Glycobiology, 2016, vol. 26, no. 3, 218–229
doi: 10.1093/glycob/cwv102
Advance Access Publication Date: 17 November 2015
Review
Review
Sweating the small stuff: Glycoproteins in human
sweat and their unexplored potential for
microbial adhesion
Robyn A Peterson2, Audrey Gueniche3, Ségolène Adam de Beaumais3,
Lionel Breton3, Maria Dalko-Csiba3, and Nicolle H Packer1,2
2
Department of Chemistry and Biomolecular Sciences, Biomolecular Frontiers Research Centre, Macquarie
University, Sydney 2109, Australia, and 3L’Oréal Research and Innovation, Aulnay sous bois 93600, France
1
To whom correspondence should be addressed: Tel: +1-612-9850-8176; Fax: +1-612-9850-8313; e-mail: nicki.packer@
mq.edu.au
Received 31 August 2015; Revised 28 October 2015; Accepted 2 November 2015
Abstract
There is increasing evidence that secretory fluids such as tears, saliva and milk play an important role
in protecting the human body from infection via a washing mechanism involving glycan-mediated
adhesion of potential pathogens to secretory glycoproteins. Interaction of sweat with bacteria is well
established as the cause of sweat-associated malodor. However, the role of sweat glycoproteins in
microbial attachment has received little, if any, research interest in the past. In this review, we demonstrate how recent published studies involving high-throughput proteomic analysis have inadvertently, and fortuitously, exposed an abundance of glycoproteins in sweat, many of which have also
been identified in other secretory fluids. We bring together research demonstrating microbial adhesion to these secretory glycoproteins in tears, saliva and milk and suggest a similar role of the sweat
glycoproteins in mediating microbial attachment to sweat and/or skin. The contribution of glycanmediated microbial adhesion to sweat glycoproteins, and the associated impact on sweat derived
malodor and pathogenic skin infections are unchartered new research areas that we are beginning
to explore.
Key words: adhesion, bacteria, glycoprotein, secretion, sweat
Introduction
Sweating is a natural feature of human metabolism that is particularly important to cool the body in hot climates and/or after vigorous
exercise. Most of the sweat that is secreted from the body surface
comes from eccrine glands. Eccrine glands are simple tubular structures that occur over much of the skin, with the highest concentration
on the palms, soles of the feet, forehead and armpits. Eccrine sweat
consists predominantly of water (99%), salts (∼47 mM Na, 8.6 mM
K, 29.7 mM Cl), amino acids, glycerol, lactic acid, proteins (0.06–
0.12 g/L) and other electrolytes (Lloyd 2008; Kanlayavattanakul
and Lourith 2011). Apocrine sweat glands are the dominant type
of sweat gland in the human axilla (underarm region) and exist
alongside axillary eccrine glands at a ratio of ∼10:1. From the
onset of adolescence, the apocrine glands secrete a milky, thick
fluid consisting of lipids, fatty acids, proteins, steroids, vitamins
and electrolytes (Burry et al. 2001; Draelos 2010; Kanlayavattanakul
and Lourith 2011).
Sweat in the eccrine gland is released by a mechanism termed
merocrine secretion, which is similar to exocytosis and does not involve significant damage to the secretory cell, nor release of cytoplasm
or cell membrane into the secretory fluid. In contrast, the apocrine
sweat glands exhibit “decapitation” secretion, in which the apical
part of the cytoplasm and cell membrane is pinched off along with
the secretory materials (Ackerman et al. 2005). The apocrine secretion
© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]
218
The potential of sweat glycoproteins for microbial adhesion
mechanism is also found in the mammary glands (Krstič 2004) and
mucus-producing goblet cells in the intestine (Gesase 2007).
Both eccrine and apocrine sweat are initially odorless upon secretion. It is the enzymatic action of the bacterial microbiome on the
sweat, and in particular on the apocrine sweat, that causes body malodor. The predominant bacterial species present in the axillary region
and the metabolic pathways by which they produce undesirable aromas have been described in detail in recent reviews (Kanlayavattanakul and Lourith 2011; Fredrich et al. 2013; James et al. 2013). Despite
a certain level of controversy about the exact interactions involved, the
enzymes produced by Corynebacterium, Staphylococcus and Propionibacterium species appear to be highly implicated in the production
process of odors. Corynebacterium appear to be the main causal species for the most potent odors caused by alcohols with a thiol group
and short to medium chain (C6–C10) volatile fatty acids, while the
Staphylococcus species are most closely associated with a milder
odor arising from short chain (C2–C5) volatile fatty acids, in particular isovaleric acid (James et al. 2013). Propionibacterium species are
most closely associated with the formation of carboxylic acids, in particular propionic acid. A schematic representation illustrating the main
microbial associations with axillary odor is shown in Figure 1.
Although the interaction of bacteria with sweat has received much
attention due to the odor association, there has been little research addressing how bacteria bind to sweat. Strong experimental evidence has
accumulated demonstrating that other secretory fluids, such as tears,
saliva and human milk adhere to bacteria and other potential pathogens, at least partly by virtue of the glycan moieties attached to the
secreted glycoproteins (Guzman-Aranguez and Argüeso 2010; EverestDass et al. 2012; Peterson et al. 2013). The conjugated glycans in the
secretory fluids have been found to mimic the glycan receptors typically used by pathogens on the epithelial cells of the host and thus act as
219
a decoy to prevent pathogen binding to the epithelial surfaces, for example on the eye or gastrointestinal tract.
