title: 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.
authors: Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Canada. navsharm@ucalgary.ca
authors: Navneet
authors: N
authors: Sharma
authors: Rajeev
authors: R
authors: Kumar
authors: Bernard
authors: B
authors: Renaux
authors: Mahmoud
authors: M
authors: Saifeddine
authors: Sandra
authors: S
authors: Nishikawa
authors: Koichiro
authors: K
authors: Mihara
authors: Rithwik
authors: R
authors: Ramachandran
authors: Morley D
authors: MD
authors: Hollenberg
authors: Derrick E
authors: DE
authors: Rancourt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D059748:Proteolysis / Q000187:drug effects; D051381:Rats; D044462:Receptors, Proteinase-Activated / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D019151:Peptide Library; D010455:Peptides; D011480:Protease Inhibitors; D044462:Receptors, Proteinase-Activated; D011994:Recombinant Proteins; D048049:Extracellular Signal-Regulated MAP Kinases; C436412:Prss28 protein, mouse; D012697:Serine Endopeptidases