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Review Article
Control of blood proteins by functional disulfide bonds
Diego Butera, Kristina M. Cook, Joyce Chiu, Jason W. H. Wong, and Philip J. Hogg
Lowy Cancer Research Centre and Prince of Wales Clinical School, University of New South Wales, Sydney, NSW, Australia
Most proteins in nature are chemically
modified after they are made to control
how, when, and where they function. The 3
core features of proteins are posttranslationally modified: amino acid side chains
can be modified, peptide bonds can be
cleaved or isomerized, and disulfide bonds
can be cleaved. Cleavage of peptide bonds
is a major mechanism of protein control in
the circulation, as exemplified by activation
of the blood coagulation and complement
zymogens. Cleavage of disulfide bonds is
emerging as another important mechanism
of protein control in the circulation. Recent
advances in our understanding of control
of soluble blood proteins and blood cell
receptors by functional disulfide bonds is
discussed as is how these bonds are
being identified and studied. (Blood.
2014;123(13):2000-2007)
Introduction
There are 3 fundamental types of posttranslational modifications of
proteins that appear to operate in all life forms.1-3 Amino acid side
chains can be covalently modified (type 1), peptide bonds can be
hydrolytically cleaved or isomerized (type 2), and disulfide bonds
can be reductively cleaved (type 3) (Figure 1). These chemical
changes are involved in all aspects of protein function and vastly
increase the sophistication of the proteome.
The type 1 modifications of side chains nearly always require an
enzyme and a cosubstrate, so these events are usually restricted to
specific intracellular environments where all 3 components are
available.1 Type 2 modifications of peptide bonds and type 3 modifications of disulfide bonds are suited to the circulation. Cleavage of
peptide or disulfide bonds usually does not require a cofactor, so the
factors that mediate these events need only to find their substrate to
function.
Mass spectrometry analysis of human plasma has identified 1929
different proteins.6 The tertiary structures of 817 of these proteins are
currently known or inferred from a homologous structure/domain;
these contain 4594 disulfide bonds. Assuming this average protein to
disulfide bond ratio of 1:5 holds for the remaining proteins with
unknown structure, the 2000 or so plasma proteins will contain about
10 000 disulfide bonds.
Most of these disulfide bonds, as with most of the peptide bonds,
perform a structural role. They stabilize the mature protein structure
and remain unchanged for the life of the protein. However, some of
the disulfide bonds—the allosteric disulfides—control the function
of the mature protein in which they reside when they are cleaved.
Functional disulfides
Disulfide bonds in blood proteins
Protein disulfide bonds are the links between the sulfur atoms of
2 cysteine amino acids (the cystine residue) that form as proteins
mature in the cell. These bonds have accrued during the evolution of
eukaryotic proteins and, once acquired, have almost always been
retained.4
The tertiary structures or partial tertiary structures of 4104 human
proteins are currently known or inferred from a homologous protein/
domain; these contain 16 538 disulfide bonds (UniProt annotation).
About half the disulfide bonds (7264) are in membrane proteins
(1987) and most of the rest (8424) are in proteins containing a
secretion signal sequence (1204). Interestingly, 587 proteins that
reside in the cytoplasm or nucleoplasm contain 935 disulfide bonds,
which are environments traditionally thought not to be conducive to
disulfide bond formation. The mechanism of formation of disulfide
bonds in cytoplasm and nucleoplasm proteins is largely unknown,
although protein disulphide isomerase (PDI) has recently been found
to interact with the actin cytoskeleton.5 These numbers are representative of the protein disulfide bonds in and on leukocytes.
Submitted January 14, 2014; accepted February 6, 2014. Prepublished online
as Blood First Edition paper, February 12, 2014; DOI 10.1182/blood-2014-01549816.
