account for its ability to bind HA. A recent study suggests
merlin, the neurofibromatosis type 2 tumor suppressor protein,
and radixin, as two novel binding partners for layilin (Bono et
al., 2005). Both proteins contain a FERM domain at their Nterminus, suggesting that layilin may mediate signals from
ECM to cell cytoskeleton through interaction with different
binding partners.
TSG-6 (tumor necrosis factor-stimulated gene-6) is a
multifunctional protein that is up-regulated in many physiological and pathological conditions associated with inflammation
K.S. Girish, K. Kemparaju / Life Sciences 80 (2007) 1921–1943
and tissue remodeling, such as in the sera and joints of arthritis
patients (Bayliss et al., 2001). Several studies using a murine
arthritis model have been demonstrated the anti-inflammatory
and chondroprotective effects of TSG-6. A number of
mechanisms might contribute to these effects. First, recombinant TSG-6 down-regulates the protease network and inhibits
matrix metalloproteinase. Second, the anti-plasmin activity of
IαI is significantly increased by TSG-6. This is supported by the
observation of inflamed paw joints of TSG-6 deficient mice
compared to wild type mice in arthritis model. Third, TSG-6
inhibits neutrophil migration in vivo, causing 50% inhibition in
an air pouch model of acute inflammation (Cao et al., 2004;
Mahoney et al., 2005) by modulating the protease network in
conjunction with IαI (Milner and Day, 2003).
TSG-6 also interacts with a broad spectrum of GAG and
protein ligands, including HA, heparin, chondroitin 4-sulfate,
aggrecan, versican, IαI, pentraxin-3, and thrombospondin-1.
The interactions of TSG-6 with HA, heparin, and the aggrecan
G1-domain are sensitive to pH. Therefore the functions of TSG6 are likely to be tissue specific and dependent on pH and GAG
content (Milner et al., 2006). In cartilage, TSG-6 may inhibit the
formation of HA–protein aggregates, promote aggregate
dissociation, or modulate CD44 function. In certain tissue
locations, TSG-6 can associate with inter-α-inhibitor (IαI), and
this TSG-6/IαI complex may be able to stabilize the ECM by
crosslinking with HA chains (Milner et al., 2006).
These studies suggest that hyaladherins and HA binding
proteins participate in several important functions, such as the
retention of HA-rich ECM and signal transduction events
mediated via cell–matrix interactions. Disruption of the
interaction between HA and its receptors/binding proteins by
HA oligosaccharides, antibodies, hyaluronidase and HA
degradation, antisense oligonucleotides can induce a cascade
of events resulting in the activation of both catabolic and
anabolic pathways.
Hyaluronan and cancer
A large amount of experimental evidence from animal
models shows that epithelial and connective tissue cancers are
associated with high levels of HA. Histological studies on
various tumors, using a specific probe, have shown greater HA
enrichment of extracellular matrices surrounding tumors than
found in parenchymal regions (Toole, 2004; Toole and Hascall,
2002; Paiva et al., 2005). In human cancers, HA concentrations
are usually higher in tumors than in normal tissues. Human
breast, lung, prostate, ovarian, nephroblastomas, and colon
cancer are considered to enriched with HA (Heldin, 2003;
Toole, 2004; Adamia et al., 2005). In these tumors, HA may
support tumor growth by stimulating anchorage-independent
growth and proliferation of tumor cells. Moreover, HA may also
actively promote tumor metastasis by promoting tumor cell
adhesion and migration and may also protect against immune
surveillance (Itano et al., 2004; Paiva et al., 2005). Additionally,
tumor cells may take advantage of HA-rich extracellular
matrices to invade more easily into the surrounding tissues.
HA-induced tissue hydration physically creates spaces through
1925
which tumor cells may migrate and invade. HA-rich matrices
within the tumor-associated stroma are also infiltrated with
newly forming blood vessels (Toole, 2004).
It is well known that HA stimulated cell migration and
signaling are accomplished through the interactions mediated
by the HA cell surface receptors CD44 and RHAMM, both of
which promote tumor progression. As mentioned earlier, the
interaction of HA with CD44 and RHAMM has been implicated
in cell proliferation, migration and angiogenesis. CD44 also
mediates a variety of intracellular signaling cascades and
interacts with cytoskeletal proteins that are essential for the
normal functioning of the cells. However, abnormal activation
of these signaling pathways can lead to malignant behavior
(Toole, 2004; Adamia et al., 2005).
Manipulation of HA biosynthesis in tumor cells has shown
its importance in metastasis (Liu et al., 2001). HA synthesis by
HAS can facilitate tumor progression through reorganization of
the cytoskeleton, including lamellipodial formation which is a
prerequisite for cell spreading and metastatic development
(Itano et al., 2004; Adamia et al., 2005; Paiva et al., 2005).
Numerous studies demonstrated the overexpression of HAS
proteins and subsequent overproduction of HA molecules in
metastatic development of fibrosarcoma, and prostate and
mammary carcinoma (Itano et al., 1999; Simpson et al., 2002;
Heldin, 2003).
Overexpression of HAS1 or HAS2 results in the synthesis of
high molecular weight HA, which may activate hyaluronidase, the
enzyme that degrades HA is up-regulated or down-regulated
during the progression of cancer (Lokeshwar et al., 2002). HYAL1
is the main hyaluronidase expressed in tumors and is active only at
acidic pHs (Stern, 2003, 2004). Shorter fragments of HA resulting
from HA degradation have been found in tumor extracts (Toole,
2004). Several studies have shown that HA oligosaccharides or
HA fragments promote angiogenesis (West et al., 1985).
These studies suggest that HA not only stimulates malignant
characteristics in cancer cells, but its degradation products
might also promote tumor progression through the stimulation
of angiogenesis.
Biological activities of HA and HA oligomers
HA is known to be involved in fundamental physiological
and pathological process such as embryological development,
migration, adhesion, proliferation and differentiation of cells
(Manzel and Farr, 1988; Heldin, 2003; Spicer and Tien, 2004),
immune surveillance, inflammation (Termeer et al., 2003;
George and Stern, 2004; Day and de la Motte, 2005; Jiang et al.,
2005), wound healing (Chen and Abatangelo, 1999), multi-drug
resistance (Toole, 2004), angiogenesis, malignant transformation, and water homeostasis and viscoelasticity of ECM (West
et al., 1985; Frost et al., 1996; McDonald and Camenisch, 2002;
Toole, 2004; Adamia et al., 2005). The biological functions
exhibited by HA depend on the chain length, molecular mass
and on the circumstances under which it is synthesized (Noble,
2002; Toole, 2004).
High and low molecular weight forms of HA exhibit
opposite effects on cell behaviour. Extracellular high molecular
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weight HA (HMWHA) inhibits endothelial cell growth, and is
thereby anti-angiogenic in nature. The HMWHA polymers have
an increased ability to bind fibrinogen; this is one of the first
reactions to occur in clot formation, which are critical in early
wound healing (Chen and Abatangelo, 1999). These HA
polymers are also anti-inflammatory and immunosuppressive
in nature (Day and de la Motte, 2005; Milner et al., 2006). Fetal
circulation and amniotic fluid contain high concentrations of
HMWHA, which may account for some of the immunosuppression in the developing fetus. HMWHA inhibits scar
formation in fetal wounds, whereas HA degradation products
contribute to scar formation (Noble, 2002). Furthermore,
production of HMWHA is increased at sites of inflammation,
often correlating with leukocyte adhesion and migration.
Several studies suggest that HMWHA can be organized to
generate wide superstructures with multiple architectures and
functional activities by its association with specific binding
proteins (Day and de la Motte, 2005; Milner et al., 2006).
Recently, it has been observed that peripheral blood monocytes
are stimulated through their binding to HMWHA chains but this
interaction induces the expression of growth factors and matrix
components rather than pro-inflammatory mediators (Day and
de la Motte, 2005). This indicates a counter-inflammatory or
anti-inflammatory role of highly crosslinked HMWHA.
Low molecular weight HA (LMWHA) fragments interact
with a different set of receptors that trigger signaling cascades
and profound changes in cell behavior. LMWHA has been
shown to promote angiogenesis in several experimental models
(West et al., 1985; Noble, 2002; Toole, 2004). Also, LMWHA
enhances the synthesis of type I and VIII collagen, which are
ECM molecules of the endothelial cell angiogenic phenotype.
Low and intermediate molecular weight (2 × 104–4.5 × 105) HA
oligosaccharides are potent stimulators of inflammatory cytokine and adhesion molecules (Noble et al., 1993). HA oligomers
increased cell migration and gene expression of MMP-9 and
MMP-13 in Lewis Lung Carcinoma (3LL) cells and in primary
embryonic fibroblasts (Fieber et al., 2003).
Studies on activated macrophages have shown that HA
fragments induce the expression of chemokine genes such as
macrophage inflammatory protein (MIP)-1α, MIP-1β, crg-2,
RANTES and monocyte chemotactic protein-1 (MCP-1), the
functions of which are crucial in initiating and maintaining the
inflammatory response (McKee et al., 1996). Furthermore,
expression of HAS-2, aggrecan, MMP-3, MMP-13 and iNOS
was stimulated by HA oligosaccharides in bovine chondrocytes
and human articular chondrocytes (Knudson and Knudson,
2004; Iacob and Knudson, 2006).
Shorter HA fragments have been reported to promote cell
proliferation of chondrocytes, endothelial cells and fibroblasts.
Iacob and Knudson (2006) have shown that the HA fragments
activate nitric oxide synthase and the production of nitric oxide
by articular chondrocytes. The activation of NO production by
HA fragments has been shown in macrophages, rat liver
endothelial cells, Kupffer cells, T-24, HeLa, MCF7, and J774
cells. In some cases, the stimulatory effects of HA fragments
were shown to be mediated by activation by nuclear factor
(NF)-κβ. Conversely, IL-10 and interferon-γ were found to
inhibit LMWHA-induced cytokine production in mouse bone
marrow-derived macrophages (Horton et al., 1999).
