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3 Osteopontin - A Distinct Transglutaminase-Catalyzed Polymer

3 Osteopontin - A Distinct Transglutaminase-Catalyzed Polymer

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134



Y. Yokosaki



MMP-3, -7



143



152



Fig. 6.1 Schematic diagram of osteopontin. There are three critical sites for osteopontin function.

There are two integrin-binding sites in the middle of the molecule, the C-terminal end of which is

cleaved by thrombin. The otherwise cryptic SVVYGLR sequence is exposed upon thrombincatalyzed cleavage, allowing integrin α9β1 access. Canonical integrin-binding tripeptide RGD is

immediately upstream of the SVVYGLR sequence. Six integrins bind to RGD, which does not

require thrombin-catalyzed cleavage. There are a few Gln residues in the N-terminal position that

serve as acyl donors for transglutaminase 2



immunomodulation, and tumor metastasis. These functions are exerted through its

receptors, the integrins and CD44. At least ten integrins are known to function as

OPN receptors, including α5β1 (Nasu et al. 1995), α8β1 (Denda et al. 1998), αvβ1

(Hu et al. 1995), αvβ3 (Miyauchi et al. 1991), αvβ5 (Hu et al. 1995), αvβ6

(Yokosaki et al. 2005), α9β1 (Smith et al. 1996), α4β1 (Bayless et al. 1998), α4β7

(Green et al. 2001), and αXβ2 (Schack et al. 2009). Interactions with these receptors

are influenced by phosphorylation and glycosylation (Christensen et al. 2007). In

addition, OPN serves as a substrate for many proteases, including thrombin, matrix

metalloproteinases, cathepsin D, and plasmin, with at least 20 cleavage sites

(Christensen et al. 2010). The thrombin cleavage site at 152R–153S was the first

of these to be discovered; the resultant N-terminal fragment (nOPN) is the most

characterized of the cleaved fragments (Senger et al. 1994). Cleavage of nOPN

results in an up-regulated capacity for cell adhesion. nOPN also serves as a niche

for glioma cells (Yamaguchi et al. 2013), is a marker of osteoarthritis (Hasegawa

et al. 2011), and gains a new role as a neutrophil chemoattractant (Nishimichi

et al. 2011). Interestingly, the two integrin-binding sites are contiguous and

followed by the thrombin cleavage site immediately in the C-terminus (Yokosaki

et al. 1999, 2005). Upon cleavage by thrombin the sequence SVVYGLR is exposed

at the C-terminus of the fragment and serves as a binding site for integrin α9β1 that

does not bind to full-length OPN (Yokasaki and Sheppard 2000). The other

integrin-binding site is RGD of canonical integrin-binding tripeptide. Binding of

α5β1 to the RGD site is up-regulated by thrombin cleavage, but binding of αvβ3,

αvβ5, and αvβ6 to the RGD site is unaffected. The regulation of integrin-OPN

interactions by enzymatic cleavage is further managed by another protease cleavage site. MMP-3 and MMP-7 share a cleavage site at 150GL151, which is within



6 A New Integrin-Binding Site on a Transglutaminase-Catalyzed Polymer



135



the integrin-binding sequence SVVYGLR (Agnihotri et al. 2001). The resulting

C-terminus SVVYG no longer supports α9β1 binding (Yokosaki et al. 2005). The

overlap of receptors for OPN does not necessarily mean that these integrins merely

provide redundancy because each cell type has different integrin repertoires and the

combinations of utilized integrins vary widely among cell types. In addition,

interactions of different integrins with a single ligand can exert distinct effects on

cell behavior in a single cell type. For example, we have previously reported that

signals generated by binding of a single ligand, tenascin-C, to αvβ3, αvβ6, or α9β1

differently affected cell adhesion, spreading, and proliferation of the colon cancer

cell line SW480 (Yokosaki et al. 1996). Furthermore, intact OPN or thrombin- or

matrix metalloproteinase-cleaved OPN interact with distinct subsets of integrins

and exhibit distinct effects on cell behavior. Collectively, some of the functional

diversity of OPN could be attributed to this multiplicity of receptors and responses.



6.3.2



Polymerization



Besides the post-translational glycosylation, phosphorylation, and enzymatic digestions, OPN undergoes a post-translational modification, polymerization catalyzed

by TG2 or factor XIII (Prince et al. 1991). In our previous mutational study, Gln34,

36, 42, and 55 were compatible with the binding residues (Nishimichi et al. 2009).

