Significance of the highly conserved gly-4 residue in human cystatin A

Kazunori Shibuya, Hiroyuki Kaji, Yukihito Ohyama, Shin ichi Tate, Masatsune Kainosho, Fuyuhiko Inagaki, Tatsuya Samejima

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13 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)635-642
Number of pages8
JournalJournal of biochemistry
Volume118
Issue number3
DOIs
Publication statusPublished - 1995 Sep
Externally publishedYes

Keywords

  • Cassette mutagenesis
  • Cystatin A
  • Cysteine proteinase inhibitor
  • Inhibitory activity
  • Papain

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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  • Cite this

    Shibuya, K., Kaji, H., Ohyama, Y., Tate, S. I., Kainosho, M., Inagaki, F., & Samejima, T. (1995). Significance of the highly conserved gly-4 residue in human cystatin A. Journal of biochemistry, 118(3), 635-642. https://doi.org/10.1093/oxfordjournals.jbchem.a124957