Human inter-α-inhibitor is a substrate for factor XIIIa and tissue transglutaminase

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Abstract

In this study, we show that inter-α-inhibitor is a substrate for both factor XIIIa and tissue transglutaminase. These enzymes catalyze the incorporation of dansylcadaverine and biotin–pentylamine, revealing that inter-α-inhibitor contains reactive Gln residues within all three subunits. These findings suggest that transglutaminases catalyze the covalent conjugation of inter-α-inhibitor to other proteins. This was demonstrated by the cross-linking between inter-α-inhibitor and fibrinogen by either factor XIIIa or tissue transglutaminase. Finally, using quantitative mass spectrometry, we show that inter-α-inhibitor is cross-linked to the fibrin clot in a 1:20 ratio relative to the known factor XIIIa substrate α2-antiplasmin. This interaction may protect fibrin or other Lys-donating proteins from adventitious proteolysis by increasing the local concentration of bikunin. In addition, the reaction may influence the TSG-6/heavy Chain 2-mediated transfer of heavy chains observed during inflammation.

Highlights

► Inter-α-inhibitor is a substrate for factor XIIIa and tissue transglutaminase. ► We demonstrate cross-linking of inter-α-inhibitor and fibrinogen by factor XIIIa. ► Inter-α-inhibitor is cross-linked to the plasma clot.

Introduction

Inter-α-inhibitor (IαI) is a proteoglycan composed of bikunin and two homologous heavy chains referred to as heavy chain 1 (HC1) and heavy chain 2 (HC2) [1]. The three components are covalently cross-linked by an under-sulfated chondroitin-4-sulfate, which originates from Ser10 of bikunin (Fig. 1) [2], [3], [4], [5]. The C-terminal carboxyl groups of the HCs form ester bonds with the C6 atoms of the internal N-acetylgalactosamines and these bonds are referred to as protein–glycosaminoglycan–protein (PGP) cross-links [3].

IαI has been implicated in several biological processes, such as ovulation, cell migration and inflammation [6]. The suggested roles of IαI are based on its interactions with hyaluronan-rich extracellular matrix (ECM) or the protease inhibitory activity associated with bikunin. The bikunin subunit is composed of two Kunitz-A domains (http://merops.sanger.ac.uk) [7] and inhibits a broad range of serine proteases, including plasmin [8]. The concentration of circulating bikunin and its Ki indicates that IαI is not an effective protease inhibitor in plasma [8]. However, several studies have indicated that the inhibitory capacity of bikunin is increased by interaction with tumor necrosis factor stimulated gene-6 protein (TSG-6) [9], [10], [11].

TSG-6 also mediates the covalent interaction between the HCs and hyaluronan. Two sequential transesterifications mediated by TSG-6/HC2 transfer the HCs from the bikunin-associated chondroitin-4-sulfate to hyaluronan [12], [13], [14]. The role of the HC–HA complexes has yet to be determined, but evidence suggests a role in arthritis, as the concentration of HC–HA covalent complexes is increased in the synovial fluid [15]. In addition, these complexes appear to play a role in the attachment of leukocytes during inflammation [16].

The transglutaminases, including factor XIIIa (FXIIIa) and tissue transglutaminase (TTG), belong to a family of calcium-dependent enzymes (EC 2.3.2.13) and catalyze the formation of Nε(γ-glutamyl)lysine cross-links [17]. The generation of these isopeptide bonds increases mechanical stability and resistance to adventitious proteolysis. FXIII circulates in the plasma as a non-covalently associated tetramer composed of two a-chains and two b-chains [18]. In the final phase of blood coagulation, thrombin-activated FXIIIa cross-links the fibrin clot into an acid and urea stable polymer [19]. Following fibrin cross-linking, FXIIIa may further covalently incorporate a number of different proteins into the fibrin clot, including α2-antiplasmin (α2AP) [20], factor V [21], thrombin-activatable fibrinolysis inhibitor (TAFI) [22], von Willebrand factor [23] and plasminogen activator inhibitor type 2 (PAI-2) [24]. FXIIIa deficiency leads to severe bleeding complications, reduced wound healing and recurring miscarriages [25].

In this study, we show that IαI is a substrate for both FXIIIa and TTG. Using a combination of transglutaminase-mediated biotin–pentylamine incorporation and mass spectrometry, we have identified sites of modification in all three IαI subunits. Furthermore, we present evidence for the cross-linking of IαI and fibrinogen and show that IαI is cross-linked to the plasma clot in a 1:20 ratio relative to the known FXIIIa substrate α2-antiplasmin. These data suggest that transglutaminases cross-link IαI and other substrate proteins in general, a reaction that is likely to introduce additional cross-links and thereby increase the mechanical stability and resistance to adventitious proteolysis. Moreover, our data indicate that IαI plays a direct role in the formation and stability of the plasma clot.