Smaller HA oligomers (6–20 kDa size range) are potent
activators of dendritic cells, the antigen presenting cells of the
immune system. Thus HA fragments tend to be angiogenic,
immuno-stimulatory, and inflammatory (Noble, 2002; Termeer
et al., 2002; Rossler and Hinghofer-Szalkay, 2002; Stern, 2003).
Angiogenic HA fragments stimulate endothelial cell proliferation, adhesion, and migration by activating focal adhesion
kinase and mitogen activated protein (MAP) kinase pathways
(Rossler and Hinghofer-Szalkay, 2002; Murai et al., 2004).
Most pro-inflammatory HA fragments can signal through TLR4 in dendritic cells and endothelial cells (Termeer et al., 2002;
Taylor et al., 2004). Recent studies confirmed that HA
oligomers require MyD88 and both toll like receptor 4
(TLR4) and TLR2 in vitro and in vivo to initiate the
inflammatory response in acute lung injury (Jiang et al., 2005).
Very small HA oligosaccharides also have unique specific
biological activities. Oligomers of six disaccharides promote
differentiation of the endothelial cells induced in response to the
angiogenic effect of larger HA fragments (Takahashi et al.,
2005). Moreover, control of migration, maturation and
signaling in skin keratinocytes is also closely associated with
small HA oligomers. Recently, Takahashi et al. investigated the
effects of HA oligomer (HA12) on differentiation of endothelial
cells at the molecular level, by using microarray approach. The
data revealed that the HA oligomer (HA12) induces 2-fold upregulation of the Vil2 gene, which encodes ezrin, the protein
that is associated with activated CD44 and participates in cell
shape changes, adhesion, motility, endocytosis/exocytosis, and
signal-transduction pathways. In addition, HA12 induces
expression of the myosin 1b gene, which belongs to myosin I
family of proteins and participates in a variety of cellular
processes, including membrane fusion/vesicle scission (Takahashi et al., 2005).
Tetra and hexasaccharides are predominant products of
hyaluronidase-mediated degradation. Tetrasaccharides induce
expression of heat shock proteins (Hsps) and are anti-apoptotic,
suppressing cell death in cultures undergoing hyperthermia (Xu
et al., 2002). Other studies have demonstrated that the addition
of tetrasaccharides inhibits anchorage-independent growth of
several tumor cell types by suppressing the phospho-inositol-3kinase/Akt survival pathway (Ghatak et al., 2002). Ohano et al.
have shown that hexasaccharides act as antagonists to
HMWHA, interfering with normal bovine chondrocyte cell–
matrix interactions such as assembly of pericellular matrix. In
addition, expression of transcription factors such as AP-1,
NFκB, p53, Sp1, and Stat4 was markedly up-regulated in
bovine articular chondrocytes after treatment with hexasaccharides (Ohno et al., 2005).
From these observations, it can be concluded that HA
catabolism depends on individual hyaluronidase activities under
pathological conditions and the products generated during
catabolic pathway with contrasting biological activities. In
addition, it would be more interesting to compare the biological
effects of HA degradation products generated by enzyme
dependent and independent pathways.
K.S. Girish, K. Kemparaju / Life Sciences 80 (2007) 1921–1943
For normal tissue organization and function, a balance
between HA synthesis and degradation is important. The
turnover of HA is relatively rapid compared to other ECM
molecules (Laurent and Fraser, 1986; Tammi and Tammi,
2006). High molecular weight tissue HA is degraded extracellularlly by chemical/mechanical means or enzymatic degradation by HA degrading enzymes. Non-enzymatically, HA is
degraded by reactive oxygen species (ROS) such as superoxide
anion radical (O2−), hydrogen peroxide (H2O2) and especially by
–OH radicals yielding several intermediate end products under
oxidative conditions (Deguine et al., 1998; Vercruysse et al.,
1999; Yamazaki et al., 2003). This type of degradation generally
occurs at sites of inflammation, tissue injury, and tumorigenesis
(Uchiyama et al., 1990). Interestingly, hyaluronidases are
endoglucosaminidases, whereas ROS degrade HA randomly
at internal glycosidic linkages.
Hyaluronidases
The hyaluronidases are a group of enzymes distributed
throughout the animal kingdom. In recent years hyaluronidases
have received attention and importance due to their regulatory
function in HA metabolism. Duran-Reynals observed that
extracts of mammalian testis and other tissues contain a
“spreading factor” which facilitated the diffusion of dyes and
antiviral vaccines injected subcutaneously (Duran-Reynals,
1928; Chain and Duthie, 1940). Karl Meyer introduced the
term “hyaluronidase” to denote the enzymes that degrade HA.
Meyer classified hyaluronidases into three different groups
based on biochemical analysis and generated end products
(Meyer, 1971). The three groups include: (1) Mammalian
hyaluronidases (testis type) (E.C. 3.2.1.35) that are endo-β-Nacetlyhexosaminidases and randomly cleave β-1-4 glycosidic
linkages in HA, chondroitin and chondroitin sulfates (A and C)
to yield even numbered oligosaccharides (tetra and hexa) as the
major end products, with N-acetylglucosamine at the reducing
terminal. Enzymes of this class have both hydrolytic and
transglycosidase activities (e.g. testicular hyaluronidase) present in mammalian spermatozoa, lysosomes and the venom of
snakes, reptiles and hymnoptera (Cramer et al., 1994). (2)
Hyaluronate-3-glycanohydrolases (E.C. 3.2.1.36) (Leech hyaluronidase) are endo-β-D-glucuronidases that cleave glucuronate
linkages of HA and are inert towards other GAGs. Tetrasaccharides and hexasaccharides are the main end products with
glucuronic acid at the reducing end of the product. This class
includes enzymes present in salivary glands of leeches and hook
worms (Hotez et al., 1992). (3) Microbial hyaluronidases (E.C.
4.2.99.1) cleave HA at β 1-4 glycosidic linkages using the βelimination process and yield Δ4–5 unsaturated oligosaccharides. These are different from the two other groups of
hyaluronidases, as they do not use hydrolysis in their activity.
Enzymes in this class include HA lyases from Streptococcus
pneumoniae (S. PHL) and S. agalactiae.
In addition, hyaluronidases are loosely classified into two
groups based on their pH dependent activity profile. Acid active
hyaluronidases are active between pH 3 and 4, and this group
includes human liver and serum hyaluronidases. Neutral active
1927
hyaluronidases are active between pH 5 to 8 and include PH-20,
snake venom, and bee venom hyaluronidases (Kreil, 1995;
Stern, 2004; Kemparaju and Girish, 2006).
Mammalian hyaluronidases
The enzymatic degradation of HA in mammals is mediated
through the coordinated activity of three separate enzymes,
namely a hyaluronidase, an endoglycosidase and two other
exoglycosidases that remove the terminal sugars, a β-glucuronidase and β-N-acetyl hexosaminadase. The initial degradation accomplished by hyaluronidase generates oligosaccharides
of different chain length and these are substrates for the two
exoglycosidases (Roden et al., 1989). Human hyaluronidases
have been reported and some of them are well characterized
from a number of tissues (Frost et al., 1996; Stern, 2003).
Six hyaluronidase like gene sequences have been identified
in humans. These hyaluronidase genes are also referred to as
hyaluronoglucosaminidase (HYAL) genes. These six paralogs
of HYAL genes are known to share about 40% of their identity
with one another. However, the expression of each gene has a
unique tissue distribution. HYAL1, HYAL2 and HYAL3 are
located on chromosome 3p21.3. Another set of three genes,
HYAL4, PHYAL1 (a pseudogene), and sperm adhesion
molecule 1 (SPAM 1) are clustered in a similar fashion on
chromosome 7p31.3. Hyal1 and Hyal2 are the major hyaluronidases expressed in human somatic tissues (Csoka et al.,
2001). In contrast, the mouse genome has seven hyaluronidaselike gene sequences (Miller et al., 2007; Reitinger et al., 2007).
Six of the seven genes are clustered in groups of three at two
chromosomal locations. Hyal1, Hyal2, and Hyal3 are located on
chromosome 9F1–F2. Hyal4, Spam1/PH-20, and Hyalp1 are
located on chromosome 6A2 (Csoka et al., 2001; Baba et al.,
2002; Zhang et al., 2005). The seventh mouse hyaluronidaselike gene, tentatively called Hyal5, is localized in close
proximity to the cluster at chromosome 6A2 (Kim et al.,
2005; Reitinger et al., 2007). Both Hyalp1 and Hyal5 share a
high degree of sequence similarity with biochemically characterized hyaluronidases. Hyal5 has a broad optimal pH range and
is active in both neutral and extracellular ionic pH (Reitinger
et al., 2007). Besides mice, Hyal5 is also present in rats
(accession number BC091219), suggesting that further gene
duplication has occurred in rodents (Zhang et al., 2005;
Reitinger et al., 2007). Mature spermatozoa of Spam1 deficient
mouse are fertile and exhibit hyaluronidase activity. This could
be due to the presence of functionally active Hyal5 on the
surface of Spam1 deficient spermatozoa. Furthermore, we can
assume both SPAM1 and HYAL5 enzymes participate in
spermatozoa fertilization of the egg.
Hyal1 was the first somatic hyaluronidase isolated and
characterized from human plasma (Csoka et al., 1999; Afify
et al., 1993). It is an acid active 57 kDa single polypeptide
glycoprotein that is also present in a processed 45 kDa form
generated by two endoprotease reactions. Only the high
molecular weight isoform is present in the circulation, and
both isoforms occur in urine, in tissue extracts and cultured cells
(Csoka et al., 1997). Hyal1 can use HA of any size as a substrate
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and generates predominantly tetrasaccharides/hexasaccharides.
