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J. Biochem, 1995, Vol. 118, No. 3 635-642
© 1995 Japanese Biochemical Society

other

Significance of the Highly Conserved Gly-4 Residue in Human Cystatin A1

Kazunori Shibuya*, Hiroyuki Kaji*,2, Yukihito Ohyama*, Shin-ichi Tate{dagger}, Masatsune Kainosho{dagger}, Fuyuhiko Inagaki{ddagger} and Tatsuya Samejima*,3

*Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University Setagaya-ku, Tokyo 157
{dagger}Department of Chemistry, Faculty of Science, Tokyo Metropolitan University Minami-ohsawa, Hachioji, Tokyo 192-03
{ddagger}Division of Molecular Physiology, The Tokyo Metropolitan Institute of Medical Science Bunkyo-ku, Tokyo 113

2 Present Address: Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Minami-ohsawa, Hachioji, Tokyo 192-03.

3 To whom correspondence should be addressed.

The expression system for human recombinant cystatin A has already been established to be a fusion protein with porcine adenylate kinase in Escherichia coli [Kaji et al. (1990) Biol. Chem. Hoppe-Seyler 371, Suppl., 145–150]. After cyanogen bromide cleavage of the fused protein expressed in E. coli, the cystatin portion could be readily isolated. The inhibitory activity of the obtained variant (Cyst A2–98)was found to be almost identical with that of the wild type, and thereafter a mutation was introduced into this variant (Cyst A2–98)called the standard variant. To elucidate the role of the Gly-4 residue, which is completely conserved in all cystatin species, this residue was substituted with 17 other amino acids by means of cassette mutagenesis. Thus 17 variants (Cyst A2–98[G4X]) obtained were examined as to their inhibitory activity towards papain. As the side chain of the substituted amino acid residue became more bulky, the inhibitory activity of the variant markedly decreased. Variants whose side chains were bulkier than a Val residue showed almost no inhibitory activity towards papain. Consequently, it was deduced that the large side chain of a substituted amino acid may cause steric hindrance, which may be responsible for the decrease in inhibitory activity. Thus, we could conclude that the 4th (Gly) residue on cystatin A must be small, because amino acids which existed on the N-terminal side of this residue could interact with a papain molecule.

1This study was partially supported by a grant from the Research Institute of Aoyama Gakuin University.


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