2000
Allosteric control is defined as a change in 1 site—the allosteric
site—that influences another site by exploiting the protein’s
flexibility.7 Thus, cleavage of an allosteric disulfide bond results in
a functional change at another site in the protein. Changes in ligand
binding, substrate hydrolysis, proteolysis, or oligomer formation
have been identified in blood proteins.2 The allosteric disulfide bonds
are reduced by the catalytic disulfides of oxidoreductases (Figure 2A)
or by thiol-disulfide exchange (Figure 2B).2,8
Oxidoreductase cleavage
Oxidoreductase active sites contain a reactive dithiol/disulfide in
a CXXC motif that can reduce or oxidize a substrate disulfide bond
(Figure 2A). Seven members of the PDI family,9 2 members of the
thioredoxin family, and glutaredoxin-110 have been identified in
human plasma6 (Table 1). Three other members of the PDI family,
transmembrane TMX3, ERp44, and ERp29, and the flavoenzyme,
Ero1a, have been detected on the platelet surface.11,12 ERp44 and
ERp29, however, do not contain a CXXC active site motif, so they
may not participate in thiol-disulfide exchange reactions.9 The roles
in the circulation of the PDI family members—PDI and ERp57—are
best understood to date. There is also emerging evidence for a role for
D.B. and K.M.C. contributed equally to this study.
© 2014 by The American Society of Hematology
BLOOD, 27 MARCH 2014 x VOLUME 123, NUMBER 13
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BLOOD, 27 MARCH 2014 x VOLUME 123, NUMBER 13
Figure 1. The 3 fundamental types of posttranslational modifications. The ribbon
structure of a model protein is shown. The amino acid side chains and the peptide
and disulfide bonds that bind the polypeptide backbone can be posttranslationally
modified. Type 1 modifications are covalent additions of a molecule to an amino acid side
chain. The side chains of 15 of the 20 common amino acids of proteins can be covalently
modified in reactions that usually involve an enzyme and a cosubstrate.1 The lysine and
tyrosine side chains are shown. The most frequent Type 1 modification in humans is
phosphorylation of tyrosine. Type 2 modifications are hydrolytic cleavage or isomerization of certain peptide bonds. Hydrolytic cleavage is catalyzed by proteases, which are
tightly regulated in space and time because the cleavage is irreversible. Isomerization of
the peptide bond on the C-terminal side of proline residues is catalyzed by peptidyl prolyl
cis-trans isomerases. Type 3 modifications are reductive cleavage of certain disulfide
bonds, known as allosteric disulfides. Allosteric disulfide bonds control the function of the
mature protein in which they reside by mediating a change when they are cleaved by
oxidoreductases or by thiol-disulfide exchange.
ERp5 in platelet function and thioredoxin in the inflammatory
response.
PDI is secreted by platelets,13 endothelial cells,14,15 and neutrophils,16 and mediates protein dithiol-disulfide exchange events in the
Figure 2. Mechanisms of cleavage of allosteric
disulfide bonds. Disulfide bond reduction occurs via
a second-order nucleophilic substitution (SN2)-type reaction mechanism in which the 3 sulfur atoms involved
must form an ;180° angle. (A) For oxidoreductase
cleavage, the active site sulfur ion nucleophile of the
oxidoreductase (green) attacks 1 of the sulfur atoms of the
allosteric disulfide bond (gray). The mixed disulfide that
forms then spontaneously decomposes, releasing oxidized oxidoreductase and the substrate protein containing a reduced allosteric disulfide. (B) For thiol-disulfide
exchange, the protein contains a sulfur ion nucleophile
that is unreactive until a conformational change brings the
sulfur ion in line with the allosteric bond, where it attacks 1
of the sulfur atoms of the disulfide, cleaving the bond.
The conformational change can be mediated by ligand
binding or by mechanical shear of the protein. Intramolecular cleavage is shown, but this can also occur
intermolecularly.
CONTROL OF BLOOD PROTEINS BY DISULFIDE BOND CLEAVAGE
2001
extracellular space.17,18 This oxidoreductase binds to b3 integrins in
the thrombus19 and avb3 integrin on endothelial cells.20 PDI inhibitors reduce platelet thrombus size and fibrin formation in both microand macrovascular murine models.14,21-23 Platelet PDI is involved in
aIIbb3 integrin–mediated platelet accumulation,24 whereas endothelial PDI is required for fibrin formation.14 Neutrophil PDI modulates
ligand binding to aMb2 integrin and neutrophil recruitment during
venous inflammation.25
The PDI family member ERp57 translocates to the platelet surface after activation, binds to b3 integrin, and is involved in platelet
activation and aggregation in vitro and incorporation of platelets into
a growing thrombus in murine models.26,27 PDI and ERp57 appear to
have distinct roles in b3 integrin function and platelet aggregation as
addition of function blocking anti-ERp57 antibodies to PDI-deficient
platelets further diminishes integrin aIIbb3 activation and platelet
aggregation.24 Another PDI family member, ERp5, is also recruited
to the platelet surface after activation in vitro, and function-blocking
anti-ERp5 antibodies inhibit platelet aggregation, fibrinogen binding, and P-selectin exposure.28 A role for ERp5 in platelet function in
vivo has not been reported to date.
