Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Human inter-α-inhibitor is a substrate for factor XIIIa and tissue transglutaminase☆
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)
- et al.
Analysis of inter-alpha-trypsin inhibitor and a novel trypsin inhibitor, pre-alpha-trypsin inhibitor, from human plasma, polypeptide chain stoichiometry and assembly by glycan
J Biol Chem
(1989) - et al.
Presence of the protein-glycosaminoglycan-protein covalent cross-link in the inter-alpha-inhibitor-related proteinase inhibitor heavy chain 2/bikunin
J Biol Chem
(1993) - et al.
Chondroitin 4-sulfate covalently cross-links the chains of the human blood protein pre-alpha-inhibitor
J Biol Chem
(1991) - et al.
Inter-alpha-trypsin inhibitor, a covalent protein–glycosaminoglycan–protein complex
J Biol Chem
(2004) - et al.
Inter-alpha-trypsin inhibitor. Inhibition spectrum of native and derived forms
J Biol Chem
(1989) - et al.
Characterization of the interaction between tumor necrosis factor-stimulated gene-6 and heparin: implications for the inhibition of plasmin in extracellular matrix microenvironments
J Biol Chem
(2005) - et al.
The TSG-6 and I alpha I interaction promotes a transesterification cleaving the protein-glycosaminoglycan-protein (PGP) cross-link
J Biol Chem
(2005) - et al.
Characterization of complexes formed between TSG-6 and inter-alpha-inhibitor that act as intermediates in the covalent transfer of heavy chains onto hyaluronan
J Biol Chem
(2005) - et al.
SHAP potentiates the CD44-mediated leukocyte adhesion to the hyaluronan substratum
J Biol Chem
(2006) Factor XIII and the clotting of fibrinogen: from basic research to medicine
J Thromb Haemost
(2005)
Factor XIII-mediated cross-linking of NH2-terminal peptide of alpha 2-plasmin inhibitor to fibrin
FEBS Lett
Factor V is a substrate for the transamidase factor XIIIa
J Biol Chem
Human procarboxypeptidase U, or thrombin-activable fibrinolysis inhibitor, is a substrate for transglutaminases, Evidence for transglutaminase-catalyzed cross-linking to fibrin
J Biol Chem
Covalent crosslinking of von Willebrand factor to fibrin
Blood
Analysis of protein and peptide mixtures: evaluation of three sodium dodecyl sulphate-polyacryl-amide gel electrophoresis buffer systems
J Chromatogr
Reversible cross-linking of alpha 2-plasmin inhibitor to fibrinogen by fibrin-stabilizing factor
Biochim Biophys Acta
Defect in SHAP-hyaluronan complex causes severe female infertility. A study by inactivation of the bikunin gene in mice
J Biol Chem
TSG-6 transfers proteins between glycosaminoglycans via a Ser28-mediated covalent catalytic mechanism
J Biol Chem
The transfer of heavy chains from bikunin proteins to hyaluronan requires both TSG-6 and HC2
J Biol Chem
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
Characterization of hyaluronan cable structure and function in renal proximal tubular epithelial cells
Kidney Int
Transglutaminases catalyze cross-linking of plasminogen to fibronectin and human endothelial cells
J Biol Chem
Chondroitin sulphate covalently cross-links the three polypeptide chains of inter-alpha-trypsin inhibitor
Eur J Biochem
Cited by (10)
Matrix-degrading protease ADAMTS-5 cleaves inter-αinhibitor and releases active heavy chain 2 in synovial fluids from arthritic patients
2019, Journal of Biological ChemistryCitation Excerpt :In addition to the main cleavage sites, there are two other regions in HC2 (308–331 and 436–438) that are susceptible to cleavage to a minor degree. The 420–438 region has previously been shown to be exposed and available for transglutaminase cross-linking (31). It has previously been shown that MMP-3, MMP-13, ADAMTS-5, and IαI are present in the cartilage and synovial fluid of arthropathic patients (27, 29, 32, 33).
Coagulation factor XIIIa substrates in human plasma: Identification and incorporation into the clot
2014, Journal of Biological ChemistryCitation Excerpt :Plasmin is the main fibrinolytic protease; by cross-linking a plasmin inhibitor such as α2-antiplasmin to the fibrin fibers, FXIIIa contributes to a significant increase in clot stability. FXIIIa furthermore cross-links a number of different proteins to the clot, including factor V (9), thrombin-activable fibrinolysis inhibitor (10), von Willebrand factor (11), complement C3 (12), inter-α-inhibitor (13), and plasminogen activator inhibitor type 2 (14). These proteins could regulate clot characteristics other than stability.
Administration of TSG-6 improves memory after traumatic brain injury in mice
2013, Neurobiology of DiseaseCitation Excerpt :TSG-6 can stabilize the extracellular matrix and thereby limits the invasion of inflammatory cells by binding to hyaluronan, heparin, heparan sulfate, thrombospondins-1 and − 2, and fibronectin (Baranova et al., 2011; Blundell et al., 2005; Kuznetsova et al., 2005, 2008; Mahoney et al., 2005). In addition, it can inhibit the cascade of proteases released by inflammation by its complex catalytic interaction with inter-α-inhibitor (Rugg et al., 2005; Scavenius et al., 2011; Zhang et al., 2012), or by forming ternary complexes with mast cell trypases and heparin (Nagyeri et al., 2011). In apparently independent interactions, TSG-6 also reduces the migration of neutrophils through endothelial cells (Cao et al., 2004), and inhibits FGF-2 induced angiogenesis through an interaction with pentraxin (Leali et al., 2012).
The Inter-α-Trypsin Inhibitor Family: Versatile Molecules in Biology and Pathology
2020, Journal of Histochemistry and CytochemistryUse of a natural modulator of inflammation (TSG-6) for the therapy of traumatic brain injury
2016, Cellular Therapy for Neurological Injury
- ☆
The work was supported by grants from the Danish Natural Science Research Council (J.J.E. and P.H.).