The protein content of human sweat is very low (0.06–0.12 g/L;
Lloyd 2008), as compared with tears (6–10 g/L; Fullard and Snyder
1990; Ng and Cho 2000), saliva (1.4–6.4 g/L; Lloyd 2008) and
human milk (8.9 g/L; Räihä 1985). Consequently, there has been little
research on sweat proteins in general, and even less about the glycosylation of these proteins. Hence, the mechanisms by which sweat could
potentially prevent the adhesion of bacteria to skin cells via a glycan
decoy mechanisms similar to that advocated for other secretory fluids
has not yet been reported. Also, less is known about the involvement
of conjugated glycans in the binding of bacteria to skin cells in comparison to other epithelial cells such as those of the human gastrointestinal tract. A rare published study demonstrated glycan involvement in
the adherence of axillary bacteria to differentiated keratinocytes in cell
culture (Romero-Steiner et al. 1990), and several patents or patent application have advocated the use of glycans to inhibit the adhesion of
bacteria to skin cells (Brachman et al. 1985; Balish et al. 1993; Ansari
and Polefka 2007; Stahl and Boehm, 2011). However, the involvement of sweat glycoproteins in mediating bacterial adhesion to skin
cells has remained unexplored.
In this literature review, we bring together numerous, thus far unconnected, studies on sweat, tears, saliva and human milk glycoproteins to reveal a high similarity of the glycoproteins in sweat to
those in other human secretions, many of which have been shown to
bind to bacteria. Consequently, we suggest that the potential for the
same glycoproteins in sweat to bind to bacteria is extremely high.
Such a scenario implicates sweat glycoproteins in the adhesion of
malodor-causing bacteria to sweaty axillary skin and hairs, as well
as sweaty clothes. The sweat glycoprotein binding mechanism could
also play a role in the removal of potential skin pathogens to prevent
Fig. 1. Schematic representation of a human underarm and the components of sweat malodor produced by the enzymatic action of axillary bacteria. Odor
components are grouped by biochemical similarity within the diagrammatic clouds, adjacent to the bacteria known to produce them (based on the reviews of
James et al. 2013 and Fredrich et al. 2013; 3M2H, 3-methyl-2-hexanoic acid; HMHA, 3-hydroxy-3-methylhexanoic acid). This figure is available in black and
white in print and in color at Glycobiology online.
220
infection, in a similar manner to other secretory fluids. Evidence for
glycoproteins in sweat and their potential to bind to bacteria is described in more detail below.
Early evidence of glycoproteins in sweat
The presence of glycosylated proteins in human sweat was reported as
early as 1957 when biochemical analysis (Brdicka reaction) revealed
the presence of “mucoproteins” (Jirka and Kotas 1957). In 1963,
Pallavicini et al. characterized carbohydrate protein complexes from
human sweat and identified galactosamine, galactose, N-acetylneuraminic acid, mannose and fucose as components by paper chromatography; cystic fibrosis patients had significantly more fucose and less
sialic acid in their sweat (Pallavicini et al. 1963).
Histochemical and lectin affinity studies also provided early evidence for the presence of conjugated glycans in sweat. An acidic protein was detected in eccrine sweat glands and named “sialomucin” as
it had similar properties to the sialomucins in salivary and mammary
glands and gastrointestinal mucus (Constantine and Mowry 1966).
Glycoconjugates with terminal α-galactose, N-acetylgalactosamine,
fucose and sialic acid moieties were also detected in the stored secretory
material in sweat gland coils (Hazen-Martin et al. 1986), correlating
with the location of periodic acid-Schiff stain reactive glycoprotein.
Later, antibodies against chondroitin sulfate proteoglycans and chondroitin sulfate glycosaminoglycans stained granules within the secretory cells of eccrine sweat gland and lectin reactivity suggested the
presence of proteoglycan or mucin-type glycans (Sames et al. 1999).
Immunohistochemical analysis was also able to localize apocrine secretion odor-binding glycoproteins (ASOB1 and ASOB2) to apocrine
sweat glands and secretions (Spielman et al. 1998). Yet, despite the evidence provided for glycosylation of sweat in these studies, no significance was attributed to the glycan moieties nor was any connection
made to their potential for bacterial adhesion.
Proteomic analysis of sweat
The first broad-scale analysis of total sweat proteins took place in
1990 and involved sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of sweat proteins followed by immunoblotting to enable protein identification (Nakayashiki 1990).
Several of the identified proteins including Zn-α2-glycoprotein, immunoglobulins and carcinoembryonic antigen (CEA) are known to
be glycosylated but elaboration on this fact was not attempted at
the time.
The advances made in mass spectrometric techniques over the last
two decades greatly enhanced the ease and coverage of protein analysis in two more recent studies on sweat. The first of these studies
used liquid chromatography tandem mass spectrometry (LC–MSMS) to identify antimicrobial peptides and proteins in sweat (Park
et al. 2011), and the second used LC–MS-MS and multiple reaction
monitoring mass spectrometry (MRM–MS) to find biomarker proteins for schizophrenia in sweat (Raiszadeh et al. 2012). Both studies
achieved their respective aims; the antimicrobial proteins lactoferrin,
dermicidin, lysozyme and psoriasin were identified (Park et al.