Section snippets

Materials

Dansylcadaverine was purchased from Biochemika, biotin–pentylamine (EZ-link) was from Pierce, immobilized monomeric avidin was from Thermo, biotin was from Fluka and dansyl-PGGQQIV-OH was from New England Peptide.

Proteins human IαI was purified from human plasma obtained from Aarhus University Hospital, Skejby, Denmark as previously described [1]. Chondroitinase ABC (EC 4.2.2.4) was from Seikagaku. Guinea pig tissue transglutaminase (EC 2.3.2.13), human fibrinogen, thrombin and trypsin were

IαI is a substrate for transglutaminases

The ability of IαI to act as a substrate for FXIIIa or TTG was tested by co-incubation of dansylcadaverine, IαI and increasing amounts of FXIIIa or TTG. The samples were incubated for 2 h at 37 °C, analyzed by SDS-PAGE and visualized under UV light (Fig. 2A and B). It is evident that both FXIIIa and TTG labeled IαI with dansylcadaverine in a concentration-dependent manner (Fig. 2A and B, lanes 1–5), indicating the existence of reactive Gln residues in IαI. To test whether IαI was able to act as

Discussion and conclusions

In this study, we show that IαI is a substrate for both TTG and FXIIIa. Incorporation of dansylcadaverine or biotin–pentylamine demonstrated the presence of reactive Gln residues (amine acceptors). We have determined the location of these residues and established that all three subunits of IαI are substrates for transglutaminases. The majority of the sites have been highly conserved throughout evolution. In addition, we presented evidence for the in vitro cross-linking of IαI and fibrinogen.

References (46)

  • A. Ichinose et al.

    Factor XIII-mediated cross-linking of NH2-terminal peptide of alpha 2-plasmin inhibitor to fibrin

    FEBS Lett

    (1983)
  • R.T. Francis et al.

    Factor V is a substrate for the transamidase factor XIIIa

    J Biol Chem

    (1986)
  • Z. Valnickova et al.

    Human procarboxypeptidase U, or thrombin-activable fibrinolysis inhibitor, is a substrate for transglutaminases, Evidence for transglutaminase-catalyzed cross-linking to fibrin

    J Biol Chem

    (1998)
  • M. Hada et al.

    Covalent crosslinking of von Willebrand factor to fibrin

    Blood

    (1986)
  • A.F. Bury

    Analysis of protein and peptide mixtures: evaluation of three sodium dodecyl sulphate-polyacryl-amide gel electrophoresis buffer systems

    J Chromatogr

    (1981)
  • A. Ichinose et al.

    Reversible cross-linking of alpha 2-plasmin inhibitor to fibrinogen by fibrin-stabilizing factor

    Biochim Biophys Acta

    (1982)
  • L. Zhuo et al.

    Defect in SHAP-hyaluronan complex causes severe female infertility. A study by inactivation of the bikunin gene in mice

    J Biol Chem

    (2001)
  • K.W. Sanggaard et al.

    TSG-6 transfers proteins between glycosaminoglycans via a Ser28-mediated covalent catalytic mechanism

    J Biol Chem

    (2008)
  • K.W. Sanggaard et al.

    The transfer of heavy chains from bikunin proteins to hyaluronan requires both TSG-6 and HC2

    J Biol Chem

    (2008)
  • C.A. de la Motte et al.

    Mononuclear leukocytes bind to specific hyaluronan structures on colon mucosal smooth muscle cells treated with polyinosinic acid:polycytidylic acid: inter-alpha-trypsin inhibitor is crucial to structure and function

    Am J Pathol

    (2003)
  • W. Selbi et al.

    Characterization of hyaluronan cable structure and function in renal proximal tubular epithelial cells

    Kidney Int

    (2006)
  • E. Bendixen et al.

    Transglutaminases catalyze cross-linking of plasminogen to fibronectin and human endothelial cells

    J Biol Chem

    (1993)
  • W. Morelle et al.

    Chondroitin sulphate covalently cross-links the three polypeptide chains of inter-alpha-trypsin inhibitor

    Eur J Biochem

    (1994)
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    The work was supported by grants from the Danish Natural Science Research Council (J.J.E. and P.H.).

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