SHEEP MAST-CELL PROTEINASE-1 AND PROTEINASE-3 - CDNA CLONING, PRIMARYSTRUCTURE AND MOLECULAR MODELING OF THE ENZYMES AND FURTHER-STUDIES ON SUBSTRATE-SPECIFICITY
Sm. Mcaleese et al., SHEEP MAST-CELL PROTEINASE-1 AND PROTEINASE-3 - CDNA CLONING, PRIMARYSTRUCTURE AND MOLECULAR MODELING OF THE ENZYMES AND FURTHER-STUDIES ON SUBSTRATE-SPECIFICITY, Biochemical journal, 333, 1998, pp. 801-809
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.