2011), and 17 proteins were found that had a 2-fold or greater abundance in the sweat of schizophrenic patients than controls, including
peroxiredoxin, thioredoxin and S100A7/psoriasin (Raiszadeh et al.
2012).
The Park et al. (2011) and Raiszadeh et al. (2012) studies are also
valuable for reasons far beyond their original aims. Each of these
R A Peterson et al.
two studies resulted in the identification of over 100 proteins in
sweat, lists that were provided only as supplementary material to
the publications because most of the identified proteins were not
of interest to the respective studies at the time. However, close inspection of the identified proteins was undertaken for this review, revealing a wealth of glycosylated proteins identified in the studies.
Many of the glycoproteins identified from sweat in these two studies
have also been identified in other secretory fluids including tears,
saliva, semen and human milk. Adhesion of bacteria to these glycoproteins from these other body fluids has been demonstrated in numerous cases.
The glycoproteins identified from sweat by Park et al. (2011) and
Raiszadeh et al. (2012), as well as those identified in other studies concentrating on particular components of sweat, are listed in alphabetical order below. Other secretory fluids that have also been found to
contain these glycoproteins and research that has demonstrated their
involvement in microbial binding are described where available. Several of the glycoproteins are known to have antibacterial activity,
others are essential for the transit or production of other molecules
or peptides known to subsequently interact with bacteria. A summary
is provided in Table I. It is acknowledged that some of these glycoproteins are not known to be secreted. Their presence might be explained
by the inclusion of some proteins from skin cells and the extracellular
matrix, and intracellular components of secretory cells which might
have been released during the process of apocrine sweat gland secretion (Ackerman et al. 2005).
Glycoproteins in sweat
Apocrine secretion odor binding proteins 2
Apocrine secretion odor binding proteins 2 is a distinct variant of apolipoprotein D (apoD), a lipid binding protein involved in the transit of
ligands such as cholesterol, and steroid hormones in plasma and a variety of human tissues (Perdomo et al. 2010). Peptides matching apoD
have been identified in the proteomic analysis of sweat (Park et al.
2011; Raiszadeh et al. 2012) and are most likely to indicate the presence of the apocrine version of the glycoprotein which has been characterized in more detail in other studies. ASOB2 is localized to the
apocrine gland and secretions and carries the precursors of odorous
compounds such as 3M2H (3-methyl-2-hexanoic acid, Figure 1) to
the skin surface (Zeng et al. 1996; Spielman et al. 1998). ASOB2 is
glycosylated at Asn65 with neutral and acidic complex glycans and
at Asn98 with high mannose glycans (Zeng 1996). This is in contrast
to human plasma apoD, which carries complex, sialylated glycans on
both sites (Schindler et al. 1995). ApoD has also been identified in
tears where it is thought to assist the distribution of lipids to protect
the cornea (Holzfeind et al. 1995). By structure, apoD is considered
part of the lipocalin family of proteins (Eichinger et al. 2007). Lipocalin 2 is known to bind to bacterial siderophores, sequester iron and
thus restrict bacterial growth (Guglani et al. 2012).
Basic proline-rich protein
Basic proline-rich protein (BPRP) is a secreted protein that has been
identified in sweat (Park et al. 2011), saliva (Levine 2011) and tears
(Zhou et al. 2006). Salivary BPRP binds to oral streptococci (Levine
2011) and HIV-1 gp120 (Robinovitch et al. 2001), and the glycoprotein is also thought to play a role in tears to remove pathogens from the
eye (Aluru et al. 2012). Salivary BPLPs carry sialylated O-linked glycans (Carpenter and Proctor 1999), and the glycoprotein also has one
potential N-linked glycan site (www.uniprot.org).
221
The potential of sweat glycoproteins for microbial adhesion
Table I. Glycoproteins in sweat, their known functions in sweat and/or other secretory fluids and their reported glycosylation features
Glycoprotein
Loc
S
T
M Known functions
Apocrine secretion
odor-binding protein 2
/ASOB2
(apolipoprotein D/
ApoD)
Sec
–
Y
–
Basic proline-rich protein
(BPRP)
Sec
Y Y
–
Carboxypeptidase A3
(A4)
Sec
–
–
–
Carboxypeptidase E
Sec
–
–
–
Carcinoembryonic (CEA)
antigen
Sec CM
Y –
–
Cathepsin D
LysES
Y Y
Y
Clusterin
(Apolipoprotein J)
Sec
Y Y
Y
Cysteine-rich secretory
protein (CRISP-3)
Sec
Y Yb Y
Deleted in malignant
brain tumors 1 protein
(DMBT1; salivary
agglutinin)
Sec
Y Y
Y
Desmoglein 1
Sk CM
Y –
–
Glycosylation
Referencesa
ASOB2—transit of 3M2H to ASOB2—one site of complex and Spielman et al. (1998), Park
skin surface in sweat via
one site of high mannose
et al. (2011), Zeng (1996),
apocrine gland (apoD—
N-linked glycans (apoD—two
Zeng et al. (1996), Schindler
lipid metabolism, transport
sites with complex glycans)
et al. (1995), Perdomo et al.
of ligands (e.g. cholesterol,
(2010)
steroid hormones) in human
plasma and tissues)
Salivary BPRPs bind to oral
One potential N-linked glycan
Park et al. (2011), Carpenter
bacteria, viruses, calcium
site; salivary BPLPs carry
and Proctor (1999),
and oral epithelial cells;
sialylated O-linked glycans
Robinovitch et al. (2001),
BPRPs in tears may remove
Zhou et al. (2006), Levine
pathogens from the eye
(2011)
Metalloprotease; Extracellular One site with N-linked glycans
Park et al. (2011), Raiszadeh
peptide processing
et al. (2012), Pallarès et al.