Thioredoxin has been implicated in the inflammatory response in
the circulation. Expression of this reductant is upregulated, and the
protein is secreted by immune cells during inflammation, leading to
a high local concentration and elevated blood levels.29 Serum levels
of thioredoxin are increased in patients with asthma, rheumatoid
arthritis, and heart failure30,31 and positively correlate with disease
activity in rheumatoid arthritis.32,33
PDI, ERp57, possibly ERp5, and thioredoxin are involved,
therefore, in thrombosis and/or inflammation in mammals. The target
disulfide bonds of these oxidoreductases are being defined. PDI is
involved in reduction or formation of disulfide bonds in some platelet
and leukocyte integrins and possibly leukocyte tissue factor (TF) (see
the following section). Thioredoxin reduces the allosteric disulfides
in b2-glycoprotein I34,35 and mast cell b tryptase36 in vitro; the role
of this reductase in vivo is being studied (see the following section).
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2002
BLOOD, 27 MARCH 2014 x VOLUME 123, NUMBER 13
BUTERA et al
Table 1. Disulfide bond oxidoreductases identified in plasma and/or on the surface of platelets or leukocytes
Class/family
PDI
Member
PDI
Alternative names
Cellular thyroid hormone–binding protein, prolyl
Gene name
P4HB
4-hydroxylase subunit b, p55
PDI A3
58-kDa glucose-regulated protein, p58, disulphide
PDIA3
isomerase ER-60, ERp57, ERp60
PDI A4
ERp70, ERp72
PDIA4
PDI A6
ERp5, PDI P5, thioredoxin domain–containing
PDIA6
Thioredoxin domain–containing protein 5
ERp46, thioredoxin-like protein p46
protein 7
Thioredoxin domain–containing protein 15
TXNDC5
TXNDC15
Thioredoxin domain–containing protein 17
TRP14, protein 42-9-9, thioredoxin-like protein 5
PDI TMX3
Thioredoxin domain–containing protein 10,
TXNDC17
TMX3
thioredoxin-related transmembrane protein 3
Thioredoxin
Endoplasmic reticulum resident protein 44
ERp44, thioredoxin domain–containing protein 4
ERP44
Endoplasmic reticulum resident protein 29
ERp29, ERp28, ERp31
ERP29
Thioredoxin
ADF, SASP
Thioredoxin-like protein 1
32 kDa thioredoxin-related protein
TXN
TXNL1
Glutaredoxin
Glutaredoxin-1
Thioltransferase-1
GLRX
Flavoenzyme
ERO1-like protein a
Ero1a, endoplasmic oxidoreductin-1–like protein,
ERO1L
oxidoreductin-1-L-a
The general question of which oxidoreductase cleaves which
allosteric disulfide will be determined by some of the same factors
that govern which protease cleaves which peptide bond. Some
proteases cleave many peptide bonds (such as thrombin), whereas
others cleave only 1 or a very restricted number (such as factor
VIIa), and the same scenario will likely apply to oxidoreductases
and allosteric disulfide bonds. Oxidoreductase substrate selectivity
will largely be driven by steric factors (accessibility of the disulfide
bond in the substrate protein and conformation of the active site
pocket of the enzyme) and by environmental context (being in the
right place at the right time). The highly directional nature of
disulfide bond cleavage will further restrict the substrate selection
of oxidoreductases. Reduction of a disulfide bond proceeds via
a second-order nucleophilic substitution (SN2)-type reaction
mechanism in which the 3 sulfur atoms involved (the sulfur ion
nucleophile and the 2 sulfur atoms of the disulfide bond) must form
an ;180° angle37,38 (Figure 2). For instance, stretching, twisting, or
pulling of proteins makes their disulfide bonds easier or harder
to cleave by changing the alignment of the 3 sulfur atoms.39,40
Another factor that influences the substrate selectivity of oxidoreductases is the redox potential of the oxidoreductase catalytic
disulfide and the allosteric disulfide. Catalytic disulfides will only
cleave allosteric disulfides with a bigger (less negative) redox
potential.