A mass-spectrometric analysis revealed them to be Gln34 and 42 (Christensen

et al. 2014). Matrix stabilization is a widely accepted role for the action of the

transglutaminase on OPN. OPN is crosslinked to fibronectin to increase matrix

integrity (Beninati et al. 1994) and is polymerized to enhance its collagen binding

activity (Kaartinen et al. 1999). OPN is also covalently linked to bone matrix

(Kaartinen et al. 2002) and urinary stones (Kohri et al. 1992; Hamamoto

et al. 2011). In the mineralized compartment of intramembranous rat bone there

are three different transglutaminase substrates: bone sialoprotein, α2

HS-glycoprotein, and OPN. Each of these substrates are polymerized to form

homopolymers that do not interact with substrates different from themselves

(Kaartinen et al. 2002). Experiments using two-dimensional gel electrophoresis

and immunoblotting analyses have clearly demonstrated the specific homopolymer

formation.

For OPN, there is evidence for in vivo polymerization. Western blotting with

anti-OPN antibody showed a smeared and high molecular weight band from rat

bone and the aorta of Matrix Gla protein-deficient mice (Kaartinen et al. 2007). We

described the polymerization of exogenous recombinant OPN 3 h after injection

into the peritoneal space of mice (Nishimichi et al. 2011). Functionally, polymeric

OPN showed dramatically enhanced cell adhesion and migration as well as focal

contact formation through interaction with RGD-binding integrins (Fig. 6.2)

(Higashikawa et al. 2007). This enhancement supports the idea that the polymerization of ligands potentiates interactions with integrins by concentrating ligandbinding sites, thereby potentiating integrin clustering. At this point, we questioned



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Y. Yokosaki



Fig. 6.2 Spreading of cells and focal contact formation on polymeric osteopontin. HUVE cells in

serum free DMEM were plated on plates coated with 1.0 μg/ml of monomeric or polymeric

osteopontin. In the course of 30 min, cells spread dramatically on the polymeric osteopontin,

where apparent focal contacts were formed. This research was originally published in FEBS

Letters (Higashikawa et al. 2007 © Federation of European Biochemical Societies)



whether polymeric OPN expresses SVVYGL on its surface, or is cryptic, as it is in

full-length OPN. Polymeric OPN did support adhesion and migration of α9transfected SW480 cells in the presence of anti-RGD-integrin antibodies. Further,

the polymeric OPN bound to recombinant α9β1 in a dose-dependent manner, as

shown by surface plasmon resonance analysis (Fig. 6.3). These interactions were all

abrogated by an anti-α9β1 blocking mAb. Thus, it was convincing that, like the

RGD site, the SVVYGLR site is exposed on the surface of the polymer and retains

its capacity to bind α9β1. It was surprising, however, that mutations in the

SVVYGLR sequence that disrupt interaction of nOPN with α9β1 (Yokosaki

et al. 1999) had no effect at all on its binding to polymeric OPN. Moreover, antiSVVYGLR antibody did not bind to polymeric OPN, although it clearly bound to

nOPN (Nishimichi et al. 2011). These results indicated that polymeric OPN is a

ligand for integrin α9β1, as is nOPN, but unlike nOPN, the binding site is not

SVVYGLR. The binding site of α9β1 in polymeric OPN has not yet been identified.



6.3.3



Gain of a New Function of Polymeric OPN Binding

to Integrin α9β1



Tissues expressing integrin α9β1 include the basal layer of epithelial cells (Palmer

et al. 1993), the valves of lymphatic vessels (Bazigou et al. 2009), and neutrophils

(Shang et al. 1999). The β2 integrins are a class of primary leukocyte integrins

involved in tethering to and rolling on endothelial cells. Beside β2 integrins, α5, α6,

and α9 subunits are also expressed on neutrophils as β1 partners, but in contrast to



6 A New Integrin-Binding Site on a Transglutaminase-Catalyzed Polymer



137



Fig. 6.3 Binding of osteopontin to integrin α9β1. Surface plasmon resonance (Biacore) analysis

of recombinant α9β1 binding to polymeric osteopontin. The vertical axis shows the surface

plasmon resonance intensity in resonance units (RU). The horizontal axis shows the duration of

flow of buffer containing 1 mM Mn2ỵ. Recombinant 91 (10 g/ml) was bound to anti-V5 that

had been coupled to the sensor chip. Polymeric osteopontin, at concentrations of 3, 10, 30, or

90 μg/ml in buffer, was added over the sensor chip at a flow rate of 10 μl/min for 180 s (This

research was originally published in The Journal of Biological Chemistry. Nishimichi et al. (2009)