(2005), Tanco et al. (2010),
Biosynthesis of neuropeptides; Two sites with N-linked glycans Baechle et al. (2006), Park et al.
generation of antimicrobial
(2011), Raiszadeh et al.
peptides in sweat
(2012), Manser et al. (1990)
Immuoglobulin superfamily;
Very highly glycosylated
Nakayashiki (1990), Park et al.
regulation of cellular
glycoprotein (∼50%
(2011), Metze et al. (1996),
differentiation,
carbohydrate), 28 potential
Hammarström (1999), Hill
proliferation, apoptosis;
N-linked glycan sites
et al. (2005), Ramachandran
binds to N. meningitides, N.
et al. (2006)
gonorrhoea, H. influenzae
and M. catarrhalis
Aspartate protease; lysosomal Two sites with complex and
Baechle et al. (2006), Park et al.
enzyme; post-secretory
phosphorylated N-linked
(2011), Raiszadeh et al.
processing of antimicrobial
glycans, one site of O-linked
(2012), Halim et al. (2013)
peptides in sweat
glycans
Seven sites with sialylated and
Park et al. (2011), Raiszadeh
Regulation of cellular
fucosylated N-linked glycans
et al. (2012), Kirszbaum et al.
interactions, involved in
(1992), Partridge et al.
protein aggregation and
(1996), Cuida et al. (1997),
apoptosis; lipid transport;
Wilson and
semen clusterin binds to
Easterbrook-Smith (2000),
DC-SIGN receptor via Lex
and Ley epitopes, prevents
Li and Ljungh (2001),
binding of HIV-1; binds to
Picariello et al. (2008)
S. aureus and S. epidermidis
Hettinga et ,al. (2011),
Sabatte et al. (2011), Li et al.
(2014)
Not fully understood; Innate
1 N-linked glycan site
Udby et al. (2002), Haendler
immune response
et al. (1999), D’Alessandro
et al. (2013), Udby et al.
(2005)
Scavenger receptor
Highly glycosylated (up to 40% Park et al. (2011), Schulz et al.
cysteine-rich protein;
of MW), 14 potential N-linked
(2002), Eriksson et al.
possible tumor suppressor;
glycan sites, numerous
(2007), Ahn et al. (2008),
immune protection; binds to
O-linked sites, extended
Ligtenberg et al. (2010),
streptococci, Helicobacter
fucosylated
Ronellenfitsch et al. (2012)
pylori and HIV; promotes/
N-acetyllactosamines, sialyl
or inhibits biofilm formation
Lewis epitopes
when adhered to surface/or
in solution
Calcium binding desmosome Three potential N-linked glycan Park et al. (2011), Raiszadeh
protein; cellular adhesion in
sites, at least one occupied in
et al. (2012), Ramachandran
epithelial cells, including the
saliva
et al. (2006), Hammers and
skin
Stanley (2013)
Continued
222
R A Peterson et al.
Table I. Continued
M Known functions
Glycosylation
Referencesa
Y –
–
Five potential N-linked sites,
keratin sulfate chains
Park et al. (2011), Lauder et al.
(1997), Oldberg et al.
(2007), Gonzalez-Begne et al.
(2011)
ES
Y Y
Y
Immunoglobulins sIgA,
IgG, IgD, IgE
Sec
Y Y
Y
Antibodies, Immune defence;
sIgA binds to E. coli and H.
pylori via glycan moieties
preventing adhesion of
bacteria to human epithelial
cells; Staphylococci have a
specific IgG binding protein
Lactoferrin (Lf )
Sec
Y Y
Y
N-sulfoglucosamine
sulfohydrolase
Lys
–
–
Antimicrobial; human milk Lf
binds to gut pathogens at
least partly via glycan
residues
Lysosomal degradation of
heparin sulfate
Glycoprotein
Loc
S
Fibromodulin
Sk ES
Galectin-3-binding
protein (G3BP)
T
–
Collagen assembly and
maintenance in interstitial
connective tissues,
particularly cartilage,
tendons, but also present in
skin
Cell adhesion; may stimulate
immune defence; binds to
adeno-associated viruses;
fish G3BP binds to bacteria
as immune defence
Yb Binds to bacterial
peptidoglycan; bactericidal
to many pathogenic
bacteria, bacteriostatic for
most gram-positive and
gram-negative bacteria
– Serpin peptidase inhibitor,
inactivates urokinase;
coagulation factor;
maintains tissue
homeostasis; adaptive
immune response
Peptidoglycan recognition Sec
proteins PGLYRP3,
PGLYRP4
Y Y
Plasminogen activator
inhibitor 2 (Urokinase
inhibitor)
Sec IC
Y Y
Polymeric
immunoglobulin
receptor (secretory
component)
Sec
Y Y
Y
Prolactin-inducible
protein (PIP)
Sec
Y Y
Y
Highly glycosylated with up to 7
N-linked glycans
Park et al. (2011), Liu et al.