An open question is whether the oxidoreductases act catalytically in the circulation to reduce several substrate disulfides or
reduce only 1 disulfide bond. Intracellular oxidoreductases act
as catalysts because factors regenerate the reduced enzyme. The
thioredoxin active site disulfide, for example, is reduced by
thioredoxin reductase and reduced NAD phosphate (NADPH) in
the cytoplasm.10 NADPH provides the hydrogens and electrons
used to reduce the oxidized thioredoxin. Thioredoxin reductase is
found in plasma,6 but NADPH is not. It is possible, and seems
likely, that a mechanism or mechanisms exist to cycle some
oxidoreductases in blood. For instance, Ero1a has been implicated in
oxidation of PDI on the platelet surface.12 On the other hand,
restricting oxidoreductases to cleavage of a single substrate disulfide bond would be an efficient mechanism of regulation of their
activity. Proteases, for instance, only require a source of water to
cleave peptide bonds, so protease inhibitors have coevolved
with proteases to control their activity.1 Controlling access to
a source of hydrogens and electrons is a simple means of restricting
oxidoreductase activity. Endogenous inhibitors of the oxidoreductases in the extracellular space have not been identified to date,
which supports the idea that oxidoreductases may function as
single-turnover enzymes.
Thiol-disulfide exchange cleavage
Cleavage of allosteric disulfides by thiol-disulfide exchange does
not require additional hydrogens and electrons (Figure 2B), so this
mechanism of cleavage is particularly suited to the circulation. All
that is required is a conformational change in the substrate protein,
which could be triggered by ligand binding and/or the mechanical
shear forces of flowing blood. The substrate protein contains a sulfur
ion that is unreactive until a change in structure brings it in line
with the allosteric bond, where it attacks and cleaves the disulfide
bond. Allosteric disulfide bonds in plasma plasminogen41 and von
Willebrand factor (VWF)42 are cleaved by thiol-disulfide exchange
(see the following section).
An important difference between peptide and disulfide bond
cleavage is that cleavage of disulfide bonds is potentially reversible;
this can provide for a fine level of protein regulation not possible with
peptide bond cleavage. Some allosteric disulfides exist in equilibrium between reduced and oxidized isoforms in the population of
protein molecules, and perturbation of the equilibrium by oxidoreductase or ligand binding, for instance, leads to a biological change.
Cleavage of other allosteric disulfides, though, is irreversible as the
downstream effects of the cleavage prevent the disulfide bond from
reforming. Both of these situations in different blood proteins are
discussed here.
Blood proteins regulated by allosteric
disulfide bonds
We have chosen to highlight 4 soluble blood proteins and a lymphocyte receptor whose functions are controlled by cleavage of
allosteric disulfide bonds. These examples are the best characterized
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BLOOD, 27 MARCH 2014 x VOLUME 123, NUMBER 13
at the molecular level so far. We also briefly discuss 7 other examples
at a more preliminary stage of characterization.
Angiotensinogen
Plasma angiotensinogen is proteolyzed by renin and angiotensinconverting enzyme to produce the angiotensin peptides that control
blood pressure and fluid homeostasis. The angiotensinogen Cys18–
Cys138 disulfide bond is cleaved in a fraction of the plasma protein,
which is significant because the oxidized protein is more efficiently
activated by renin than the reduced protein.43 The ratio of oxidized to
reduced angiotensinogen in blood is approximately 60:40 in healthy
volunteers (independent of age or gender). This ratio increases in
pregnant females with preeclampsia, which correlates with increased
cleavage by renin and elevated blood pressure. It has been suggested
that exposure of maternal angiotensinogen to reactive oxygen species
present in the placenta increases the level of oxidized angiotensinogen,
contributing to the development of preeclampsia.43
b2-glycoprotein I
Plasma b2-glycoprotein I is an autoantigen in the antiphospholipid syndrome, which is associated with vascular thrombosis,
accelerated atherosclerosis, and recurrent miscarriages.44 The
b2-glycoprotein I–antibody complex appears to prime the arterial
and venous vasculature for thrombosis through effects on blood
cells and coagulation and fibrinolysis proteins. b2-glycoprotein I
consists of 4 complement modules and a unique fifth domain that
contains a disulfide bond linking Cys288 and the C-terminal
residue, Cys326.
