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Accessible Unlicensed Requires Authentication Published by De Gruyter January 10, 2007

Activation profiles of the zymogen of aspergilloglutamic peptidase

Xiang-Ping Huang, Yutaka Yabuki, Masaki Kojima, Hideshi Inoue and Kenji Takahashi
From the journal

Abstract

Aspergilloglutamic peptidase produced by Aspergillus niger var. macrosporus belongs to the novel glutamic peptidase family. Its zymogen is autocatalytically activated under acidic conditions to the mature enzyme with a two-chain structure. Analyses by SDS-PAGE and mass spectrometry of the activation products of the recombinant zymogen showed that the major pathway of activation includes initial fast cleavage at Glu12-Ala13, followed by stepwise cleavages in the N-terminal and intervening propeptide regions. Essentially the same activation profile was obtained with the recombinant zymogen lacking the N-terminal 12-aa sequence. The missing region includes the most prominent cluster of basic residues of the propeptide, indicating low importance of this cluster for activation and refolding of the zymogen.

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References

Fujinaga, M., Cherney, M.M., Oyama, H., Oda, K., and James, M.N.G. (2004). The molecular structure and catalytic mechanism of a novel carboxyl peptidase from Scytalidium lignicolum. Proc. Natl. Acad. Sci. USA101, 3364–3369.Search in Google Scholar

Huang, X.-P., Kagami, N., Inoue, H., Kojima. M., Kimura, T., Makabe, O., Suzuki, K., and Takahashi, K. (2000). Identification of a glutamic acid and an aspartic acid residue essential for catalytic activity of aspergillopepsin II, a non-pepsin type acid proteinase. J. Biol. Chem.275, 26607–26614.Search in Google Scholar

Inoue, H., Kimura, T., Makabe, O., and Takahashi, K. (1991). The gene and deduced protein sequences of the zymogen of Aspergillus niger acid proteinase. J. Biol. Chem.266, 19484–19489.Search in Google Scholar

Kataoka, Y., Takada, K., Oyama, H., Tsunemi, M., James, M.N., and Oda, K. (2005). Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases. FEBS Lett.579, 2991–2994.Search in Google Scholar

Kubota, K., Nishii, W., Kojima, M., and Takahashi, K. (2005). Specific inhibition and stabilization of aspergilloglutamic peptidase by the propeptide: identification of critical sequences and residues in the propeptide. J. Biol. Chem.280, 999–1006.Search in Google Scholar

Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227, 680–685.Search in Google Scholar

Oda, K. (2004). Scytalidopepsin B. In: Handbook of Proteolytic Enzymes, 2nd Edition, Vol. 1, A.J. Barrett, N.D. Rawlings, and J.F. Woessner, eds. (London, UK: Academic Press), pp. 219–221.Search in Google Scholar

Oda, K., Takahashi, S., Shin, T., and Murao, S. (1995). Pepstatin-insensitive carboxyl proteinases. In: Aspartic Proteinases: Structure, Function, Biology, and Biomedical Implications, K. Takahashi, ed. (New York, USA: Plenum Press), pp. 529–542.Search in Google Scholar

Sasaki, H., Nakagawa, A., Muramatsu, T., Suganuma, M., Sawano, Y., Kojima, M., Kubota, K., Takahashi, K., and Tanokura, M. (2004). The three-dimensional structure of aspergilloglutamic peptidase from Aspergillus niger. Proc. Jpn. Acad. Ser. B80, 435–438.Search in Google Scholar

Sasaki, H., Kojima, M., Sawano, Y., Kubota, K., Suganuma, M., Muramatsu, T., Takahashi, K., and Tanokura, M. (2005). A proposed catalytic mechanism of aspergilloglutamic peptidase from Aspergillus niger. Proc. Jpn. Acad. Ser. B81, 441–446.Search in Google Scholar

Takahashi, K. (1995). Proteinase A from Aspergillus niger. Methods Enzymol.248, 146–155.Search in Google Scholar

Takahashi, K. (1997). The specificity of peptide bond cleavage of acid proteinase A from Aspergillus niger var. macrosporus toward oxidized ribonuclease A. Biosci. Biotechnol. Biochem.61, 381–383.Search in Google Scholar

Takahashi, K. (2004). Aspergillopepsin II. In: Handbook of Proteolytic Enzymes, 2nd Edition, Vol. 1, A.J. Barrett, N.D. Rawlings, and J.F. Woessner, eds. (London, UK: Academic Press), pp. 221–224.Search in Google Scholar

Takahashi, K., Inoue, H., Sakai, K., Kohama, T., Kitahara, S., Takishima, K., Tanji, M., Athauda, S.B.P., Takahashi, T., Akanuma, H., Mamiya, G., and Yamasaki, M. (1991). The primary structure of Aspergillus niger acid proteinase A. J. Biol. Chem.266, 19480–19483.Search in Google Scholar

Takahashi, K., Kagami, N., Huang, X.-P., Kojima, M., and Inoue, H. (1998). Aspergillus niger acid proteinase A. Structure and function. In: Aspartic Proteinases. Retroviral and Cellular Enzymes, M.N.G. James, ed. (New York, USA: Plenum Press), pp. 275–282.Search in Google Scholar

Yabuki, Y., Kubota, K., Kojima, M., Inoue, H., and Takahashi, K. (2004). Identification of a glutamine residue essential for catalytic activity of aspergilloglutamic peptidase by site-directed mutagenesis. FEBS Lett.569, 161–164.Search in Google Scholar

Published Online: 2007-01-10
Published in Print: 2007-01-01

©2007 by Walter de Gruyter Berlin New York