(2005), Ramachandran et al.
(2006), Acera et al. (2011),
Denard et al. (2012), Chan
et al. (2013), Chen et al.
(2013), Kratz et al. (2014)
sIgA—two sites with N-linked
Herrmann and Habbig (1976),
glycans, five sites with O-linked
Okada et al. (1988),
glycans
Nakayashiki (1990), Park
et al. (2011), Wold et al.
(1990) Falk et al. (1993),
Schroten et al. (1998),
Marcotte and Lavoie (1998),
Willcox and Lan (1999),
Burman et al. (2008), Zauner
et al. (2013)
Three sites with sialylated and
Park et al. (2011), Waszkiewicz
fucosylated N-linked glycans
et al. (2012), Flanagan and
Willcox (2009), Barboza
et al. (2012)
Five potential N-linked glycan
Park et al. (2011), Anson and
sites, at least two occupied
Bielicki (1999), Scott et al.
(1995), Zhang et al. (2003),
Chen et al. (2009)
PGLYRP3—one potential
Lu et al. (2006), Wang et al.
N-linked glycan; PGYRP3—
(2007), Dziarski and Gupta
five N-linked glycans
(2010)
Secreted form is glycosylated (up
to three N-linked glycan sites)
Park et al. (2011), Wang and
Jensen (1998), Medcalf and
Stasinopoulos (2005),
Virtanen et al. (2006),
Csutak et al. (2008), Lobov
et al. (2008). Schroder et al.
(2011)
Binds polymeric IgA and IgM Seven sites with N-linked glycans Park et al. (2011), Huff (1990),
and transports to apical
Eiffert et al. (1984), Asano
surface; Binds to
et al. (2004), Asmat et al.
S. pneumonia
(2011), Balasubramanian
et al. (2013), Sakaguchi et al.
(2013)
Regulates water transport in
One site with N-linked glycans
Park et al. (2011), Raiszadeh
apocrine and eccrine glands;
et al. (2012), Schenkels et al.
(1997), Lee et al. (2002),
plasma PIP binds to IgG;
Chiu and Chamley (2003),
immune defence; salivary
Hassan et al. (2008), Moi
PIP binds to Streptococcus
et al. (2012)
species; bacterial
aggregation and biofilm
formation
Continued
223
The potential of sweat glycoproteins for microbial adhesion
Table I. Continued
Glycoprotein
Loc
S
T
M Known functions
Thrombospondin
Sec
Y –
–
Zinc-α2-glycoprotein
Sec
Y Y
Y
Glycosylation
Adhesive glycoprotein,
C-mannosylation and
mediates cellular
O-fucosylation, as well as
interactions; binds to the
N-linked glycan sites
peptidoglycan of
gram-positive S. pneumonia,
S. pyogenes, Staphylococcus
aureus and L.
monocytogenes; binds to
H1V1 virus
Lipid metabolism and
Four sites with N-linked glycans
mobilization (binds to
polyunsaturated fatty acids);
regulation of cell cycle
Referencesa
Park et al. (2011), Raiszadeh
et al. (2012), Bégány et al.
(1994), Crombie et al.
(1998), Rennemeier et al.
(2007)
Nakayashiki (1990), Park et al.
(2011), Raiszadeh et al.
(2012), Ramachandran et al.
(2006), McDermott et al.
(2006), Hassan et al. (2008),
Sörensen-Zender et al.
(2013)
The expected location (Loc) and glycosylation of the glycoproteins is provided according to their annotation at www.uniprot.org and in the references provided
(Sec, secreted; ES, secreted to extracellular space only; CM, cell membrane bound; IC, intracellular). Presence of the proteins in saliva (S), tears (T) and human milk (M)
is indicated by Y; a dash indicates no experimental evidence.
a
References in italics relate to studies in which the glycoprotein was identified in human sweat. Other references contain information relating to known functions,
glycosylation or evidence of the protein in other secretory fluids (S, T and M only); further information is in the text.
b
Glycoprotein was identified in the indicated secretory fluid of a mammal other than a human (camel milk and mouse tears).
Carboxypeptidase A3
Carboxypeptidase A3 (A4) is a metalloprotease that has been identified in sweat (Park et al. 2011; Raiszadeh et al. 2012) and is involved
in extracellular peptide processing (Tanco et al. 2010). The glycoprotein has at least one site carrying N-linked glycans (Pallarès et al.
2005).
Carboxypeptidase E
Carboxypeptidase E is another metalloprotease in sweat (Baechle
et al. 2006; Park et al. 2011; Raiszadeh et al. 2012) that may be involved in the generation of antimicrobial peptides (Baechle et al.
2006). The glycoprotein has two sites of N-linked glycosylation (Manser et al. 1990).