The Cys288–Cys326 disulfide bond is cleaved in a fraction of
plasma b2-glycoprotein I, and the bond is reduced in the purified
protein by PDI and thioredoxin.34,35 The ratio of oxidized to
reduced b2-glycoprotein I is elevated in the blood of patients with
antiphospholipid syndrome compared with healthy volunteers
and with patients with vascular thrombosis and no antiphospholipid antibodies.45 There is also more oxidized b2-glycoprotein I
in the blood of patients with antiphospholipid syndrome with both
anti–b2-glycoprotein I antibodies and lupus anticoagulant than
patients with only anti–b2-glycoprotein I antibodies.45
The elevated oxidized b2-glycoprotein I in patients with
antiphospholipid syndrome is associated with increased immunogenicity of the protein and increased thrombosis. 45 Anti–b2glycoprotein I antibodies from mice and rabbit plasma and
autoantibodies from the plasma of patients with antiphospholipid
syndrome bind more avidly to oxidized b2-glycoprotein I than
reduced protein. The increased thrombosis may also relate to loss of
anti-thrombotic properties of reduced b2-glycoprotein, such as
protection of endothelial cells from oxidative stress.34
IL receptor subunit g
Interleukin (IL) receptor subunit g or CD132 is the common g
subunit of the cytokine receptors for IL-2, -4, -7, -9, -15, and -21 on
the surface of lymphocytes. The CD132 Cys183–Cys232 disulfide
bond is cleaved on the surface of cultured T cells by protein
reductants and on the surface of thymocytes in mice after an inflammatory challenge.46 The disulfide bond is located at the subunit
surface close to the IL-2 binding site.47 Reduction of the disulfide46
or replacement of Cys183 and Cys232 with alanine or serine48
inhibits IL-2 binding to the receptor complex and therefore receptor
signaling and cell proliferation. Notably, Cys183 and Cys232
mutants are among those identified in patients with X-linked severe
combined immunodeficiency.49,50
CONTROL OF BLOOD PROTEINS BY DISULFIDE BOND CLEAVAGE
2003
Plasminogen
Plasma plasminogen is the zymogen form of plasmin. The zymogen
consists of an N-terminal Pan-apple domain followed by 5 kringle
domains and a C-terminal serine protease domain. Plasminogen is
converted to plasmin by urokinase or tissue plasminogen activator
through cleavage of the Arg561–Val562 peptide bond in the serine
protease domain and the Pan-apple domain is autoproteolytically
released to produce mature plasmin. Plasmin activates other zymogens
and cleaves the fibrin meshwork during dissolution of the thrombus
and components of the extracellular matrix.
The Cys462–Cys541 disulfide bond in the kringle 5 domain is
cleaved in a fraction of plasma plasminogen, and the level of reduced
plasminogen varies in healthy individuals.41,51 Fragments of plasminogen containing the kringle domains are generated in plasma; these
fragments function as endogenous inhibitors of tumor angiogenesis.52
The kringle fragments are produced from the reduced form of the
zymogen.41 Generation of the kringle fragments from reduced
plasminogen involves conversion to reduced plasmin, reduction
of an additional kringle 5 disulfide bond, and subsequent proteolysis
of up to 3 peptide bonds.41,53-55 Plasmin ligands such as tumorderived phosphoglycerate kinase56 induce a conformational change
in reduced kringle 5 that leads to attack by the Cys541 thiolate on the
Cys536 sulfur atom of the Cys512–Cys536 disulfide bond, resulting
in reduction of the bond by thiol-disulfide exchange (Figure 2B).
Cleavage of the Cys512–Cys536 disulfide bond leads to a conformational change in plasmin and exposure of the peptide backbone to
proteolysis on the C-terminal side of residues Lys486, Arg474, and
Arg530. The proteolysis can be catalyzed by plasmin itself or other
serine- or metalloproteases.