Moreover, the mouse ortholog has also been cloned, expressed
and was observed to be 73% identical to the human enzyme
(Stern, 2004; Csoka et al., 1998).
Hyal2, also acid active, is anchored to plasma membrane by
a glycosylphosphatidyl-inositol (GPI) link (Lepperdinger et al.,
1998, 2001). It also occurs in a processed soluble form. Hyal2
cleaves high molecular weight HA to intermediate sized
fragments (approximately 20 kDa). Hyal1 and Hyal2 have
similar structures but produce different reaction products. Hyal2
is considered the more important enzyme of the two, Hyal2 null
mutation in the mouse is embryonically lethal and the Hya1
mutation is not. However, recent evidence indicates that the
Hyal2 knockout mice is not an embryonic lethal and that
knockout mouse has now been generated (Flamion B., personal
communication). Hya12 also functions as a receptor for
Jaagsiekte sheep retrovirus (JRSV) and enzootic nasal tumor
virus (ENTV). Human Hyal2 binds to the envelope proteins of
these viruses and serves as a functional receptor. Hyal2 from
mice, however, does not bind to envelope proteins nor does it
mediates entry of either virus (Duh et al., 2005).
Hyal3 is widely expressed, but no activity can be detected
using the available hyaluronidase assays (Csoka et al., 2001). It
occurs in chondrocytes, testis and bone marrow, and its
expression increases when fibroblasts undergo chondrocyte
differentiation (Flannery et al., 1998; Nicoll et al., 2002). Hyal3
and Hyal2 are up-regulated by inflammatory cytokines such as
IL-1 and tumor necrosis factor-alpha (TNF-α), whereas Hyal1
is not (Flannery et al., 1998).
The biochemical and functional characteristics of hyaluronidase genes were reviewed by Robert Stern (Stern, 2003).
Recently, Jedrzejas and Stern proposed the 3D model structures
for human Hyal enzymes Hyal1–4 and PH-20 (Jedrzejas and
Stern, 2005). The catalytic cleft and the active sites of these
enzymes are highly conserved. However, the C-terminal
domain differs both in sequence and structural functionality
among all the Hyal enzymes. All known hyaluronidases are
active at acidic pH, consistent with a lysosomal location, except
PH-20 and Hyal2, which are active over a broad optimal pH
range (Vigdorovich et al., 2005). In addition, Gatphayak et al.
(2004) characterized a cluster of porcine three hyaluronidase
genes (HYAL1, HYAL2 and HYAL3). The porcine hyaluronidase genes share approximately 40% homology with one
another. The porcine cDNA and protein sequences share
homologies to human orthologs of 85% and 81% for HYAL1,
87% and 89% for HYAL2 and 86% and 83% for HYAL3
respectively. Research on these enzymes was barely investigated until recently due to their instability and other associated
problems such as isolation, purification and activity assays.
The vertebrate enzymes are hydrolases, while the enzymes
from microbial source are eliminases. The microbial hyaluronidases can be assayed by spectrophotometric methods that detect
the formation of an unsaturated bond during the enzymatic
degradation of substrates (Baker et al., 2002; Jedrzejas, 2004;
Stern and Jedrzejas, 2006). The hydrolases are relatively deserted
in comparison with other glycosidase, most likely due to the
unavailability of simple and sensitive assay method that measure
degradation of substrate (Manzel and Farr, 1988; Afify et al.,
1993; Kemparaju and Girish, 2006). The most commonly used
hyaluronidase assays were based upon the measurement of the
generation of new reducing N-acetyl glucosamine or loss of
viscosity or turbidity. These methods are either less sensitive or
lack of specificity. In addition, there are problems associated with
the presence of hyaluronidase inhibitors and proteins present in
medium, cell and tissue extracts. However, recent superior
detection procedures for vertebrate hyaluronidase activity such as
substrate gel assay, ELISA like assay and fluorescence-based
assays have facilitated their identification and isolation (Afify
et al., 1993; Csoka et al., 1997; Jedrzejas and Stern, 2005; Stern
and Jedrzejas, 2006).
Among the mammalian hyaluronidases, testis hyaluronidases are very well studied and characterized. Mammalian
sperm have a GPI-anchored hyaluronidase which is known as
PH-20 and also as SPAM-1. PH-20 is located on the sperm
surface and in the lysosome-derived acrosome, where it is
bound to the inner acrosomal membrane. PH-20 facilitates
penetration of sperm through the cumulus ECM and zona
pellucida of the ovum, and is also necessary for fertilization.
Several studies have confirmed that PH-20 is the only
hyaluronidase in mammalian sperm, including the sperm of
guinea pigs, rats, macaques and humans (Cherr et al., 2001;
Zheng et al., 2001). It is originally synthesized as a polypeptide
with an apparent molecular weight of 64 kDa. During the course
of sperm maturation, part of PH-20 is processed into two
fragments that are linked through disulfide bridges, such as one
at N-terminal domain of 41–48 kDa and a C-terminal domain of
27 kDa. PH-20 is unique among hyaluronidases, in that it shows
enzyme activity at both acidic and neutral pH and that these
activities appear to involve two different domains in the protein
(Gmachl and Kreil, 1993; Cherr et al., 2001; Oettl et al., 2003).
Using more sensitive techniques involving PCR analysis, PH20 have been detected in the epididymis, seminal vesicles,
prostate, female genital tract, breast, placenta and fetal tissue, as
well as in certain malignancies (Beech et al., 2002; Zhang and
Martin-DeLeon, 2003; Oettl et al., 2003). These studies have
further confirmed that PH-20 is a multifunctional enzyme that is
not sperm-specific and also has functions in addition to its
hyaluronidase activity.
Venom hyaluronidases
Hyaluronidases are often found in all types of animal
venoms. Hyaluronidase activity has been detected in the venom
of snakes (Kudo and Tu, 2001; Girish et al., 2004b; Kemparaju
and Girish, 2006), bees (Gmachl and Kreil, 1993; MarkovicHousley et al., 2000), stonefish (Poh et al., 1992; Ng et al.,
2005), scorpions (Ramanaiah et al., 1990; Pessini et al., 2001;
Morey et al., 2006), spiders (Rash and Hodgson, 2002;
Nagaraju et al., 2006), lizards (Tu and Hendon, 1983), wasps
(Kreil, 1995), caterpillars (da C B Gouveia et al., 2005) and
hornets (Lu et al., 1995). In all animal venoms, the
hyaluronidase causes local tissue damage and is generally
referred to as a spreading factor. The spreading property of the
enzyme is presumed to be the critical event in spreading toxins
K.S. Girish, K. Kemparaju / Life Sciences 80 (2007) 1921–1943
from the site of injection to systemic circulation. This process is
accomplished by the degradation of HA and the eventual loss of
integrity in the ECM of soft connective tissues surrounding the
blood vessels, leading to the easy diffusion of the toxic
components of venom (Girish et al., 2004b; Kemparaju and
Girish, 2006). Many venom hyaluronidases have a sequence
homology of about 36% with that of spermatozoan PH-20
(Table 4).
The first marine hyaluronidase was isolated and cloned from
venom of the stonefish Synanceja horrida. The purified enzyme
is a β-endo-N-acetyl hexosaminidase, acts on β-1–4 glycosidic
linkages and yields tetrasaccharides as the major end product.
The enzyme is a 62 kDa glycoprotein with a pI of 9.2 and is
non-toxic in nature (Poh et al., 1992; Ng et al., 2005).
Hyaluronidase from lizards, scorpions, spiders, caterpillars,
wasps and hornets have been isolated and characterized to some
extent (Kemparaju and Girish, 2006, references therein).
Among these, the enzyme from bee venom has been
investigated in depth, being the first eukaryotic hyaluronidase
cloned via cDNA by using the N-terminal sequence. The mature
enzyme is a globular protein composed of 349 amino acids with
a calculated molecular mass of 40.746 kDa (Gmachl and Kreil,
1993). It is secreted as a basic glycoprotein and has 7%
carbohydrate content when compared to the total protein mass.
It contains four cysteine residues that are present as disulfide
bonds in the extracellular enzyme. The enzyme is acid-active
and has three potential N-glycosylation sites, though only one
site appears to be glycosylated. This enzyme is derived from a
precursor containing a signal peptide of 24 or 28 amino acids
and a short pro-segment. Bee hyaluronidase is an endo-Nacetyl-D-glucosaminidase and yields tetrasaccharides as a major
end product (Gmachl and Kreil, 1993). Expression of the cloned
cDNA in E. coli resulted in a polypeptide with hyaluronidase
Table 4
Biochemical and biophysical properties of animal venom hyaluronidases
Venom
Molecular
pI
weight (kDa)
Optimum Optimum PAS
pH
temp. °C staining
A. acutus
A. contortrix contortrix
N. naja
(isoforms)
H. fulvipes
T. serrulatus
A. mellifera
S. horrida
H. horridum horridum
L. oblique
(isoforms)
L. reclusa
(isoforms)
D. maculata
V. germanica
C. limpidus limpidus
P. regosus
V. vulgaris
Palamneus gravimanus
33
59.2
70.4
52
82
51
41
62
63
49
53
33
63
39
42
47.5
36
43
52
5
6
5
5
4
6
6
6
5
6–7
6–7
5–6.6
5–6.6
5–6
5–6
5–6
5–6
5–6
4.5
10.3
9
9.2
9.7
ND
ND
9
9.2
5.1
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
37
37
37
37
30
40
37
37
Sensitive
37
37
37
37
37
37
37
37
37
37
+
ND
−
−
ND
ND
+
ND
+
ND
ND
ND
ND
ND
ND
ND
ND
+
ND
PAS = periodic acid staining; ND = not determined; + = positive; − = negative.