© the American Society for Biochemistry and Molecular Biology)



the unchanged expression levels of α5 and α6 upon neutrophil activation, expression of α9 is markedly up-regulated and plays a synergistic role with β2 (Mambole

et al. 2010). To find if there is any biological significance to the interaction of α9β1

with polymeric OPN, we focused on the neutrophil response to polymeric OPN. In

our previous study (Fig. 6.4), neutrophil migration was first observed with a

horizontal migration apparatus, which has a 5-μm-deep, flat channel where

chemoattractants form a gradient between one chamber and the other. Neutrophils

collected from human blood migrated through the gradient of polymeric OPN. This

activity was inhibited by anti-α9β1 antibody, but not by an anti-β2 blocking mAb

(Nishimichi et al. 2009). Similar results were obtained by random migration

analysis video-captured in the presence or absence of homogeneous concentrations

of polymeric or monomeric full-length OPN (Nishimichi et al. 2011). nOPN also

elicited random migration, which was anticipated because, unlike full-length OPN,

nOPN interacts with α9β1 (Nishimichi et al. 2011). Next, we observed the chemotactic effect of polymeric OPN in vivo (Nishimichi et al. 2011). As expected, intraperitoneal injection of polymeric OPN-induced accumulation of neutrophils

peaking at 3 h after injection. The control full-length monomeric OPN (intact

OPN), however, unexpectedly induced the accumulation. Although the peak of

the increase was delayed more than with polymeric OPN, the induction was in

contrast to the effect in vitro. Therefore, we hypothesized that intact OPN would



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Y. Yokosaki



Fig. 6.4 Neutrophil migration induced by polymeric osteopontin. (a) the numbers of neutrophils

that had migrated into and were present within the 5-μm horizontal channels containing gradients

of polymeric osteopontin (upper panel) or fMLP (lower panel) at each time point during 60 min.

Neutrophils were pre-incubated in the presence or absence of antibodies as indicated at the right.



6 A New Integrin-Binding Site on a Transglutaminase-Catalyzed Polymer



139



Fig. 6.5 Polymerization-incompetent mutant osteopontin. Western blotting of the transglutaminase 2-catalyzed polymerization reaction of human osteopontin. Lane 1 of each osteopontin

variant shows a reaction with no transglutaminase 2; lane 2, 5 μg/ml; lane 3, 10 μg/ml; and lane

4, 20 μg/ml. Wild type and three mutant osteopontins (mouse recombinant) in which one, two, or

four Gln residues, as indicated above, are replaced with Ala were polymerized and probed with an

anti-OPN mAb that recognizes both monomeric and polymeric osteopontin (This research was

originally published in The Journal of Biological Chemistry. Nishimichi et al. (2011) © the

American Society for Biochemistry and Molecular Biology)



have been polymerized in vivo. To test this hypothesis of in vivo polymerization,

we generated a mutant OPN that does not polymerize (Fig. 6.5) and injected it into

mice peritoneal spaces. Neutrophil accumulation was of significantly lower magnitude than with intact OPN. To observe the in vivo polymerization directly, the

peritoneal space was washed by PBS 3 h after injection of polymeric OPN, and

analyzed by western blotting for immunoreactivity of OPN. The blot showed a high

molecular weight smeared band characteristic of the appearance of polymerized

OPN. Further ELISA measurement showed that the mutant did not decrease as

ä



Fig. 6.4 (continued) (b) chemokinesis of neutrophils induced by three different forms of

osteopontin. Each dot represents the distance of human neutrophil migration induced by OPN*

(full-length monomeric osteopontin, n ¼ 20), nOPN (N-terminal fragment of thrombin-cleaved

osteopontin, n ¼ 22), or polymeric osteopontin (n ¼ 20) with negative (medium alone; n ¼ 20) and

positive (fMLP; n ¼ 21) controls. Migrations of 20 or more randomly selected neutrophils were

video-captured, and cell tracks were traced for 20 min. Cells that did not migrate were excluded as

non-viable. Cells were kept at 37  C in a humidified atmosphere of 5 % CO2 during the migration.

(c) representative computer-traced cell tracks for three cells induced by fMLP or medium alone.

Bars, 100 μm (This research was originally published in The Journal of Biological Chemistry.