VWF
VWF is a plasma protein produced by vascular endothelial cells and
megakaryocytes that chaperones blood coagulation cofactor factor
VIII and tethers platelets to the injured blood vessel wall. It is
a large glycoprotein that circulates as a series of multimers containing
variable numbers of 500-kDa dimeric units. VWF structure is
influenced by the mechanical shear forces in blood, where it transitions
from a loosely coiled ball to elongated fibers that bind platelets.57-63
Fiber formation involves covalent self-association of VWF
mediated by thiol-disulfide exchange in the VWF C2 domains.64,65
Both the C2 Cys2431–Cys2453 and nearby Cys2451–Cys2468
disulfide bonds are involved in fiber formation. Reduction of the C2
domain Cys2431–Cys2453 disulfide bond in 1 molecule, presumably
by an oxidoreductase in blood, creates a Cys2431 thiolate anion that
attacks the Cys2431 sulfur (of the Cys2431–Cys2453 disulfide bond)
in another VWF molecule, resulting in a disulfide-linked dimer.42 The
Cys2451–Cys2468 disulfide-dithiol then mediates formation of
trimers and higher order oligomers.
Other blood proteins
Three blood cell receptors and 4 other soluble plasma proteins have
been found to contain putative allosteric disulfides. These examples
lack either in vivo evidence for a role for the bond and/or there is
uncertainty as to the identity of the allosteric disulfide or disulfides in
the protein. Their classification as allosteric disulfides should be
considered preliminary at this stage.
Integrins are transmembrane, heterodimeric cell-adhesion receptors that mediate interactions between the cytoskeleton of cells and
extracellular ligands and also play a role in signal transduction.
Integrin activation has been associated with reduction of 1 or more
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2004
BUTERA et al
disulfide bonds in the receptors.18,20,66-77 There are 18 a integrin
subunits and 8 b subunits in mammals, but the majority of the redox
studies have been conducted on the b3 subunit of the platelet aIIbb3
(fibrinogen) receptor and the platelet and endothelial avb3 (vitronectin) receptor. There are 4 epidermal growth factor (EGF)-like
domains in the extracellular region of b3.78,79 The disulfides in the
EGF domains are generally important for maintaining the inactive
state of b3 because mutation of a single cysteine for most of the
disulfide bonds within the EGF domains results in a constitutively
active b3.80-82 A recent study of the function of an atypical disulfide
bond in b3 EGF domains is revealing.83 The disulfide bond in each of
the 4 EGF domains in both aIIbb3 and avb3 was investigated by
mutating the cysteines to serines. An allosteric role for the EGF-4
Cys560–Cys583 disulfide in both aIIbb3 and avb3 and for the
EGF-3 Cys523–Cys544 bond only in avb3 was identified. For
instance, activation of the C523S/C544S avb3 mutant by an
antibody and a reducing agent is impaired. These disulfides are
possible PDI19,20 and/or ERp5726,27 substrates.
TF is a transmembrane cofactor for factor VIIa. The TF/VIIa
complex proteolytically activates factor X to initiate blood coagulation
and provide the thrombin burst required for a stable thrombus. Intravascular TF is found on monocytes and neutrophils and exists mostly
in a noncoagulant or cryptic form bound nonproductively to VIIa.
Acute events lead to local decryption of TF, which appears to involve
both formation of a disulfide bond between unpaired Cys186 and
Cys209 in cryptic TF and exposure of phosphatidylserine on the cell
surface (reviewed in Chen and Hogg84). Thioredoxin has been implicated in the maintenance of cryptic TF by cleaving the TF
Cys186–Cys209 disulfide,84,85 whereas PDI has been implicated in
TF decryption.86,87 The molecular mechanism of action of PDI in TF
decryption is not known. Both the oxidoreductase and chaperone functions of PDI are possibly involved (reviewed in Langer and Ruf88).
Two circulating serine proteases appear to be regulated by
allosteric disulfides. Proteolytic activation of coagulation factor XI
contributes to the intrinsic/amplification phase of blood coagulation.