1929
acitivity. Markovic-Housley et al. (2000) co-crystallized bee
venom hyaluronidase with HA tetramer. The crystal structure
analysis revealed the presence of an unusual overall fold, a (β/
α)7 barrel instead of a regular (β/α)8 barrel. The HA binding site
is situated at the C-terminal end of the β barrel and is lined with
many conserved amino acids. Moreover, the structure of
complex strongly suggests an acid-base catalytic mechanism,
with Glu113 acting as a proton donor and the N-acetyl carboxyl
groups of HA acting as the nucleophilic base. The enzyme is
closely related to human PH-20 and human plasma hyaluronidase. The amino acid sequence of hornet venom hyaluronidase
has also been determined via cDNA cloning. The enzyme is
comprised of 331 amino acids, of which 56% are identical to
bee venom hyaluronidase (Lu et al., 1995).
Hyaluronidase, an ubiquitous enzyme in snake venom
originally known as “spreading agent”, has not been well
studied. Recently, we reported the purification of two
hyaluronidase isoforms from Indian cobra (Naja naja) venom
and provided systematic evaluation of the spreading property of
the enzyme. The hyaluronidases NNH1 and NNH2 were
purified through gel permeation and ion exchange chromatography. The molecular mass NNH1 was found to be 70.406 kDa
by MALDI-TOF mass spectrometry, and it has a pI of 9.2. Both
the isoforms are active at pH 5 to 7 and belong to the group of
neutral active enzymes. Enzymes show absolute specificity for
HA and generate tetrasaccharides as the final end product
having N-acetylglucosamine at the reducing terminal. Hyaluronidases cleave β 1,4-glycosidic linkage and belong to a group
of endo-β-N-acetyl hexosaminidases. The enzyme indirectly
potentiates the myotoxicity of VRV-PL-VIII, a phospholipolytic
myotoxin, and also the hemorrhagic potency of hemorrhagic
complex-I. Localization of hyaluronan in human skin section
and selective degradation by venom hyaluronidase NNH1 and
NNH2 corroborate the plausible in vivo degradation of
hyaluronan in the ECM, resulting in the easy dissemination of
VRV-PL-VIII myotoxin and hemorrhagic complex-I (Girish
et al., 2004b; Girish and Kemparaju, 2005a).
Hyaluronidase is a major allergen of scorpions, bees, hornets
and wasps that can induce serious and occasionally fatal
systemic IgE-mediated anaphylactic reactions in humans (Lu
et al., 1995; Kolarich et al., 2005). Knowledge of the structural
determinants responsible for its allergic potency is expected to
have important clinical implications. Furthermore, it will be
exiting to understand the in vivo fragmentation of HA by venom
hyaluronidases and the subsequent effects on pathophysiology
of envenomation.
Microbial hyaluronidases
In microbes, HA lyases are virulence factors involved in
pathogenesis and disease progression caused by the pathogen.
Often HA lyases directly interact with host tissues or conceal
the bacterial surface from host-defense mechanisms. The
enzymatic degradation of ECM components of host tissues
facilitates the invasion of pathogens (Table 5). Enhanced tissue
permeability caused by the action of HA lyases on ECM
appears to play a major role in wound infections, pneumonia
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K.S. Girish, K. Kemparaju / Life Sciences 80 (2007) 1921–1943
and other types of sepsis such as bacteremia and meningitis
(Sutherland, 1995; Matsushita and Okabe, 2001; Jedrzejas,
2001, 2004; Makris et al., 2004). The HA degrading activities of
microbes may facilitate adhesion, colonization and provide
nutrients (Hynes and Walton, 2000). Disaccharides are the
major end products of HA degradation and these can be
transported and metabolized intracellularlly to supply needed
nutrients (as a carbon source) for a pathogen as it replicates and
spreads (Matsushita and Okabe, 2001; Jedrzejas, 2001, 2004;
Makris et al., 2004). In host tissues, high molecular mass HA is
involved in the regulation of immune system and is antiinflammatory in nature (Toole, 2004). In contrast, HA
oligomers generated by microbial HAases are potent inflammatory agents and promote microbial-friendly environment
(Hynes and Walton, 2000).
HA lyases are produced by many different genera of bacteria.
In gram-positive bacteria, the HA lyases are often secreted and
are thought to have a role in pathogenesis (Hynes and Walton,
2000; Spellerberg, 2000; Makris et al., 2004). It has been noted
that all gram-positive bacteria that produce HA lyases appear to
be capable of causing infections in animals (Table 6). Grampositive organisms capable of producing HA lyases include
various species of Streptococcus, Staphylococcus, Peptostreptococcus, Propionibacterium, Streptomyces and Clostridium.
HA lyase production has been reported in Streptococci groups
A, B, C and G as well as S. pneumoniae (Fitzgerald and Gannon,
1983; Berry et al., 1994; Gunther et al., 1996). HA lyase is also
produced by gram-negative bacteria but is less likely to play a
role in pathogenesis. HA lyase and chondroitin lyase activities
have been reported from Aeromonas, Vibrio, Beneckea and
Proteus. Bacteroides fragilis, Bacteroides vulgatus, B. ovatus,
B. melaninogenicus and Fusobacterium mortiferum are also
reported to produce hyaluronidase. The pathogens Treponema
pallidum and T. pertenue both produce surface associated HA
lyases, whereas the non-pathogenic T. denticola and T. vencentii
do not produce such an enzyme (Linhardt et al., 1986; Sting
et al., 1990). Generally, HA lyases from gram positive and
Table 5
Biochemical and biophysical properties of HA lyases
Source
Molecular
pI
weight (kDa)
Streptomyces
33
hyalurolyticus
Streptococcus pyogenes 39.5
H4489A
Clostridium perfringens 48
Streptomyces
121
agalactiae
S. dysgalactiae
55
S. intermedicus
115
Staphylococcus aureus
Propionibacterium
acnes
Peptostreptococcus sps
84
85
160
pH
Temp. Substrate
°C
specificity
10.3
5
37
HA
9.28
6
37
HA
4.4
8.9
5.7–6.1 37
6.3
37
∼ 5.0
9.3
5.8
5
40
37
7.4–7.9 4.8–6
ND
5–5.8
37
37
ND
37
6.5
HA, C0S
HA, C0S,
C6S
HA, C0S
HA, C0S,
C4S, C6S
HA
HA. C4S,
C6S
HA, C4S,
C6S
HA = hyaluronan; C0S = chondroitin sulfate; C4S = chondroitin 4 sulfate;
C6S = chondroitin 6 sulfate; ND = not determined.
Table 6
List of hyaluronate lyase producing microorganisms
Gram-negative bacteria
Aeromonas, Vibrio, Beneckea, Proteus vulgaris, Bacteroides fragilis,
B. vulgatus, B. ovatus, B. melaninogenicus, B. asaccharolyticus,
B. tgetaiotaomicron, Fusobacterium mortiferum, Treponema pallidum and
T. pertenue.
Gram-positive bacteria
Streptococcus pneumoniae, S. intermedicus, S. constellatus, S. dysgalactiae,
S. uberis, S. suis, Staphylococcus hyicus subsp. hyicus, S. aureus.
Clostridium difficile, C. perfringens, C. septicum, C. chauvoei.
Mycoplasma alligatoris, M. crocodyli, Propionibacterium acnes,
P. granulosum, Streptomyces coelicolor, S. hyalurolyticus, S. griseus.
Peptostreptococcus.
Bacteriophage
Streptococcus pyogenes, S. equi
Fungi
Candida albicans, C. tropicalis, C. guillermondii, C. parapsilosis, C. krusei.
Paracoccidioides brasiliensis, Pygocelis adeliae, Dasypus novemcinctus
negative organisms are high molecular weight proteins compared to phage related hyaluronidases. The well-characterized
hyaluronidases reported thus far have varied molecular weight,
as represented by S. pneumoniae (107 kDa) S. agalactiae
(121 kDa), C. perfringens (114 kDa), S. aureus (92 kDa) P.
acnes (82 kDa) and two enzymes from Streptomyces sp (77 and
84 kDa). The molecular weight of other non-sequenced HA
lyases also vary from 50 to 160 kDa (Sting et al., 1990;
Kostyukova et al., 1995; Hynes and Walton, 2000). HA lyases
are known to be involved in spreading toxins and pathogens in
various disease conditions, including gas gangrene, meningitis,
synovitis, hyperplasia, nephritis, mycoplasmosis, periodontal
disease, mastitis, pneumonia, septicemia, syphilis and toxic
shock syndrome (Li et al., 2000b; Spellerberg, 2000; Hynes and
Walton, 2000; Jedrzejas, 2001; Makris et al., 2004).
Among microbial hyaluronidases, the HA lyases from
S. pneumoniae (S. PHL) and P. agalactiae are well characterized. The 3D X-ray crystal structure of S. PHL indicates that the
enzyme is a globular protein with two distinct structural domains
connected by a short peptide linker. The two domains are
spherical and are of similar size. The N-terminal α-helical
domain contains the first 361 amino acid residues of the
functional form of the enzyme. The C-terminal β-sheet domain
contains 347 residues, and Asn 349, His 399 and Tyr 408 are
identified as catalytic residues in the active enzyme. Moreover, a
recent X-ray crystallography report on the structure of
S. agalactiae HA lyase has revealed the similar structure of
S. PHL in the architecture of the entire enzyme as well as in the
active site geometry (Jedrzejas, 2001, 2000; Stern and Jedrzejas,
2006). The active center of these HA lyases is composed of two
main parts, a catalytic group responsible for substrate degradation and an aromatic patch responsible for the selection of
cleavage sites on the substrate chains (Jedrzejas, 2000). The
hyaluronate lyases from S. pneumoniae, S. agalactiae and
S. aures showed a high degree of similarity, with global
similarities above 65% and local similarities of about 80%
(Jedrzejas, 2000; Baker et al., 2002; Stern and Jedrzejas, 2006).