CEA
CEA is a member of the immunoglobulin family involved in regulation
of cellular differentiation, proliferation and apoptosis. The protein is
very highly glycosylated (∼50% carbohydrate) with multiantennary
complex type glycans and is expressed at high levels in gastrointestinal
tumors (Hammarström 1999). It has been identified in sweat
(Nakayashiki 1990; Park et al. 2011) and in sweat glands (Metze
et al. 1996), where it may play a role in binding to pathogens to prevent them from progressing to the epithelial cell binding sites of the
skin (Hammarström 1999). Respiratory tract CEA binds to Neisseria
meningitides, Neisseria gonorrhoea, Haemophilus influenzae and
Moraxella catarrhalis (Hill et al. 2005). The glycoprotein is also present in saliva (Ramachandran et al. 2006).
Cathepsin D
Cathepsin D is an aspartate protease that is present in the lysosome of
cells. Secretion of the enzyme has also been demonstrated by its identification in human sweat (Baechle et al. 2006; Park et al. 2011;
Raiszadeh et al. 2012), saliva (Minarowska et al. 2007), tears (de
Souza et al. 2006) and milk (El Messaoudi et al. 2000). Cathepsin
D is considered to play a role in post-secretory processing of antimicrobial peptides in sweat. The glycoprotein has two sites with complex
and phosphorylated N-linked glycans (Baechle et al. 2006). O-linked
glycosylation of Cathepsin D from human cerebrospinal fluid has also
been demonstrated (Halim et al. 2013).
Clusterin (Apolipoprotein J)
Clusterin (Apolipoprotein J) is a highly glycosylated protein found in
sweat (Park et al. 2011; Raiszadeh et al. 2012), saliva (Cuida et al.
1997), tears (Li et al. 2014), human milk (Hettinga et al. 2011), plasma (Thambisetty et al. 2010), urine (Dieterle et al. 2010) and semen
(Sabatte et al. 2011). The glycoprotein has many proposed functions
including the regulation of cell interactions, apoptosis and lipid transport (Wilson and Easterbrook-Smith 2000). Experimental evidence
for seven sites of N-linked glycosylation has been provided, all of
which are rich in sialic acid (Kirszbaum et al. 1992; Picariello et al.
2008). Clusterin in human semen carries abundant fucose-containing
blood antigens (Lewisx and Lewisy) which serve as ligands for dendritic
cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), thus preventing the binding of human immunodeficiency virus type 1 (HIV-1)
(Sabatte et al. 2011). Staphylococcus aureus also binds to clusterin
from human serum (Partridge et al. 1996), as does S. epidermidis
(Li and Ljungh 2001), although glycan involvement was not investigated in either of these studies.
Cysteine-rich secretory protein
Cysteine-rich secretory protein (CRISP-3) has been quantified in
human sweat (0.15 µg/mL), saliva (21.8 µg/mL) and plasma (6.3 µg/mL;
Udby et al. 2002). The protein has also been identified in human milk
(D’Alessandro et al. 2013) and seminal fluid (Udby et al. 2005) and
murine tears (Haendler et al. 1999). Both glycosylated and nonglycosylated forms of CRISP are known, and there is one potential site of
N-linked glycosylation. The role of CRISP-3 is not yet fully clear,
224
although the protein has sequence similarity to pathogenesis-related
proteins and may play an antimicrobial role (Udby et al. 2002).
Deleted in malignant brain tumors 1 protein
Deleted in malignant brain tumors 1 protein (DMBT1; salivary agglutinin) belongs to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins and appears to be a tumor suppressor. DMBT1
has been identified in human sweat (Park et al. 2011), saliva (Eriksson
et al. 2007), tears (Schulz et al. 2002) and milk (Ronellenfitsch et al.
2012). DMBT1 is highly glycosylated accounting for up to 40% of its
molecular weight, although various forms of the glycoprotein exist
that differ in their glycosylation (Ligtenberg et al. 2010). DMBT1
from tears has been shown to carry core 1 and core 2, neutral or
monosialylated O-linked glycans extended by fucosylated oligo-Nacetyllactosamine units with sialyl-Lewisx or Leb/Ley epitopes (Schulz
et al. 2002). An additional 14 potential N-linked glycosylation sites
are also predicted (www.uniprot.org). The glycosylation of salivary
and lung DMBT1 differs, with further difference between those of
the blood types classified as secretors compared with non-secretors,
who are known to glycosylate their proteins differently (Eriksson
et al. 2007). DMBT1 is thought to play a role in immune protection
since several studies have provided evidence that the protein binds to
pathogens, e.g., streptococci, Helicobacter pylori and HIV, a process
that appears to be at least partly mediated by protein glycosylation
(reviewed in Ligtenberg et al. 2010). In saliva, DMBT1 (salivary agglutinin) either increases biofilm formation (when it adheres to a surface),
or reduces biofilm formation (when in solution) (Ahn et al. 2008).
Interestingly, an upregulation of the protein is found in human milk
upon neonatal infection (Ronellenfitsch et al. 2012).
Desmoglein 1
Desmoglein 1 is a calcium binding desmosome protein-mediating cellular adhesion in epithelial cells, including in the skin (Hammers and
Stanley 2013). Natural desquamation of skin cells could explain the
presence of the protein in sweat (Park et al. 2011; Raiszadeh et al.
