SIGNIFICANCE OF THE HIGHLY CONSERVED GLY-4 RESIDUE IN HUMAN CYSTATIN-A

The expression system for human recombinant cystatin A has already bee n established to be a fusion protein with porcine adenylate kinase in Escherichia coli [Kaji et al, (1990) Biol, Chem. Hoppe-Seyler 371, Sup pl,, 145-150], After cyanogen bromide cleavage of the fused protein ex pressed in E. coli, the cystatin portion could be readily isolated, Th e inhibitory activity of the obtained variant (Cyst A(2-98)) was found to be almost identical with that of the wild type, and thereafter a m utation was introduced into this variant (Cyst A2(-98)), called the st andard variant, To elucidate the role of the Gly-4 residue, which is c ompletely conserved in all cystatin species, this residue was substitu ted with 17 other amino acids by means of cassette mutagenesis. Thus 1 7 variants (Cyst A(2-98) [G4X]) Obtained were examined as to their inh ibitory activity towards papain, As the side chain of the substituted amino acid residue became more bulky, the inhibitory activity of the v ariant markedly decreased, Variants whose side chains were bulkier tha n a Val residue showed almost no inhibitory activity towards papain. C onsequently, it was deduced that the large side chain of a substituted amino acid may cause steric hindrance, which may be responsible for t he decrease in inhibitory activity, Thus, we could conclude that the 4 th (Gly) residue on cystatin A must be small, because amino acids whic h existed on the N-terminal side of this residue could interact with a papain molecule.