Nishimichi et al. (2011) © the American Society for Biochemistry and Molecular Biology)



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Y. Yokosaki



intact OPN did in the peritoneal space. These result indicate that intact OPN is

polymerized in vivo and attracts neutrophils. But these result do not preclude the

possibility that nOPN was also generated in the peritoneal space and contributed to

the neutrophil accumulation. nOPN specific ELISA measurement indicated that

nOPN was there, although it was at the level of ~1 ng/ml while intact OPN was at

~1 μg/ml. Then we generated thrombin cleavage-incompetent mutant OPN to

determine whether nOPN attracts any neutrophils after full-length OPN injection.

There was no difference in neutrophil accumulation between injections of wild-type

and mutant OPN, excluding the possibility that the small amount of nOPN generated support the neutrophil accumulation. In these context, neutrophils were

attracted by polymeric OPN but not by intact OPN or thrombin-cleaved OPN.

The requirement of polymerization for OPN to attract neutrophils suggests the

recruitment was mediated by α9β1, as it is in vitro. However, in vivo there would be

other factors for polymeric OPN than the interaction with α9β1. For example,

polymeric OPN may stimulate cells to produce chemoattractants more avidly

than monomeric OPN, including peritoneal mesothelial cells and monocyte/macrophages,. To assess the contribution of polymeric OPN, we generated mice lacking

integrin subunit α9 expression on all leukocytes. We then compared neutrophil

recruitment into the peritoneal space of leukocyte-α9-null mice induced by intact

OPN, or polymerization-incompetent mutant OPN with that of wild-type mice

(Fig. 6.6). Neutrophil recruitment induced by injection of intact OPN was



Fig. 6.6 Neutrophil accumulation in wild-type and neutrophil α9-null mice by injection of WT,

polymerization-incompetent mutant, or thrombin cleavage-incompetent osteopontin. One of three

species of osteopontin shown below was injected into the peritoneal space of mice that lack the

integrin α9 subunit on their neutrophils. Neutrophils in the peritoneal space were counted 4 h after

injection. The numbers of mice are indicated below each bar. Values represent the mean Ỉ S.D.

(error bars). OPN*, full-length monomeric osteopontin (This research was originally published in

The Journal of Biological Chemistry. Nishimichi et al. (2011) © the American Society for

Biochemistry and Molecular Biology)



6 A New Integrin-Binding Site on a Transglutaminase-Catalyzed Polymer



141



significantly reduced in mice that lack the integrin α9 subunit on their leukocytes. In

contrast, recruitment in response to the polymerization-incompetent mutant was

reduced in wild-type mice, but was unaffected by loss of the integrin α9 subunit on

leukocytes. These results indicate that intact OPN-induced neutrophil accumulation

is mediated, at least in part, by α9β1 and that polymerization is critical for the

recruitment.

Before the description of the requirement of polymerization, OPN was already

regarded as a chemoattractant for neutrophils. Two independent groups had

reported that neutrophil chemotaxis was a consequence of peritoneal OPN injection

(Carrigan et al. 2007; Koh et al. 2007). It should be noted, however, that there are

few data supporting the chemotactic activity of OPN in vitro. The only report that

claimed to show OPN’s in vivo neutrophil chemotactic activity used OPN purified

from cells of a macrophage cell line, not recombinant OPN (Carrigan et al. 2007),

where polymerized OPN could be involved. All of the previous publications are

consistent with the finding that OPN stimulates chemotaxis after polymerization.



6.3.4



The Specific Integrin α9β1 Binding Site on Polymeric

OPN



There must be a specific conformation that fits with the α9β1 ligand-binding pocket

on the surface of the polymer. Two possibilities can explain how the conformation

is provided upon polymerization; either an otherwise cryptic structure is exposed,

or a new conformation arises as consequence of the atomic interactions, particularly

at molecular interfaces. The relevance of SVVYGLR has been excluded, as

described above. However, another cryptic binding sequence could be exposed by

polymerization. The other possibility, i.e., generation of a new site, might be

associated with the regulated enzymatic reaction as discussed in Sect. 6.2. In fact,

there are few mAbs, including 81D4, that react with the isopeptide bond Nε-(γ-Lglutamyl)-L-lysine (el Alaoui et al. 1991; Thomas et al. 2004), which indicates that

the isopeptide bond has a specific conformation that serves an epitope of these

mAbs. Further, a polymer-specific antiserum for amyloid β protein does not recognize the monomeric fibril (Kayed et al. 2003). The authors further developed

monoclonal antibodies that recognize generic epitopes on amyloid β oligomers

that do not depend on a specific linear amino acid sequence. These mAbs display

distinct preferences for different subsets of prefibrillar oligomers (Kayed

et al. 2010). The presence of such epitopes on the amyloid β polymer supports

the hypothesis that polymeric OPN has a specific binding site for integrins. Note

that amyloid β serves as a substrate for transglutaminase (Hartley et al. 2008;

Schmid et al. 2011).

