Elastase Inhibitor Elafin Is a New Type of Proteinase Inhibitor Which Has a Transglutaminase-Mediated Anchoring Sequence Termed “Cementoin”1

Abstract

Elafin was shown to be a new type of proteinase inhibitor which has an anchoring sequence. Human elafin, a potent inhibitor specific for elastase and proteinase 3, has a unique repeating sequence in its prosegment that is rich in Gln and Lys residues. The prosegment, termed "cementoin," exhibits high homology with the repetitive element of seminal vesicle clotting protein, which is known as a good substrate for prostate transglutaminase. The cross-linking of cementoin by tissue transglutaminase showed that the cementoin moiety is indeed a preferable substrate for transglutaminase. In addition, transglutaminase-mediated cross-linking between cementoin and laminin was observed in vitro, suggesting that cementoin has the ability to covalently attach to other extracellular matrix proteins. To determine whether or not this type of covalent gluing of elafin through the cementoin moiety occurs in vivo, we determined the molecular size of cementoin-elafin in the trachea mucous epithelium by Western blotting; the rationale of this approach is that (i) the trachea is the richest source of cementoin-elafin, as shown below, and (ii) if cementoin-elafin is covalently associated with other proteins, it should migrate as a higher M(r) species on SDS-polyacrylamide gel electrophoresis; cementoin-elafin immunoreactivity was indeed detected at a position corresponding to 50 kDa, a value much higher than that of its monomeric form. RNase protection analysis and immunohistochemical staining revealed that cementoin-elafin is densely distributed in the skin and trachea, and moderately in the stomach, duodenum and small intestine. These sites of localization are consistent with the locations where elastic fibers are abundant.(ABSTRACT TRUNCATED AT 250 WORDS)

Elafin, an Elastase-specific Inhibitor, Is Cleaved by Its Cognate Enzyme Neutrophil Elastase in Sputum from Individuals with Cystic Fibrosis

“…In the cornified cell envelope of the epidermis, both molecules are cross-linked to several proteins (including involucrin, keratin-1, loricrin, cystatin ␣, filaggrin) and are thought to function as cross-bridging molecules (47). In tracheal epithelium, trappin-2/elafin is also found in a complexed form (4). Previous in vitro studies showed that trappin-2 and/or elafin can covalently bind to a number of ECM proteins including elastin, fibronectin, laminin, fibrinogen, collagen, and ␤-crystallin by transglutamination (4,5,48).…”

“…In tracheal epithelium, trappin-2/elafin is also found in a complexed form (4). Previous in vitro studies showed that trappin-2 and/or elafin can covalently bind to a number of ECM proteins including elastin, fibronectin, laminin, fibrinogen, collagen, and ␤-crystallin by transglutamination (4,5,48). The transglutaminase-mediated cross-linking of trappin-2 and elafin to fibronectin and elastin was demonstrated to preserve their antiprotease activity and to protect the associated ECM molecule against proteolysis mediated by NE (5,48).…”

“…In addition to its ability to inhibit porcine pancreatic elastase, elafin is a potent inhibitor of two neutrophil serine proteases, neutrophil elastase (NE) and proteinase 3 (1,2), and is therefore thought to protect tissue from degradation by these enzymes. Elafin is released by proteolytic cleavage from a larger molecule called trappin-2 or pre-elafin, which possesses at the N terminus of the whey acidic protein domain a cementoin domain containing several motifs having the consensus sequence GQDPVK that can act as transglutaminase substrate, allowing the cross-linking of the inhibitor to extracellular matrix proteins (3)(4)(5). It has been shown that tryptase, a mast cell-derived protease, may be involved in the proteolytic processing of trappin-2 into elafin (6).…”

Identification and Sequence Analysis of Two New Members of the SKALP/elafin and SPAI-2 Gene Family

“…Until now porcine SPAI-2 (2) and the proteinase inhibitor SKALP/elafin/elastase-specific inhibitor were described in the literature (1, 7, 17, 37). For the NH 2 -terminal TGase substrate domain a separate name, cementoin, was coined by Nara et al (20). Here we propose to give these proteins the acronym Trappin (Trappin ϭ TRansglutaminase substrate and WAP motif-containing ProteIN) as a new nomenclature for this protein family.…”

“…The NH 2 -terminal domain, containing the TGase substrate motifs, is homologous to the guinea pig seminal vesicle protein-1 (19) and the human semenogelins (15). We showed that the NH 2 -terminal TGase substrate domain, for which the name "cementoin" was coined by others (20), is actually used in vivo and in vitro for cross-linking to stratum corneum proteins. Recently, it was shown by direct sequencing of cross-linked peptides from human foreskin epidermis that SKALP/elafin is cross-linked in vivo to loricrin and cytokeratin-1 (21), which are structural proteins of the terminally differentiating keratinocyte.…”