SHEEP MAST-CELL PROTEINASE-1 AND PROTEINASE-3 - CDNA CLONING, PRIMARYSTRUCTURE AND MOLECULAR MODELING OF THE ENZYMES AND FURTHER-STUDIES ON SUBSTRATE-SPECIFICITY

Sheep mast-cell proteinase-l (sMCP-1) is a serine proteinase expressed predominantly by mucosal mast cells, with specificity for cleavage C- terminal to basic and hydrophobic amino acid residues. A cDNA encoding sMCP-1 has been cloned using reverse transcriptase (RT)-PCR. It appea rs to be translated as a pre-proenzyme with a 17-amino-acid signal pep tide, a basic 2-amino-acid propeptide and a 226-amino-acid catalytic d omain. A second cDNA, encoding a serine proteinase 90% identical with sMCP-1, was also cloned and named sMCP-3. Molecular models were constr ucted for both enzymes using coordinates for the refined X-ray structu res of human cathepsin G, chymase and rat mast-cell proteinase-2. The model for sMCP-1 suggests that the acidic Asp-226 side chain extends i nto the substrate-binding pocket, hydrogen-bonding with Ser-190 on the opposite side and bisecting the pocket. The location of an acidic moi ety in this position would favour interaction with basic substrate res idues and binding of aromatic residues is rationalized by interaction of the positively charged equatorial plane with Asp-226. The balance b etween chymotryptic and tryptic activities of sMCP-1 was found to be s ensitive to salt concentration, with increasing univalent cation conce ntration favouring chymotryptic activity relative to the tryptic. Usin g a peptide substrate representing residues 36-59 of the human thrombi n receptor, increasing salt concentration favoured cleavage at Phe-43 rather than at Arg-41.