Although there are many Gln and Lys residues in the OPN sequence, only four

candidate Gln residues and nine candidate Lys residues have been reported as

potential TG2 substrates. These residues could theoretically form a maximum of



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36 different isopeptide bonds between OPN molecules. However, not all of the

combinations may occur because the preferences of the enzyme for substrate

sequences are not the same among the Gln and Lys residues. If there are one or a

few dominant combinations selected by the enzyme, the identical conformation

would be scattered on the surface of the polymer.

In biological terms, this polymerization also provides us with an intriguing

question. Native, full-length non-polymerized OPN does not bind to integrin

α9β1, but it does bind after a post-translational modification: thrombin-catalyzed

cleavage. Further, another post-translational modification, polymerization, also

confers the capacity to bind to α9β1, even creating a binding site different from

SVVYGLR. From these consequences, the interaction of OPN with α9β1 is thought

to be so critical that two ways of regulation are provided. As shown above, OPN

acquires chemotactic activity when interacting with α9β1 after either cleavage or

polymerization. How are these two ways differentially utilized? One answer provided by our experimental work is that polymerization is overwhelmingly dominant

over cleavage when recombinant, full-length monomeric OPN is injected into the

peritoneal space of mice (Nishimichi et al. 2011). In this case, polymerization

occurs systemically, rather than locally. Whether the activated thrombin that

cleaves OPN is concentrated locally, such as on blood vessels as in thrombosis,

may need to be determined.



6.4



Regulation of Polymerization by TG2



Recently, it was found from crystal structure studies that there are at least two

different conformations of TG2: active open and inactive folded (Pinkas

et al. 2007). As with many other proteins, Ca2ỵ is one of the most critical regulators

of TG2 activation; it is also involved in the regulation of the conformational change

of TG2 (Belkin 2011; Kiraly et al. 2011). TG2 works in both the extra- and

intracellular environments. To exert catalytic function under both conditions,

TG2 changes its own Ca2ỵ sensitivity. The Ca2ỵ concentration displays a huge

gradient of >10,000 between the extra- and intracellular spaces; the cytoplasmic

environment is highly reductive, with low Ca2ỵ and high GTP concentrations. GTP

is another major regulator of TG2 that binds to TG2 between its core and β-barrel

domain. The redox system also changes the properties of TG2, including its Ca2ỵ

sensitivity. There are three Cys residues in the catalytic center of the core domain

that reversibly form disulfide bonds in response to redox status (Stamnaes

et al. 2010). In addition, other mechanisms are further involved in the regulation

of extra- and intracellular activation of TG2, including S-nitrosylation of Cys

residues, mechanical forces on the molecule, glycosaminoglycans, and the buffering effects of other Ca2ỵ-binding proteins. Regulation of the intracellular Ca2ỵ

signal is also tightly controlled through chelation, sequestration, or removal

(Clapham 2007).



6 A New Integrin-Binding Site on a Transglutaminase-Catalyzed Polymer



143



Although regulatory compartments have been identified, there is little information about the systemic control of TG2 activation and polymerization. Many

emerging questions still remain, particularly about regulation of the polymerization

of a variety of substrates. For example, there are multiple transglutaminase-polymerized proteins that regulate apoptosis. When the multiple substrates for polymers

that cause opposing effects on apoptosis are in place, how does transglutaminase

manage these polymerizations? As mentioned in the first section, a transcription

factor, Sp1, no longer initiates transcription of c-Met upon polymerization and

initiate apoptosis in hepatocytes. Contrarily, the cell cycle regulator Rb enhances its

protective effect (Boehm et al. 2002) against calphostin C-induced apoptosis upon

polymerization (Oliverio et al. 1997). Is it possible for transglutaminase to specifically polymerize Sp1 while leaving Rb monomeric, thus inducing apoptosis in

hepatocytes? Alternatively, DLK up-regulates its kinase activity upon polymerization and activates JNK in cells undergoing calphostin C-induced apoptosis

(Robitaille et al. 2004), thus promoting apoptosis. DLK polymerization leads to

an effect obviously opposed to the effect of Rb polymerization. In cells undergoing

calphostin C-induced apoptosis, should transglutaminase be active or remain silent

to regulate the apoptosis? These three kinds of apoptosis-related polymers illustrate

the complexity of the regulation of transglutaminase-catalyzed polymerization.

