Sheep mast-cell proteinases-1 and -3: cDNA cloning, primary structure and molecular modelling of the enzymes and further studies on substrate specificity.
Sheep mast-cell proteinase-1 (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 appears to be translated as a pre-proenzyme with a 17-amino-acid signal peptide, a basic 2-amino-acid propeptide and a 226-amino-acid catalytic domain. A second cDNA, encoding a serine proteinase 90% identical with sMCP-1, was also cloned and named sMCP-3. Molecular models were constructed for both enzymes using coordinates for the refined X-ray structures 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 into the substrate-binding pocket, hydrogen-bonding with Ser-190 on the opposite side and bisecting the pocket. The location of an acidic moiety in this position would favour interaction with basic substrate residues and binding of aromatic residues is rationalized by interaction of the positively charged equatorial plane with Asp-226. The balance between chymotryptic and tryptic activities of sMCP-1 was found to be sensitive to salt concentration, with increasing univalent cation concentration favouring chymotryptic activity relative to the tryptic. Using a peptide substrate representing residues 36-59 of the human thrombin receptor, increasing salt concentration favoured cleavage at Phe-43 rather than at Arg-41.
Department of Veterinary Clinical Studies, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian, EH25 9RG, Scotland, UK.
S M
SM
McAleese
A D
AD
Pemberton
M E
ME
McGrath
J F
JF
Huntley
H R
HR
Miller
D000595:Amino Acid Sequence; D000818:Animals; D053818:Chymases; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D016133:Polymerase Chain Reaction; D051381:Rats; D018179:Receptors, Thrombin / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012756:Sheep; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D014158:Transcription, Genetic
D018076:DNA, Complementary; D010446:Peptide Fragments; D018179:Receptors, Thrombin; D012965:Sodium Chloride; D012697:Serine Endopeptidases; C092818:mast cell protease 3; D053818:Chymases