One or more factor XI disulfide bonds are reduced in a fraction of the
zymogen in blood, and the Cys362–Cys482 and Cys118–Cys147
disulfides are reduced by thioredoxin in the isolated enzyme.89 The
reduced protein is more efficiently activated by thrombin, factor
XIIa, or factor XIa than the oxidized protein, and blood levels of the
reduced factor XI are elevated in patients with antiphospholipid
syndrome,89 which may contribute to the thrombosis in this syndrome. Mast cell bII-tryptase is associated with pathological inflammation. The bII-tryptase Cys220–Cys248 disulfide bond exists in
oxidized and reduced states in the secreted enzyme and the bond is
reduced by thioredoxin in the isolated enzyme.36 The oxidized and
reduced isoforms have different specificity and catalytic efficiency for hydrolysis of substrates.
The activity of 2 growth factors is also regulated by disulfide bond
cleavage. Platelets contain abundant transforming growth factor-b1
(TGF-b1) that functions in a variety of physiologic and pathologic
events. TGF-b1 is secreted as an inactive form in complex with
latency-associated peptide and latent TGF-b–binding protein 1.
Platelet TGF-b1 is activated by shear-induced disulfide bond
cleavage in the growth factor,90 and platelet PDI has been implicated
in this event.91 The identity of the functional disulfide bond or bonds
in TGF-b1 is not known at present. The structures of the lymphangiogenic growth factors, vascular endothelial growth factor
(VEGF)-C and VEGF-D, are also regulated by disulfide cleavage.92
The VEGFs function as disulfide-linked antiparallel homodimers, and
dimer formation of VEGF-C and VEGF-D is controlled by a unique
unpaired cysteine that exchanges with the interdimer disulfide bond.93
BLOOD, 27 MARCH 2014 x VOLUME 123, NUMBER 13
Figure 3. Configurations of allosteric disulfide bonds. (A) Classification of
disulfide bonds based on their geometry.95,96 The geometry is defined by the 5 bond
angles (x angles) linking the 2 a-carbons of the cystine residue. Ca, main chain carbon
atom; Cb, side chain carbon atom of each cysteine residue. The x angles are recorded
as being either positive or negative. The 3 basic types of bond configurations (spirals,
hooks, and staples) are based on the signs of the central 3 angles, and they can be
either RH or LH depending on whether the sign of the x3 angle is positive or negative,
respectively. These 6 bond types expand to 20 when the x1 and x19 angles are taken
into account. (B) Examples of the structures of the emerging allosteric configurations:
–RHStaple, –LHHook, and 2/1RHHook.
Detection of allosteric disulfide bonds in
blood proteins
Allosteric disulfides are being identified using bioinformatics and
experimental screens, and both approaches are proving useful for
identifying these bonds in circulating proteins.
Bioinformatic approach
The bioinformatic technique relies on high-resolution 3-dimensional
protein structures and a dataset of allosteric disulfides from which
common features can be derived. The configuration, surface exposure,
and secondary structural motifs that the disulfide links can be useful
measures for the identification of allosteric bonds. These and other
disulfide bond parameters are easily extracted from any protein
structure in Protein Data Bank format.94 The most informative
disulfide bond measure at this time is the configuration of the cystine
residue. The geometry of cystine is defined by 5 dihedral or x angles,
which are calculated by the rotation around the bonds linking the
6 atoms (Figure 3A).95 There are 20 possible different cystine configurations, and all the types have been identified in protein crystal
structures.95,96 Of the 20 different configurations, the right-handed
(RH) –RHStaple, left-handed (LH) –LHHook, and 2/1RHHook
bonds are emerging as allosteric configurations (Figure 3B).2,8 The
configurations of allosteric disulfide bonds in blood proteins and their
mechanism of cleavage, where known, are summarized in Table 2.
It is important to keep in mind, though, that the protein backbone
and the cystines that link it can change shape when in solution. That
is, the crystal structure might represent only 1 of a number of different possible structures in solution. For instance, the same disulfide
bond can exist in different configurations in different crystal
structures.96 Dynamic isomerization of allosteric disulfide bonds is
probably an important aspect of their function. Cleavage of the bond
may only occur when the cystine adopts a particular configuration,
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BLOOD, 27 MARCH 2014 x VOLUME 123, NUMBER 13
CONTROL OF BLOOD PROTEINS BY DISULFIDE BOND CLEAVAGE
2005
Table 2. Configurations of allosteric disulfide bonds in blood proteins and their mechanism of cleavage
Disulfide cysteines
Disulfide bond configuration
(Protein Data Bank identifier)
Cleavage mechanism
Angiotensinogen
18-138
2/1RHHook (2WXW)
Oxidoreductase
43
b2-glycoprotein I
288-326
2/1RHHook (1C1Z)
Oxidoreductase
34, 35
IL-2 receptor subunit g (CD132)
183-232
2LHHook (2ERJ)
Oxidoreductase
46
Plasminogen
462-541
2/1RHHook (4DUR)
Oxidoreductase?