One of the simplest explanations for the contradiction represented by the proand anti-apoptotic effects of polymeric Sp1 and Rb, respectively, may be cell type

specificity. Because the effect of Sp1 polymerization is exhibited through loss of

c-Met expression, such apoptosis would be exerted only in cells where the hepatocyte growth factor (HGF) signal is essential for survival. Another simple rationalization is the differential activation of TG2 so that it polymerizes one, but does not

polymerize the other, i.e., an activation state of TG2 that specifically polymerizes

one among multiple substrates. Because Sp1 is polymerized by ethanol-activated

TG2, whereas Rb is polymerized by calphostin C-activated TG2, it is conceivable

that these TG2-activated states are not the same. In this case, TG2 holds multiple

activation states that can discriminate between the polymerization of Sp1 and Rb

proteins. However, for dual polymerizations of DLK and Rb, the other specific

example, since TG2 is activated through the same activator calphostin C it is less

likely that TG2 could be differentially activated and exhibit specific polymerization

of DLK or Rb. One possibility is that polymerization activity is dependent on each

TG2 binding sequence of the substrate. This conflicting polymerization may be

attributed to spatial or temporal activation of transglutaminase. If cells are in

situations that promote apoptosis, activation of transglutaminase may occur locally

within the region of the cell where pro-apoptotic DLK functions, but not in the

region where anti-apoptotic Rb is concentrated. This could be the case because

DLK phosphorylates MAPK kinase in the cytoplasm, whereas Rb supports cell

survival by regulating cell cycles in the nucleus. Further speculation can be

developed; transglutaminase may polymerize DLK, first, to activate the JNK

pathway and initiate c-Jun to transcript pro-apoptotic molecules such as Bim, and

then transglutaminase translocates into the nucleus to polymerize Rb to protect

from apoptosis until Bim expression has been established. These differential



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Y. Yokosaki



activation states that allow specific polymerization among many substrate proteins,

as well as spatial and temporal regulation of TG2 activation in total, conduct the

polymerization orchestrating molecular instruments.

On comparing the regulation of TG2 polymerization with that of protein expression, although transcriptional regulation appears to be much more sophisticated, the

principle of regulation may be shared. It is recognized that not all of the proteins

expressed are required. The expression of superfluous proteins, in addition to the

protein of interest, is allowed as far as they are non-deleterious and have no

opposing effects on the protein of interest (Erickson 1993). This is probably

because, in view of cellular economy, sharing the switch to turn on the expression

of several proteins is preferable to preparing one switch for each protein. As far as it

is not toxic and not opposing to the required function of the polymer being

generated, superfluous polymerization could be allowed. For example, three substrates for TG2-polymerization were identified from bone extract, OPN, bone

sialoprotein, and α2 HS-glycoprotein (Kaartinen et al. 2002). This list might

include one or two biologically nonfunctional or needless polymers, but one or

more of the polymers is required. Every transglutaminase-induced polymers found

in vivo may not necessarily be functional or required at the time of polymerization;

some of them may exist without a justified raison d’etre.



6.5



Summary and Future Direction



We have shown in vivo and in vitro experimental evidence for polymerization of

OPN. Besides, human secreted OPN was reported to be highly polymerized in the

airways but less polymerized in patients with asthma (Arjomandi et al. 2011). Since

the magnitude of polymerization is associated with the disease state, the polymerization has to be determined in various diseases in the view of magnitude and

disease state. The elucidation of the mechanisms underlying the polymerization and

roles in pathological states help understand and manage the diseases.

Increasing numbers of substrates for transglutaminases have been identified.

Although many of them are polymerized by the same transglutaminase enzyme,

functional alterations resulting from the reaction are diverse, including

up-regulation, down-regulation, loss of function, prolonged half-life, and even a

gain of new function. Although the in vivo polymerization of some of the polymers

has been confirmed, there are many polymers that are reportedly functional in vitro.

This may arise because in vitro conditions where high concentrations of transglutaminase are present could produce many polymers that are not polymerized under

physiological conditions. Nevertheless, collecting evidence for polymerization

in vitro would be of importance because in pathological conditions transglutaminase could be highly activated locally. The identification of the conditions

that are specifically seen in any disease would be intriguing.

OPN is one of the substrates for which there is evidence of in vivo polymerization (Kaartinen et al. 2002). Further, OPN gains a new function, becoming a



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