2012). Desmoglein 1 has three potential N-linked glycan sites and
has also been identified in saliva (Ramachandran et al. 2006).
Galectin-3-binding protein/G3BP (Mac-2 binding protein)
Galectin-3-binding protein/G3BP (Mac-2 binding protein) is a secreted protein present in sweat (Park et al. 2011), saliva (Kratz et al.
2014), tears (Acera et al. 2011) and human milk (Chan et al. 2013).
G3BP is highly glycosylated with seven N-linked glycosylation sites for
which occupancy has been shown for at least six (Liu et al. 2005;
Ramachandran et al. 2006). G3BP is involved in integrin-mediated
cell adhesion, signal transduction and cellular defence response. Binding of human G3BP to recombinant adeno-associated viruses has been
reported (Denard et al. 2012) and binding of fish G3BP to pathogenic
bacteria as an immune defence response facilitating host phagocytosis
has been demonstrated in Cynoglossus semilaevis (Chinese tongue
sole fish; Chen et al. 2013).
Immunoglobulins
Immunoglobulins that have been identified in sweat include secretory
IgA, IgG, IgD, IgE (Herrmann and Habbig 1976; Okada et al. 1988;
Nakayashiki 1990; Park et al. 2011). As antibodies the immunoglobulins play a major role in immune defence in the blood and tissue fluids,
as well as in secretions such as saliva (Marcotte and Lavoie 1998), and
tears (Willcox and Lan 1999). Numerous studies have demonstrated
R A Peterson et al.
the ability of the N-linked glycans of secretory IgA from human
milk to inhibit bacterial binding to human epithelial cells, e.g. sIgA
high mannose glycans inhibited binding of E. coli to human intestinal
cells (Wold et al. 1990); sIgA fucosylated glycans inhibited binding of
Helicobacter pylori to human gastric cells (Falk et al. 1993); and sialylated glycans inhibited the binding of E. coli to buccal cells (Schroten
et al. 1998). A staphylococcal immunoglobulin-binding protein has
also been identified that binds to IgG (Burman et al. 2008).
Lactoferrin
Lactoferrin was found at a low concentration of 21 ng/mL in sweat
(Park et al. 2011). The glycoprotein has three sites of N-linked glycosylation containing sialylated and fucosylated glycans and is found in
human milk (Barboza et al. 2012), saliva (Waszkiewicz et al. 2012),
tears (Flanagan and Willcox 2009) and seminal fluid (Buckett et al.
1997). Lactoferrin has antibacterial properties and has been found
to bind to gut pathogens to prevent the adhesion to intestinal cells;
the binding is at least partly modulated by the glycan moieties (Barboza
et al. 2012).
N-sulfoglucosamine sulfohydrolase
N-sulfoglucosamine sulfohydrolase is usually reported as an intracellular protein found in lysosomes where it is involved in the degradation of the glycosaminoglycan heparin sulfate. Specifically, the
enzyme catalyzes the hydrolysis of an N-linked sulfate from the nonreducing terminal glucosamine residues (Anson and Bielicki 1999).
Five potential N-linked glycan sites have been identified on the protein
(Scott et al. 1995), at least two of which have been proven experimentally (Zhang et al. 2003; Chen et al. 2009). Park et al. (2011) identified
a protein with high similarity to N-sulfoglucosamine sulfohydrolase in
sweat but it has not been reported in other secretory fluids to our
knowledge.
Peptidoglycan recognition proteins
Peptidoglycan recognition proteins (PGRP) are innate immunity molecules that work synergistically with antibacterial peptides. PGLYRP3
and PGLYRP4 have been identified in human sweat (Lu et al. 2006;
Wang et al. 2007), saliva, mucous membranes, the skin, gastrointestinal
tract and the cornea of the eye (Dziarski and Gupta 2010), as well as in
camel’s milk (Sharma et al. 2011). PGLRP3 has one potential
N-linked glycosylation site, PGLRP4 has five potential N-linked glycosylation sites (www.uniprot.org) and glycosylation of the proteins has
also been demonstrated experimentally following reduction in protein
size in SDS-PAGE following enzymatic release of the glycans (Lu et al.
2006). PGLYRPs have been found to be bactericidal to pathogenic
bacteria but not to commensals, which may have evolved mechanisms
to withstand the molecules. However, PGRPs are bacteriostatic for
most gram-positive and gram-negative bacteria suggesting a possible
anti-adhesive role (Lu et al. 2006).
Plasminogen activator inhibitor 2/PAI-2 (Urokinase
inhibitor)
Plasminogen activator inhibitor 2/PAI-2 (Urokinase inhibitor) is a serpin peptidase inhibitor in human serum, which inactivates urokinase
to maintain tissue homeostasis. It also acts as a coagulation factor
(Lobov et al. 2008) and may have a role in the adaptive immune response (Schroder et al. 2011). The glycoprotein has also been identified in sweat (Park et al. 2011), saliva (Virtanen et al. 2006) and tears
(Csutak et al. 2008). Two forms of PAI-2 have been described: a
225
The potential of sweat glycoproteins for microbial adhesion
47 kDa intracellular nonglycosylated form and a glycosylated 60 kDa
secreted form (Medcalf and Stasinopoulos 2005) with three potential
N-linked glycosylation sites (www.uniprot.org). Human keratinocytes
produce both glycosylated and unglycosylated forms of PIA-2 during
differentiation (Wang and Jensen 1998).