Through X-ray crystallography studies including (a) native
enzyme, (b) complexes between the enzyme and degradation
K.S. Girish, K. Kemparaju / Life Sciences 80 (2007) 1921–1943
products such as disaccharides, tetrasaccharides and hexasaccharides, and (c) site-directed mutagenesis, Jedrzejas (Jedrzejas,
2000; Stern and Jedrzejas, 2006) proposed a detailed catalytic
mechanism called the proton acceptance and donation (PDA)
mechanism. This mechanism involves an acceptance of proton
by the enzyme followed by the donation of a different proton
from the enzyme to the β 1-4 glycosidic oxygen. The
degradation process consists of five distinctive steps:
(1) enzyme binds negatively charged substrate in the binding
cleft using electropositive residues constituting the
hydrophobic patch;
(2) a catalytic step involving catalytic residues Asn 349, His
399, and Tyr 408 results in the cleaving of β 1-4 bond
with the generation of disaccharide end product;
(3) hydrogen exchange between enzyme and the water
microenvironment in order to return the enzyme to its
natural state ready for the next round of catalysis;
(4) the irreversible step of the release of the disaccharide
product by utilizing negative patch in the cleft, and finally
(5) a translocation of the remaining HA by one disaccharide
unit towards the substrate's reducing end.
Several bacteriophages from the HA-encapsulated group A
Streptococci are known to synthesize a bound form of HA
lyase (Baker et al., 2002). It has been suggested that the
biological function of bacteriophage HA lyase is to degrade the
HA capsule of the bacterial host cell, thereby facilitating
attachment of phage to the cell wall. Phage HA lyases are low
molecular weight (25 to 45 kDa) enzymes compared to bacterial
hyaluronidases (50–160 kDa). Several bacteriophages from
HA-capsulated group A streptococci are known to produce
hyaluronidases and nucleotide sequences of hyaluronidase
genes hylPp1 and hylPp2 from two bacteriophage of Streptococcus pyogenes H4489A and 10403 respectively, have been
reported. The sequences of hylPp1 and hylPp2 are similar but
showed no sequence homology with the HA lyases from
S. pneumoniae and S. agalactiae. Phage HA lyases degrade HA
and other GAGs to a limited extent (Hynes and Ferretti, 1989;
Jedrzejas, 2001; Baker et al., 2002; Jedrzejas et al., 2002).
Candida and Paracoccidioides species are the most
prevalent invasive fungal pathogens affecting human. The
fungal infection affects lungs and can invade other organs and
tissues, forming secondary lesions in the mucous membranes,
skin, lymph nodes and adrenal glands. These strains produce
various enzymes, including HAases. Reports indicate that
HAases and chondroitinases play an important role in
pathogenecity of fungi, particularly species of genus Candida
and Pracoccidiodes (Shimizu et al., 1995; de Assis et al., 2003).
However, none of the enzymes from the fungal species have
been well characterized.
Hyaluronidase inhibitors
As mentioned earlier, the key role of hyaluronidases has been
recognized in a number of physiological and pathological
processes such as embryogenesis, angiogenesis, inflammation,
1931
disease progression, wound healing, bacterial pathogenesis and
the diffusion of systemic toxins/venoms. Therefore, identification and characterization of hyaluronidase inhibitors would be
valuable for developing contraceptives, anti-tumor agents, antimicrobial and anti-venom/toxin agents.
Inhibitors of hyaluronidases are potent, ubiquitous regulating
agents which are involved in maintaining the balance between
the anabolism and catabolism of HA. In general, documented
hyaluronidase inhibitors are of different chemical forms namely
proteins, glycosaminoglycans, polysaccharides, plant derived
bioactive components and synthetic organic compounds
(Table 7) (Mio and Stern, 2002; Khanum et al., 2005; Salmen
et al., 2005; Girish and Kemparaju, 2005a,b; Isoyama et al.,
2006; Machiah et al., 2006).
Haas, for the first time reported the existence of a circulating
inhibitor of hyaluronidase in serum (Haas, 1946). Furthermore,
a number of clinical studies report increased levels of inhibitor
in the serum of patients with cancer, liver disease and
dermatological disorders (Grais and Glick, 1948; Snively and
Glick, 1950; Newman et al., 1955; Kolarova, 1975). Based on
these studies, Mio et al. (2000) purified a high molecular mass
(120 kDa), thermolabile glycoprotein hyaluronidase inhibitor
from mouse serum. The inhibitory activity was protease
sensitive and magnesium dependent and showed maximum
inhibition at pH between 6 and 8. The inhibitor also inhibits
bovine testis, snake and bee venom enzymes to a varied extent,
but no inhibition towards Streptomyces HA lyase. The level of
serum inhibitor in mice was increased following CCl4 or IL-1
injection, inducers of the acute phase response. This inhibitor
has tentatively been identified as a member of inter-α-inhibitor
family (Mio et al., 2000).
Heparin is an acidic, negatively charged and highly sulfated
GAG having repeating disaccharide units of glucuronate-2sulfate (or Iduronic-2-sulfate) and N-sulfo-D-glucosamine-6sulfate linked by an α(1–4) glycosidic linkage, and is a known
and well-characterized inhibitor of hyaluronidase (Wolf et al.,
1984). It inhibits the HA degradation activity of hyaluronidase
through a non-competitive mechanism, suggesting that heparin
does not bind to the catalytic site of the enzyme. This was
further confirmed by chemical modification of hyaluronidase by
aldehyde dextran (Maksimenko et al., 2001). In this study, the
inhibitory effect of heparin decreased with increased modification of surface aminogroups of hyaluronidase, indicating that
the inhibitor does not bind to the catalytic site but instead
interacts with surface amino groups of the enzyme. At lower
concentrations, heparin inhibits venom hyaluronidases more
efficiently than bovine enzyme. In contrast, leech and Streptomyces hyaluronidases were not sensitive to heparin (Mio and
Stern, 2002). In addition, heparan sulfate and dermatan sulfate
inhibit human serum and venom hyaluronidase (Afify et al.,
1993; Girish and Kemparaju, 2005a,b). Furthermore, heparin
also inhibits several snake venom PLA2 enzymes through the
formation of electrostatic interactions between negatively
charged GAG and basic PLA2 enzymes (Melo and Ownby,
1999; Mors et al., 2000).
Toida et al. (1999) reported the inhibitory activity of Osulfated HA fragments on hyaluronidase. Their inhibitory
1932
K.S. Girish, K. Kemparaju / Life Sciences 80 (2007) 1921–1943
Table 7
List of different class of hyaluronidase inhibitors
Type of compound
Compounds
Source of hyaluronidase
Alkaloids
Antioxidants
Anti-inflammatory
drugs
Terpenoids/
flavonoids
Synthetic
compounds
Snake venom
Snake venom, microbial
Snake venom, bee venom,
testis
Snake venom, testis, microbial
Glycosaminoglycans
and glycosides,
Fatty acids
Polysaccharides/
oligosaccharides
Other proteins
Aristolochic acid, ajmaline, reserpine
Ascorbic acid, NDGA, N-propyl gallate, BHT, chlorogenic acid, curcumin, tannic acid
Dexamethasone, indomethacin, sodium cromoglycate, salicylates, tranilast, sodium aurothiomalate,
myocrisin, gossypol,
Flavone, Fenoprofen, Quercetin, Apigenin, Kaempferol, Silybin, Luteolin, Hesperidin, Triterpenes,
Rutin, Myricetin, Glycyrrhizin, Glycyrrhetinic acid
PS53 (Hydroquinone-sulfonic acid-formaldehyde polymer), phosphorylated hesperidin, polymer of
poly (styrene-4-sulfonate), sodium cellulose sulfate, 1-tetradecane sulfonic acid, L-arginin derivatives,
traxanox, norlignane, urolithin B, aescin, diphenylacrylic acids, diphenyl propionic acids, indole
derivatives, chalcone derivatives.
Heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate (A, C, D), O-sulfated HA, linamarin,
amygdalin,
Saturated (C10:0 to C22:0), cis-unsaturated fatty acids (C14:1 to C24:1)
Chitosans, dextran sulfate, sodium alginate, planteose derivatives, hydrochinone digalactoside,
2-hydroxyphenyl manolactobioside, sulphated neomycin, verbascose, lanostanoids
Withania somnifera glycoprotein (WSG), Serum hyaluronidase inhibitor
Other reagents
HCN, L-NAME, L-arginine, Guanidium HCl
strength is greater than other known inhibitors and O-sulfated
GAGs. Inhibition increased with increasing size of the sulfate
oligomer. Small sulfated oligosaccharides such as tetra to
decasaccharides did not show any significant inhibition, while
oligomers (16–20) inhibit the enzyme through competitive and
non-competitive basis. These results confirmed that the
inhibition is not only due to the electrostatic interaction but
also depends on the chain length of O-sulfated HA
oligosaccharides.
Recently, Isoyama et al. (2006) evaluated the activity of 21
different HAase inhibitors against HYAL-1, testicular, honeybee and Streptomyces HAases. Among the inhibitors tested,
polymers of poly (styrene-4-sulfonate) and O-sulfated HA
derivatives were found to be most effective. HYAL-1 and bee
venom HAase were found more sensitive than testicular HAase.
