Implantation Serine Proteinase 1 Exhibits Mixed Substrate Specificity that Silences Signaling via Proteinase-Activated Receptors

Abstract

Implantation S1 family serine proteinases (ISPs) are tryptases involved in embryo hatching and uterine implantation in the mouse. The two different ISP proteins (ISP1 and ISP2) have been detected in both pre- and post-implantation embryo tissue. To date, native ISP obtained from uterus and blastocyst tissues has been isolated only as an active hetero-dimer that exhibits trypsin-like substrate specificity. We hypothesised that in isolation, ISP1 might have a unique substrate specificity that could relate to its role when expressed alone in individual tissues. Thus, we isolated recombinant ISP1 expressed in Pichia pastoris and evaluated its substrate specificity. Using several chromogenic substrates and serine proteinase inhibitors, we demonstrate that ISP1 exhibits trypsin-like substrate specificity, having a preference for lysine over arginine at the P1 position. Phage display peptide mimetics revealed an expanded but mixed substrate specificity of ISP1, including chymotryptic and elastase activity. Based upon targets observed using phage display, we hypothesised that ISP1 might signal to cells by cleaving and activating proteinase-activated receptors (PARs) and therefore assessed PARs 1, 2 and 4 as potential ISP1 targets. We observed that ISP1 silenced enzyme-triggered PAR signaling by receptor-disarming. This PAR-disarming action of ISP1 may be important for embryo development and implantation.

Implantation serine proteinase 2 is a monomeric enzyme with mixed serine proteolytic activity and can silence signalling via proteinase activated receptors

“…Our results demonstrate that recombinant ISP2 exists in a monomeric form similar to recombinant ISP1 (Sharma et al 2011). The studies done with a random hexameric library of phage-displayed peptides revealed mixed substrate specificity for rISP2, showing chymotrypsin-like as well as elastase-like specificity of the recombinant enzyme.…”

“…Thus, for purifying active recombinant ISP2, we turned to the use of conventional ion exchange chromatography followed by gel filtration, as was previously done for the purification of the native ISP complex from biological fluids (Sharma et al 2006) and recombinant ISP1 (Sharma et al 2011).…”

“…The purification of recombinant ISP2 was performed with ion exchange (DEAE) and gel filtration (Superdex-75) column chromatography using the Duo Flow FPLC system (Bio-Rad), just as was done in case of recombinant ISP1 (Sharma et al 2011). Although the full length cDNA sequence of ISP2 suggests that it is secreted as a pro-enzyme, we only detected the mature (truncated) active form within a heterodimeric complex together with ISP1 within uterine fluid (Sharma et al 2006).…”

“…Thus, we have been interested to express each of the ISPs in isolation to determine their individual substrate preferences. To this end, we have recently reported that recombinant ISP1, which is secreted from Pichia pastoris as a monomeric serine proteinase, displays a mixed tryptic-chymotryptic substrate cleavage property (Sharma et al 2011). We have now turned to the evaluation of the substrate selectivity of recombinant ISP2.…”

“…These truncated receptors nonetheless remain responsive to PAR-APs but are unable to signal in a physiological setting (Ramachandran and Hollenberg 2008). Because of their sensitivity to serine proteinases, we wished to determine if ISP2, like ISP1 (Sharma et al 2011), might affect PAR function.…”