Polymeric immunoglobulin receptor ( poly Ig receptor)
secretory component
Polymeric immunoglobulin receptor ( poly Ig receptor) secretory component (SC) binds to and facilitates the secretion of immunoglobulins
through the epithelium and subsequently forms the secretory component of sIg, such as IgA and IgM. It has been identified in human sweat
(Huff 1990; Park et al. 2011), bovine saliva (Sakaguchi et al. 2013),
human tears (Balasubramanian et al. 2013) and human milk (Asano
et al. 2004). The SC of the poly Ig receptor contains seven sites with
N-linked glycans (Eiffert et al. 1984). The poly IgA receptor also binds
to Streptococcus pneumonia and translocates the bacterium through
nasopharyngeal and respiratory epithelial cells (Asmat et al. 2011).
Prolactin-inducible protein/PIP (gross cystic disease fluid
protein/GCDFP-15)
Prolactin-inducible protein/PIP (gross cystic disease fluid protein/
GCDFP-15) has been identified in sweat (Park et al. 2011; Raiszadeh
et al. 2012), saliva (Ramachandran et al. 2006), tears (Zhou et al.
2009), human milk (Picariello et al. 2008), seminal fluid (Chiu and
Chamley 2003) and plasma (Thambisetty et al. 2010). The glycoprotein appears to regulate water transport in apocrine glands and eccrine
glands (Mazoujian et al. 1983). Plasma PIP binds to IgG and plays a
role in immunomodulation (Chiu and Chamley 2003). Salivary PIP
binds to streptococcus species (Schenkels et al. 1997; Lee et al.
2002) and is involved in bacterial aggregation and biofilm formation
(Moi et al. 2012). One site containing N-linked glycans has been identified (Hassan et al. 2008).
Thrombospondin
Thrombospondin is an adhesive glycoprotein that mediates cellular interactions. It has been found in sweat (Park et al. 2011; Raiszadeh
et al. 2012), around the sweat glands in the epidermis (Bégány et al.
1994), in saliva (Crombie et al. 1998) and in human ocular surface
epithelium (Sekiyama et al. 2006). Glycosylation of thrombospondin
involves C-mannosylation and O-fucosylation (Hofsteenge et al.
2001), as well as the more typical N-linked glycans (Ramachandran
et al. 2006). Gram-positive bacteria including S. pneumonia,
S. pyogenes, Staphylococcus aureus and Listeria monocytogenes
bind to thrombospondin via the bacterial peptidoglycan (Rennemeier
et al. 2007). The H1V1 virus binds to thrombospondin in saliva
(Crombie et al. 1998).
Zinc-α2-glycoprotein
Zinc-α2-glycoprotein is a multifunctional protein identified in sweat
(Nakayashiki 1990; Park et al. 2011), tears (Versura et al. 2012),
human milk (Picariello et al. 2008), saliva (Wu et al. 2009), seminal
fluid (Hassan et al. 2008), plasma (Nakayashiki et al. 1992) and
urine (Jain et al. 2005). The function of the glycoprotein is not yet
fully understood, but it seems to have a role in lipid metabolism and
mobilization, cell cycle regulation and cancer progression (SörensenZender et al. 2013). Experimental evidence has been provided for
four sites on the protein which carry N-linked glycans (Ramachandran et al. 2006, Hassan et al. 2008). The protein has a binding site
for hydrophobic ligands, most probably polyunsaturated fatty acids
(McDermott et al. 2006).
Conclusions
From the above analysis, there is a very high similarity between the glycoproteins in sweat and those in other secretory fluids, including tears,
saliva and milk. Concomitantly, there is accumulating evidence that
these glycoproteins can act as a decoy for pathogen binding, allowing
removal via the washing mechanism of the secretory fluid, and inhibiting pathogen attachment to epithelial cells of the cornea, oral and
digestive tract, respectively. It thus appears likely that sweat glycoproteins may act via a similar mechanism. The potential of sweat glycoproteins to bind to bacteria is undoubtedly high and warrants further
investigation.
Acknowledgements
We thank L’Oreal Research and Innovation, France, for partial funding of this
review.
Conflict of interest statement
None declared.
Abbreviations
apoD, apolipoprotein D; ASOB1, apocrine secretion odor-binding glycoprotein
1; ASOB2, apocrine secretion odor-binding glycoprotein 2; BPRP, basic
proline-rich protein; CEA, carcinoembryonic antigen; CRISP-3, cysteine-rich
secretory protein; DC-SIGN, dendritic cell-specific ICAM-3-grabbing nonintegrin; DMBT1, deleted in malignant brain tumors 1 protein; G3BP, galectin-3binding protein; HIV-1, human immunodeficiency virus type 1; LC–MS-MS,
liquid chromatography tandem mass spectrometry; Lf, lactoferrin Lf; MRM–
MS, multiple reaction monitoring mass spectrometry; PGRP, peptidoglycan
recognition proteins; PIP, prolactin-inducible protein; SC, secretory component;
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SRCR,
scavenger receptor cysteine-rich.
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