In contrast, Streptomyces HAase showed resistance to all
inhibitors except PSS 990.000 and VERSA-TL 502 (PSS
10 6 Da). Other well-known HAase inhibitors, including
heparin, gossypol, fenoprofen, sodium-aurothiomalate, 1-tetradecane sulfonic acid, and glycerrhigic acid, were not effective.
Among these, gossypol and fenoprofen did not show any effect
on all the HAases. Furthermore, 1-tetradecane sulfonic acid and
glycerrhigic acid only weakly inhibited HYAL-1 activity. Both
PSS and O-sulfonated HA derivatives showed mixed inhibition
mechanisms (Compitative and uncompitative) towards HAases.
This report demonstrated that the HAase inhibitors showed
selectivity towards acidic and basic HAases.
Chitosan is a positively charged homopolymer of glucosamine units with β(1-4) linkages and is a deacetylated
derivative of chitin. This polysaccharide mediates a variety of
biological effects, including antiangiogenesis (Harish Prashanth
and Tharanathan, 2005; Kato et al., 2005), antimicrobial (Rabea
et al., 2003), and immune enhancing activities (Singla and
Chawla, 2001; Senel and McClure, 2004). In addition, chitosan
inhibits HA degradation by venom and bovine testicular
hyaluronidases. The inhibitory property is directly related to
the chain length and inhibition varies in proportion to molecular
Snake venom, bee venom,
testis, Hyal-1
Snake venom, human serum,
bee venom, testis
Microbial, testis
Snake venom, testis, bee
venom, HA lyase
Snake venom, bee venom,
Hyal-1, testis
HA lyase
weight: high molecular weight N medium molecular weight N
low molecular weight of chitosan (Girish and Kemparaju,
2005a). Denuziere et al. (2000) reported the complex formation
between chitosan and HA that resulted from the electrostatic
interaction between the NH3+ functionalities of chitosan and
COO− groups of HA. The complex formation limits the
substrate available for the enzyme, and this could possibly be
the reason for exertion of in vitro inhibition. Furthermore,
Asada et al. (1997) examined the effect of various types of
alginic acid consisting of L-glucuronic acid and D-mannuronic
acid, on the bovine testicular hyaluronidase. These studies were
stimulated by the structural resemblance of alginic acid to HA.
Hyaluronidase inhibition also depends on the molecular mass of
sodium alginate, as inhibition increases with increasing mass.
Suzuki et al. (2002) screened saturated and unsaturated fatty
acids for hyaluronidase and chondroitinase inhibition. Among
the studied fatty acids, saturated fatty acids did not show any
inhibition of ovine testis, Streptomyces hyalurolyticus and
Streptococcus dysgalactiae hyaluronidases. In contrast, cisunsaturated fatty acids containing one double bond inhibit the
hyaluronidase of S. dysgalactiae and four chondroitinases from
microbial sources. However, the fatty acids did not inhibit testis
and Streptomyces hyaluronolyticus hyaluronidases. Moreover,
isomers of oleic acid such as eicosatrienoic acid and nervonic
acid inhibit hyaluronidases and chondroitinases through a noncompetitive mechanism. These results clearly indicate that
the cis-unsaturated fatty acids can inhibit the enzyme and existence of the double bond is essential for inhibition. However,
the position of the double bond and the cis-trans form of the
fatty acid molecule did not greatly influence inhibition, and
fatty acids bind to a different site other than active site of
enzyme.
Zaneveld et al. (2002) identified and showed that the
polymer obtained from mandelic acid condensation polymer
inhibits the hyaluronidase activity as well as acrosin. The
authors claimed that the polymer is not cytotoxic to Lactobacilli
and it has no effect on the percentage of moving spermatozoa
K.S. Girish, K. Kemparaju / Life Sciences 80 (2007) 1921–1943
(sperm motility). It is an effective inhibitor of HIV, herpes
viruses 1 and 2 (HSV-1 and HSV-2), Neisseria gonorrhoeae
and has some effect on Chlamydia trachomatis. It is not
mutagenic, has low acute and oral toxicity and it is safe in the
rabbit vaginal irritation assay. Authors considered the mandelic
acid condensation polymer to be a very safe contraceptive with
broad-spectrum antimicrobial activity. Recently, Garg et al.
(2005) showed that sulphonated hesperidin, a citrus flavonoid
glycoside, completely inhibits the sperm enzyme hyaluronidase.
It was also found to inhibit various sexually transmitted
pathogens, including HIV and HSV-2.
Ascorbic acid is a well known antioxidant and is involved in
many biological processes. Botzki et al. (2004) reported the
inhibition of HA lyase from S. pneumoniae (S. pnHL) by
ascorbic acid and also reported the crystal structure of the
enzyme with vitamin C. It indicates that only one vitamin C
molecule binds S. pnHL at the active site and forms about 25
interactions with seven residues (mainly from the N-terminus
and only one from the C-terminal domain) of S. pnHL. In
addition, Okorukwu and Vercruysse (2003) reported inhibition
by structural analogs of vitamin C on bovine testis and S.
hyalurolyticus HAases. Among those tested, L-ascorbic acid
and D-isoascorbic acid inhibited both the enzymes but showed
stronger inhibitory capacity towards the HA lyase. But these
analogs were observed to degrade the substrate, HA, by
themselves. In contrast, D-saccharic 1–4 lactone did not inhibit
testicular enzyme but exhibited over 50% inhibition towards
HA lyase and chondroitinase ABC without affecting the
physico-chemical stability of HA. Since these are virulence
factors produced by a number of causative pathogenic
organisms, saccharic acid can serve as a potent inhibitor of
HA lyases and as a novel antibacterial agent, thus providing an
alternative to antibiotics. Furthermore, vitamin C did not inhibit
N. naja venom and ovine testicular hyaluronidases up to the
studied concentration of 500 mM (Girish and Kemparaju,
2005a,b).
Akhtar and Bhakuni (2003) screened Gdn HCl (guanidine
hydrochloride), L-arginine, C-NAME (nitro-L-arginine methyl
ester) and GdnHSCN (guanidine isothiocyanate) for its antihyaluronate lyase activity. All these compounds inhibited the
enzymatic activity of S. pnHL in a dose dependent manner.
Guanidine HCl completely inhibited the enzyme activity (about
350 mM) without altering structural properties of the enzyme.
These observations suggested that GdnHCl, L-arginine, LNAME, and GdnHSCN affect the active site of the enzyme at a
concentration much lower than that required for bringing out
any significant structural change in the enzyme. Therefore, the
authors have claimed that these studies present a new chemical
entity that can be exploited for better designing of inhibitors for
S. pnHCL.
Aristolochic acid (8-methoxy-6-nitrophenanthro (3,4,D-)
1,3-dioxole-5-corboxylic acid) is a nitro compound with a
phenanthrene nucleus. Aristolochic acid completely neutralizes ovine testis and snake venom hyaluronidases. In
contrast, ajmaline and reserpine inhibits the enzyme activities
considerably less than aristolochic acid. It completely inhibits
the hyaluronidases from N. naja venom through a non-
1933
competitive mechanism. The interaction of aristolochic acid
with hyaluronidase was confirmed by spectoflurophotometer
analysis in which the aristolochic acid quenched the pronounced fluorescence intensity of hyaluronidase. The quenching was found to increase with increasing concentration. From
these studies, it is clear that the aristolochic acid does not bind
to the catalytic site but interacts with exposed tyrosine and
tryptophan residues of hyaluronidase. In addition, the aristolochic acid completely neutralized the enzyme-mediated
potentiation of toxicity of VRV-PL-VIII myotoxin and
hemorrhagic complex-I. Inhibition of HA degradation by
hyaluronidase in human skin and muscle tissue sections by
aristolochic acid supports the possible contribution of in vivo
degradation of HA in hyaluronidase mediated enhanced
toxicity of toxins. Interestingly, aristolochic acid increased
the survival time of experimental mice injected with N. naja
venom (Girish and Kemparaju, 2005a, 2006).
Certain anti-inflammatory drugs including salycylates,
indomethacin and dexamethasone also exert anti-hyaluronidase
activity (Mio and Stern, 2002; Girish and Kemparaju, 2005a,b).
These drugs may prevent HA degradation by hyaluronidases,
though the degradation products of HA are potent inducers of
inflammatory cytokines (Horton et al., 1998; Noble, 2002).
Anti-allergic drugs such as disodium cromoglycate, sodium
aurothiomalate, and transilist were evaluated and found to
possess hyaluronidase inhibitory activity. Sodium cromoglycate
and sodium aurothiomalate completely inhibited venom
hyaluronidase. In addition, these two drugs neutralized the
local (edema, hemorrhage) as well as systemic (myotoxicity,
survival time) toxicity exhibited by Naja kauothia and Calloselasma rhodostoma venoms (Yingprasertchai et al., 2003).
Dexamethasone, phenyl butazo indomethacin, oxybutazone and
fenofrofen inhibited snake venom hyaluronidase in a dose
dependent manner (Szary et al., 1975; Girish and Kemparaju,
2005a,b). These inhibitors of snake venom hyaluronidases can
be used as first-aid components for snakebite therapy.
Generally, plant derived bioactive compounds are used to
block hyaluronidase activity. These include flavonoids (Kuppusamy and Das, 1991; Pessini et al., 2001; Hertel et al., 2006),
tannins (Kakegawa et al., 1985), antioxidants (Girish and
Kemparaju, 2005a), pectins, curcumins (Tonnesen, 1989),
coumarins, gylcyrrhizin (Furuya et al., 1997), hydrogenols
from hydrangea (Kakegawa et al., 1988), glycoproteins
(Machiah et al., 2006), anti-inflammatory compounds and
alkaloids (Szary et al., 1975; Girish and Kemparaju, 2005a).