41, 51
512-536
2LHHook (4DUR)
Thiol-disulfide exchange
523-544
2LHHook (3FCS)
Oxidoreductase?
83
560-583
2LHHook (3FCS)
523-544
2RHStaple (3IJE)
Oxidoreductase?
83
560-583
2LHHook (3IJE)
Protein
b3 integrin (aIIbb3)
b3 integrin (avb3)
Reference
Tissue factor
186-209
2RHStaple (1BOY)
Oxidoreductase
98
Factor XI
362-482
2/1RHHook (2F83)
Oxidoreductase
89
bII-tryptase
220-248
2RHHook and
Oxidoreductase
36
Thiol-disulfide exchange
93
2RHSpiral (3FPZ)
VEGF-C
2LHHook (2X1W)
156-165
which could be controlled by ligand binding or the mechanical shear
in the circulation.
Experimental approach
A method based on differential cysteine labeling and mass spectrometry is currently the best experimental screen for allosteric
disulfides.36,41,42,97 The approach is based on the principle that an
allosteric disulfide will exist in equilibrium between reduced and
oxidized states in a blood protein, such as observed in angiotensinogen, b2-glycoprotein I, and plasminogen. The technique measures
the proportion of reduced and oxidized protein using different thiol
alkylating agents. One alkylator is used to label the reduced portion
of the allosteric disulfide bond in blood, or a fraction thereof, without
or with previous treatment with a candidate oxidoreductase. The
remaining disulfide bonds in the protein are then reduced using
dithiothreitol and the new unpaired cysteines labeled with a second
alkylator. Mass spectrometry is used to determine the identity of the
protein and the ratio of differentially labeled cysteines. Typically, the
cysteines showing significant increase in the ratio of the first to
second alkylator after oxidoreductase treatment are allosteric disulfide
candidates.
thrombosis and hemostasis, blood pressure, and inflammation in the
circulation, and some bonds have been linked to disease. The pace of
discovery of new allosteric disulfides in blood proteins is likely to
increase during the coming years as new and better techniques for
identifying and characterizing these bonds are developed. In terms of
the oxidoreductases that cleave these bonds, the generation of specific inhibitors and cell-specific oxidoreductase-deficient mice has
and will prove valuable for identifying the environments in which the
enzymes work and their substrate disulfide bonds. Some allosteric
and catalytic bonds will also likely be valid pharmaceutical targets
and progress is being made on ways of targeting these bonds with
small molecules or biologicals (reviewed in Hogg8).
Acknowledgments
This study was supported by grants from the National Health and
Medical Research Council of Australia and the Cancer Council New
South Wales.
Summary
Authorship
The functions of many blood proteins are controlled by cleavage of
peptide bonds. For example, the blood coagulation and complement
cascades are exquisitely regulated by discrete proteolysis. There are
an increasing number of blood proteins found to be controlled by
cleavage of the next most frequent covalent bond linking the polypeptide backbone of proteins: the disulfide bond. The allosteric and
catalytic functional disulfide bonds contribute to the control of
Contribution: P.J.H. conceived the review and all authors contributed to the writing of the manuscript.
Conflict-of-interest disclosure: The authors declare no competing
financial interests.
Correspondence: Philip Hogg, Level 2, Lowy Cancer Research
Centre, University of New South Wales, Sydney 2052, Australia;
e-mail: [email protected].
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2014 123: 2000-2007
doi:10.1182/blood-2014-01-549816 originally published
online February 12, 2014
Control of blood proteins by functional disulfide bonds
Diego Butera, Kristina M. Cook, Joyce Chiu, Jason W. H. Wong and Philip J. Hogg
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