Secretory Leukocyte Protease Inhibitor (SLPI) Is, like Its Homologue Trappin-2 (Pre-Elafin), a Transglutaminase Substrate

Abstract

Human lungs contain secretory leukocyte protease inhibitor (SLPI), elafin and its biologically active precursor trappin-2 (pre-elafin). These important low-molecular weight inhibitors are involved in controlling the potentially deleterious proteolytic activities of neutrophil serine proteases including elastase, proteinase 3 and cathepsin G. We have shown previously that trappin-2, and to a lesser extent, elafin can be linked covalently to various extracellular matrix proteins by tissue transglutaminases and remain potent protease inhibitors. SLPI is composed of two distinct domains, each of which is about 40% identical to elafin, but it lacks consensus transglutaminase sequence(s), unlike trappin-2 and elafin. We investigated the actions of type 2 tissue transglutaminase and plasma transglutaminase activated factor XIII on SLPI. It was readily covalently bound to fibronectin or elastin by both transglutaminases but did not compete with trappin-2 cross-linking. Cross-linked SLPI still inhibited its target proteases, elastase and cathepsin G. We have also identified the transglutamination sites within SLPI, elafin and trappin-2 by mass spectrometry analysis of tryptic digests of inhibitors cross-linked to mono-dansyl cadaverin or to a fibronectin-derived glutamine-rich peptide. Most of the reactive lysine and glutamine residues in SLPI are located in its first N-terminal elafin-like domain, while in trappin-2, they are located in both the N-terminal cementoin domain and the elafin moiety. We have also demonstrated that the transglutamination substrate status of the cementoin domain of trappin-2 can be transferred from one protein to another, suggesting that it may provide transglutaminase-dependent attachment properties for engineered proteins. We have thus added to the corpus of knowledge on the biology of these potential therapeutic inhibitors of airway proteases.

Cross-Linking of SPINK6 by Transglutaminases Protects from Epidermal Proteases

“…In SPINK6, three out of four glutamines (Q4, Q11, and Q30) follow a QxxF motif (where x is any amino acid and F a hydrophobic acid). This motif was also observed for the cross-linked protease inhibitors trappin-2 and secretory leukocyte protease inhibitor (Guyot et al, 2005;Baranger et al, 2011). Our data support the hypothesis that a hydrophobic residue þ 3 to glutamine is a common motif for TGMs.…”

“…To our knowledge, this is a previously unreported description that a protein is protected from proteolytic cleavage by TGMmediated cross-linking. Cross-linked secretory leukocyte protease inhibitor and trappin-2 inhibit proteases such as elastase and proteinase 3 as well (Guyot et al, 2005;Baranger et al, 2011). Moreover, cystatin alpha is covalently bound to the stratum corneum by transglutaminase (Takahashi et al, 1994), suggesting that cross-linkage of protease inhibitors might be a general mechanism in human skin to control proteolytic enzymes.…”

A Functional Variant of Elafin With Improved Anti-inflammatory Activity for Pulmonary Inflammation

“…As mentioned previously, elafin can be cross-linked to extracellular matrix proteins such as fibronectin via the action of TG. [19][20][21][22] GG-elafin exhibited comparable binding to fibronectin when compared to WT-elafin in the presence of the TG (Figure 3b). Furthermore, the QQ-elafin variant demonstrated a significant increase in binding to fibronectin when compared to WT-elafin (P < 0.05).…”

“…Transglutamination reactions were performed as previously described 22 with minor modifications. Briefly, Greiner high binding 96-well plates were coated with 1 mg/ml of human fibronectin (Sigma-Aldrich) in 0.1 mol/l sodium carbonate buffer (pH 9.5) overnight at 4 °C.…”

“…4,5,17,18 Trappin-2 and elafin have a number of transglutaminase (TG) reactive residues and therefore can be linked covalently to various extracellular matrix proteins such as fibronectin by tissue transglutaminases and retain potent antiprotease activity. [19][20][21][22] Due to its cationic nature, it is postulated that trappin-2 displays antibacterial properties via disruption of bacterial cell membranes. 7,10 In addition, it has been demonstrated that trappin-2 and mature elafin can bind and neutralize lipopolysaccharide (LPS) 23,24 thereby suppressing macrophage TNF-α production.…”