These compounds have traditionally been used as contraceptives, to promote wound healing, as anti-inflammatory
agents and to treat snakebite, presumably through their ability to
inhibit hyaluronidase activity in respective conditions. Some of
the plant extracts are also used in folk medicines to block
several hyaluronidases (Joyce et al., 1986; Mio and Stern, 2002;
Girish et al., 2004a; Ushanandini et al., 2006).
Medical applications of hyaluronan
HA plays an important physiological role in many organisms
and it has been used principally in biomedical applications
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(Larsen and Balazs, 1991; Vercruysse and Prestwich, 1998;
Moreland, 2003; Liao, 2005). It is an extremely attractive
polymer material because it is a natural product that degrades
into simple sugars. Its hygroscopic and highly viscoelastic
nature makes it suitable for various medical and pharmaceutical
applications. This section details the medical and pharmaceutical uses of HA.
Treatment of osteoarthritis
The viscoelasticity of the HA is responsible for lubrication
and mechanical support of joints. However, in osteoarthritic
joints, HA molecular mass and concentration can decline as a
result of the pro-inflammatory cytokines and free radicals
(Balazs and Denlinger, 1993; Stern, 2003). Changes in the
viscoelasticity of the synovial fluid results in joint related
problems. Intra-articular injection of HA is approved for the
treatment of osteoarthritis in many countries including the
United States, Canada, Japan and some part of the European
Union. Commercially available products for this application
contain sodium HA with different molecular mass, for example
Hyalgan® (Fidia, Italy), Artz® (Seikagaku, Japan) and
Orthovisc® (Anika, USA). The therapeutic effects of HA
injection products on osteoarthritic joints have been extensively
investigated in vitro, in vivo and clinically (Altman, 2000;
Uthman et al., 2003; Hamburger et al., 2003). Results from
these studies indicate that HA suppresses cartilage degeneration
and release of proteoglycans from the extracellular matrix in
cartilage tissues, protects the surface of articular cartilage,
normalizes the properties of synovial fluids, and reduces pain
perception. The analgesic effect obtained after administration of
HA was found to be comparable or superior to either
corticosteroids or nonsteroidal anti-inflammatory drugs (Altman, 2000; Kirwan, 2001). However, the mechanism of these
effects of HA on osteoarthritic joints is unclear (Ghosh and
Guidolin, 2002). Many researchers believe that HA can confer
mechanical and lubricating effects and protect joint tissues by
its viscosupplementation, viscoaugmentation and viscoprotection properties, which are independent of its molecular mass.
The pharmacological role of HA may be related to its antiinflammatory and immunoregulatory properties.
Surgery and wound healing
HA is commonly used in eye surgery in conjunction with the
implantation of artificial intraocular lens and other commercially available products. HA is also used in surgical procedures
as a viscoelastic gel to aid healing and regenerative processes of
surgical wounds. In the case of cataract surgery, HA gels are
used to maintain operative space (depth of the anterior chamber)
and to protect the endothelial layer of the cornea (acting as a
lubricant) or other tissues from physical damage (Ghosh and
Guidolin, 2002; Ghosh and Jassal, 2002). Furthermore, HA
appears to promote corneal, diabetic foot, tendon, bone, nasal
mucosal, and venous leg ulcer wound healing (Miller et al.,
1997; Liao, 2005). Miyazaki et al. (1996) suggested that HA
promoted corneal wound healing is partially due to its ability to
promote the growth of corneal epithelial cells. In addition, HA
is used as an anti-adhesion and anti-scar drug in general surgery.
It acts as an antioxidant for inflamed body tissues, protecting
body tissue from free radical damage and supporting immune
function by inhibiting germs and viruses from moving between
cells and getting into cells. In addition, HA also has been
reported to decrease neutrophil elastase promoted alveolar
injury after inhalation (Liao, 2005). The study suggested that
the protective mechanism was due to specific binding between
HA and lung elastic fibers. However, the molecular mechanism
of HA-induced wound healing has yet to be identified.
Embryo implantation
Higher concentrations of HA during estrus and ovulation
suggest that HA has a function prefertilization through
embryonic development. HA has been found to be a suitable
replacement for albumin in culture media for in vivo fertilization
(Simon et al., 2003). The presence of HA as an alternate to
albumin has not resulted in any harmful effects on the
development of frozen-thawed mouse and sheep embryos. In
addition, the presence of HA in culture media could act as a
cryopreservative of human embryos by maintaining their
viability during thawing. More studies have investigated the
effect of HA in murine, porcine and bovine embryo development and viability after culture in HA-based media (Figueiredo
et al., 2002; Abeydeera, 2002; Jang et al., 2003). Simon et al.
(2003) reported that HA-based human embryo transfer medium
showed a better pregnancy and implantation rate than albuminbased medium. Recently, studies reported that exogenous HA
significantly increased the expression of insulin like growth
factor-II gene and reduced the expression of apoptosis (Bax),
oxidative stress (SOX), and embryo cell-to cell adhesion
associated with embryo compaction (Ecad) genes. Furthermore,
the general fine structure of embryos cultured with HA was
noticeably improved in comparison with the respective controls.
Based on these studies, it is confirmed that supplemental HA
could improve the quality of the embryo and developmental
rates of blastocytes (Palasz et al., 2006).
As a disease indicator
HA occurs in many forms, circulating freely in the blood
stream and lymphatic system and loosely/tightly interacting
with ECM. Normally, low concentrations of HA circulate in the
blood stream, whereas increases in tissue bound and circulating
HA occur very early in wound healing and during periods of
rapid cell turnover following tissue injury (Fraser et al., 1997;
Lee and Spicer, 2000). Rapid increases in circulating HA also
occur in response to major stress, such as blood loss, shock,
ischaemic stroke, and septicemia following massive trauma,
major surgical procedures and extensive burns (Berg, 1997;
George and Stern, 2004; Al'qteishat et al., 2006). Increased
serum HA is also one of the earliest indicators of liver cirrhosis,
liver fibrosis, knee osteoarthritis and rejection following liver
transplantation. Clearly, increased HA provides a rapid response
survival mechanism following major injuries and several
K.S. Girish, K. Kemparaju / Life Sciences 80 (2007) 1921–1943
diseases (George and Stern, 2004). HA, as a biomarker has the
potential for use clinically to detect the early changes following
injuries and diseases as well as for screening drugs.
Hyaluronan in drug delivery
In recent years, HA has had significant use in the
development of dermal and transdermal drug delivery systems.
The outermost layer of the skin acts as a protective barrier and
prevents the access of drug molecules into and across the skin.
The beneficial effect of HA on the dermal delivery of diclofenac
has been thoroughly investigated in vitro, in vivo and in clinical
studies (Wolf et al., 2001; Brown et al., 2002). In vitro studies
reported that HA significantly enhances both the partitioning of
diclofenac into human skin and its retention and localization in
the epidermis when compared with a control and other
commonly used pharmaceutically acceptable gelling agents
(Brown and Martin, 2001). HA also has been reported to
produce similar effects with cyclosporine, ibuprofen, and
clindamycin phosphate (Nazir et al., 2001; Brown et al., 2002).
HA has been used as a vehicle for topical ophthalmic and
nasal drugs due to its viscoelasticity and mucoadhesive capacity
(Lapcik et al., 1998; Castellano and Mautone, 2002). Several
studies have reported that HA is capable of the prolonging
residence and/or increasing the bioavailability of molecular
drugs such as pilocarpine, vasopressin, 1-deamino-8-D-arginine
vasopressin, tropicamide, timolol, xylometazoline, gentimycin,
arecaidine propargyl ester and [S]-aceclidine in solution
(Morimoto et al., 1991; Liao, 2005). The increased bioavailability of drugs conferred by HA was found to be molecular
mass dependent, with the high molecular mass HA fractions
promoting an increase in bioavailability while low molecular
mass HA had no effect (Morimoto et al., 1991). Mucoadhesive
and viscoelastic properties of HA are thought to be primarily
responsible for the prolonged precorneal residence of the drugs.
Furthermore, a HA-based microparticulate delivery system
showed increased mucoadhesive properties compared to
chitosan-based microparticulates. In addition, the mucoadhesive properties of microparticulates were not affected by the
presence of gentamycin. The bioavailability of intranasally
administered gentamycin was significantly higher in HA/
chitosan microspheres compared to drug particles or chitosanbased particles and HA-based particles alone. Therefore,
researchers assumed that HA mainly acts as a mucoadhesive
agent whereas chitosan had a penetration enhancement function
(Lim et al., 2000, 2002).
The non-immunogenic nature of HA was used in parental/
pulmonary drug delivery systems to obtain constant release or
to prolong the retention of drugs/hormone/anticancer agents.
Direct conjugates of low molecular mass HA to cytotoxic drugs
such as butyric acid, paclitaxel, and doxorubicin have been
reported (Luo et al., 2000). It has been shown that these
bioconjugates are internalized into cancer cells through
receptor-mediated endocytosis followed by intracellular release
of active drugs, thus restoring their original cytotoxicity For
example, taxol, an anticancer drug, when chemically linked to
HA was found to selectively target human colon, ovarian and
1935
breast cancer cell lines in vitro (Luo et al., 2000). In addition,
HA enhances the anti-inflammatory activity of conjugated
proteins in mice. The HA-modified liposomes provided more
efficiently encapsulated of anticancer drug, doxorubicin and
epidermal growth factor relative to unmodified liposomes (Peer
et al., 2003; Liao, 2005). Recently, HA has been used to prepare
DNA–HA matrices and microspheres with the goal of
achieving sustained gene delivery (Yun et al., 2004).
