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

Abstract

Implantation serine proteinase 2 (ISP2), a S1 family serine proteinase, is known for its role in the critical processes of embryo hatching and implantation in the mouse uterus. Native implantation serine proteinases (ISPs) are co-expressed and co-exist as heterodimers in uterine and blastocyst tissues. The ISP1-ISP2 enzyme complex shows trypsin-like substrate specificity. In contrast, we found that ISP2, isolated as a 34 kDa monomer from a Pichia pastoris expression system, exhibited a mixed serine proteolytic substrate specificity, as determined by a phage display peptide cleavage approach and verified by the in vitro cleavage of synthetic peptides. Based upon the peptide sequence substrate selectivity, a database search identified many potential ISP2 targets of physiological relevance, including the proteinase activated receptor 2 (PAR2). The in vitro cleavage studies with PAR2-derived peptides confirmed the mixed substrate specificity of ISP2. Treatment of cell lines expressing proteinase-activated receptors (PARs) 1, 2, and 4 with ISP2 prevented receptor activation by either thrombin (PARs 1 and 4) or trypsin (PAR2). The disarming and silencing of PARs by ISP2 may play a role in successful embryo implantation.

Blastocyst collapse as an embryo marker of low implantation potential: a time-lapse multicentre study

“…TEPs are cytoplasmic extensions of trophectodermal cells that protrude through the ZP into the extra-embryonic environment (Gonzales et al, 1996). In addition, it is known that implantation serine proteinase 1 (ISP1) and serine proteinase 2 (ISP2) play a critical role in the processes of embryo hatching and implantation, at least in mouse model (O'Sullivan et al, 2001;Sharma et al, 2013). ISPs are necessary for blastocyst hatching in vitro and the initiation of invasion.…”