To obtain optimal properties for mechanical strength, drug
delivery or biostability, a variety of HA-grafted co-polymers
have been prepared. For example, collagen crosslinking with
polyethylene glycol (PEG)-grafted-HA co-polymer, polypyrrole-functionalized HA polymer, benzyl-functionalized HA
polymer, and methacrylic anhydride-functionalized HA polymer were developed for the delivery of drugs and water-soluble
peptides. In addition, HA has been utilized to develop an
implantable delivery to achieve long-term delivery of antibiotic
and anti-inflammatory drugs. Surini et al. (2003) produced a
chitosan and HA implant containing insulin and achieved
programmed release profiles of insulin. The release rate of
insulin was dictated by chitosan-to-HA mixing ratios and the
amount of insulin loaded. Moreover, several strategies have
been developed to make implants of HA-fibronectin and HAcollagen/gelatin to enhance cell interactions with HA (Ghosh
et al., 2006). Recently, Ji et al. (2006) fabricated a threedimensional (3D) modified HA nanofibrous scaffold based on
electrospinning to mimic the architecture of natural ECM.
When NIH 3T3 fibroblasts were seeded on a fibronectinadsorbed HA scaffold for 24 h in vitro, fibroblasts attached to
the scaffold and spread, demonstrating an extended dendritic
morphology within the scaffold. This suggests potential
application of HA scaffolds in cell encapsulation and tissue
regeneration (Ji et al., 2006). In another study, Lepidi et al.
(2006) implanted a HA-based graft (HYAFF 11™ tube,
diameter 2 mm, length 1.5 cm) in an end-to-end fashion in
the abdominal aorta of rats and allowed for 7, 21, and 90 days.
The data indicated that the HA-based graft allowed complete
regeneration of a newly formed vascular tube in which all the
cellular and extracellular components are present and organized
in a well-defined architecture similar to the native artery (Lepidi
et al., 2006).
Hydrogels (hylans) based on crosslinked HA are potentially
good biomaterials for soft-tissue engineering applications
because they are highly non-antigenic and non-immunogenic.
The previous biomedical uses for hylans have capitalized on
their bio-inert characteristics; a potentially useful future
application for these highly biocompatible natural matrixderived materials is as cell scaffolds for soft tissue engineering,
for example vascular remodeling (Hoffman, 2001; Ramamurthi and Vesely, 2003, 2005; Shi et al., 2002; Hou et al.,
2005; Hahn et al., in press). To overcome the high rate of in
vivo turn over, crosslinking methods have been used to
stabilize the HA polymers. Some of these methods include
water-soluble carbodimide crosslinking, polyvalent hydrazide
crosslinking, disulfide crosslinking, divinyl sulfone crosslinking, and photocrosslinking hydrogels through glycidyl
methacrylate–HA conjugates. Covalent crosslinking of HA
1936
K.S. Girish, K. Kemparaju / Life Sciences 80 (2007) 1921–1943
provides the ability to improve the desirable biological and
mechanical properties of HA. The various crosslinking models
of HA prolong the in vivo degradation and provide long-term
stability. Thus, crosslinking HA polymers results in different
pore sizes and degradation profiles that have been used for
biomedical applications such as orthopedics, cardiovascular
medicine, and dermatology.
HA gels crosslinked with divinyl sulfone (DVS) are highly
biocompatible and can be structurally modified to obtain
desired mechanical properties. However, some studies have
indicated that the high anionic nature of hylan gels results in
poor interaction with cells in vitro (Ramamurthi and Vesely,
2003). Additionally, crosslinked hylan gels are highly (N95%
w/w) hydrated and exhibit smooth surfaces that oppose cell
attachment and bioactivity. Ramamurthi and Vesely (2003)
observed significantly higher levels of attachment and proliferation of neonatal rat smooth muscle cells on the surface of
ultraviolet (UV) irradiated and dehydrated-irradiated hylan gels
compared to unmodified gels. This modification micro-textures
the smooth gel surface to provide sites for cell anchorage and
causes limited scission of native long-chain HA, yielding
smaller fragments. In addition, when neonatal rat aortic smooth
muscle cells were cultured on hylan gels coated with matrix
molecules such as collagen I, matrigel, laminin, and fibronectin
for 4 weeks, significant cell attachment was seen on coated gels
compared to uncoated gels. Moreover, the matrix molecules
coating the hylan gels and the surface topography of the gels
influenced cell morphology. These results indicate that cell
attachment and proliferation can be enhanced by surface
texturizing and subsequent surface treatment with matrix
molecules (Ramamurthi and Vesely, 2002).
Recently, Amarnath et al. (2006) studied the interaction
between UV-irradiated hylan gels and human blood cells (RBCs,
platelets) and coagulation proteins at physiological temperature.
Relative to the positive controls, both un-irradiated and UVirradiated gels prolonged the formation of coagulum through
extrinsic and intrinsic pathways. UV-irradiated gels also induced
lower contact activation and also very low levels of hemolysis.
Furthermore, Joddar and Ramamurthi (2006) evaluated the
fragment size and dose specific effects of HA on matrix
synthesis by vascular smooth muscle cells. These findings
suggest that the HA derived grafts and implants can be used as
tissue engineering scaffolds to modulate vascular regeneration.
HA is a unique molecule with viscoelastic, hydrophilic, nonimmunogenic and lubricious properties that make possible its
use in various medical and pharmaceutical applications. It is a
distinctive biomaterial, which provides itself to crosslinking and
immobilizing in numerous ways to produce biocompatible
grafts and implants. Ongoing research developments regarding
incorporation of HA into biomaterials for biomedical applications have the potential to transform the clinical future of drug
delivery and tissue engineering.
Medical application of hyaluronidase
The biomedical applications of hyaluronidase are limited
compared to HA. Hyaluronidase is an endoglycosidase. By
cleaving HA in tissues, it increases the membrane permeability,
reduces viscosity and renders tissues more permeable to
injected fluids (spreading effect). Thus, these enzymes could
be used therapeutically to increase the speed of absorption, to
promote resorption of excess fluids, to increase the effectiveness of local anesthesia and to diminish tissue destruction by
subcutaneous and intramuscular injection of fluids (Frost et al.,
1996; Farr et al., 1997). Hyaluronidase has been used to reduce
the extent of tissue damage following extravasation of parental
nutrition solution, electrolyte infusions, antibiotics, aminophyline, mannitol and chemotherapeutic agents including Vinca
alkaloids (vincristine, vinblastine and vinorelbine).
Hyaluronidase can be used to decrease myocardial infarction
size, as demonstrated in biomedical studies on animals. Borrelli
et al. shown that combined administration of hyaluronidase
(2500 IU/kg) with urokinase (40,000 IU/kg) into rats with
myocardial infarction significantly decreased mortality data,
where the lethal index for combination of preparations was
12.5% against 29.5% for urokinase (Muckenschnabel et al.,
1997).
Hyaluronidases are widely used in many fields, including
orthopaedia, surgery, ophthalmology, internal medicine, oncology, dermatology and gynaecology. Sperm hyaluronidase plays
an important role in successful fertilization in most mammals,
including human (Manzel and Farr, 1988; Frost et al., 1996;
Borrelli et al., 1986; Primakoff et al., 1985; Lin et al., 1994;
Klocker et al., 1995). Hyaluronidase has been investigated as an
additive to chemotherapeutic drugs for augmentation of
anticancer activity (Muckenschnabel et al., 1997, 1998; Spruss
et al., 1995; Baumgartner, 1998). There is evidence that
hyaluronidase may have intrinsic anticancer effects and can
suppress tumor development. Furthermore, it was reported that
treatment with hyaluronidase blocks lymph node invasion by
tumor cells in an animal model of T cell lymphoma (Zahalka
et al., 1995).
Concluding remarks
HA is an abundant polysaccharide in the ECM and is
involved in many physiological and pathological processes.
The biosynthesis, uptake and degradation of HA are well
balanced under normal homeostasis. Despite the intensive
studies carried out thus far, our understanding of the complete
function of HA in eukaryotes is unclear. HA appears to be a
very important molecule since its aberrant production and
turnover results in many pathological and inherited disease
conditions. Furthermore, HA–cell interactions and cellular
signaling induced by HA and its degraded products from the
metabolism of HA have a major impact on normal and diseased
cell behavior.
HA degradation by hyaluronidases is also critical in a
number of important regulatory processes that range from embryonic development to wound healing. Thorough knowledge
of enzyme dependent HA degradation will be helpful in
understanding the complete process of HA catabolism. The
molecular mechanism of HA degradation by vertebrate
hyaluronidase is still under investigation; however, it will
K.S. Girish, K. Kemparaju / Life Sciences 80 (2007) 1921–1943
be interesting to know the in vivo hyaluronidase dependent
HA degradation products under normal and pathological
conditions. It will also be exciting to know the in vivo fragmentation of HA by venom and microbial hyaluronidases and
its subsequent effects on pathophysiology of envenomation/
pathogenesis.
Identification of inhibitors associated with hyaluronidase
during the HA degradation may be helpful in understanding
regulation of HA metabolism, crucial changes in tissues/
lymphatic system and in the search for rational therapeutic
approaches to prevent several disorders. In addition, identification
and characterization of hyaluronidase inhibitors from natural
sources may lead to valuable antimicrobial contraceptives, antitumor agents and alternatives to complement anti-venom as well
as first aid agents in venomous bite/hymenoptera sting therapy.
Acknowledgements
We thank Prof. B.S. Vishwanath, Department of Biochemistry, University of Mysore for his valuable suggestions during
the preparation of the article. We are also grateful to Prof.
Periasamy Selvaraj, Dr. K.D. Machaiah, Dr. M. Vedavathi, and
Dr. R. Shasidharamurthy and S. Nagaraju for their suggestions
and critical reading of the manuscript. KSG extend sincere
thanks to the Council of Scientific and Industrial Research
(CSIR), New Delhi, India, for the financial assistance. We also
apologize to the authors of many interesting studies that were
omitted due to limited space.
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