Identification of preferred substrate sequences of microbial transglutaminase from Streptomyces mobaraensis using a phage-displayed peptide library

https://doi.org/10.1016/j.abb.2008.06.014Get rights and content

Abstract

Microbial transglutaminase (TGase) from Streptomyces mobaraensis (MTG) has been used in many industrial applications because it effectively catalyzes the formation of covalent cross-linking between glutamine residues in various substrate proteins and lysine residues or primary amines. To better understand the sequence preference around the reactive glutamine residue by this enzymatic reaction, we screened preferred peptide sequences using a phage-displayed random peptide library. Most of the peptides identified contained a consensus sequence, which was different from those previously found for mammalian TGases. Of these, most sequences had a specific reactivity toward MTG when produced as a fusion protein with glutathione-S-transferase. Furthermore, the representative sequence was found to be reactive even in the peptide form. The amino acid residues in the sequence critical for the reactivity were further analyzed, and the possible interaction with the enzyme has been discussed in this paper.

Section snippets

Transglutaminases

The mature form of microbial transglutaminase was produced and purified from S. mobaraensis as described previously [12], [25]. Guinea pig liver TGase 2 (Sigma Chemical Co., MO) and human factor XIII (Fibrogammin RP; ZLB Behring, Marburg, Germany) were purchased. Factor XIII was activated (Factor XIIIa) by treatment with bovine thrombin (Sigma Chemical Co., MO) and then thrombin was inactivated by addition of phenylmethylsulfonyl fluoride (PMSF). In order to include as a similar enzymatic

Screening for preferred substrate sequences for MTG

A total of 1.5 × 1011 phage clones were incubated with Bio-Cd in the presence of MTG. Following the catalytic reactions, phage clones that bound Bio-Cd covalently were selected by avidin-affinity purification. These phages were subjected to four additional transamidation reaction/selection/amplification cycles. Of the total phage clones selected through five rounds of screening, 27 were randomly selected. Then, the peptide sequence displayed by each clone was determined and was aligned based on

Discussion

Initially, during the TGase catalytic reaction, the enzyme forms an intermediate with the γ-carboxyamide group of the glutamine residues in the substrate via the cysteine residue present in the active site domain. After the acyl transfer reaction to a nucleophilic substrate, such as ε-amino group of the lysine residue, this process results in the formation of intermolecluar isopeptide bond. TGase is generally more selective with regard to the participating glutamine residue in the substrate,

Acknowledgments

This work was supported by Grant-in-Aid for Scientific Research (C) No. 19580103 (to K.H.), and Grant-in-Aid for Young Scientists Research No. 186701 (to Y.S.). The first author is a research fellow at the Japan Society for the Promotion of Science.

References (31)

  • L. Fésüs et al.

    Trends Biochem. Sci.

    (2002)
  • J.S.K. Chen et al.

    Int. J. Biochem. Cell Biol.

    (1999)
  • L. Fésüs et al.

    FEBS Lett.

    (2005)
  • G. Schmidt et al.

    J. Biol. Chem.

    (1998)
  • D. Plácido et al.

    Protein Expr. Purif.

    (2008)
  • T. Kanaji et al.

    J. Biol. Chem.

    (1993)
  • J. Cortez et al.

    J. Biotechnol.

    (2005)
  • Y. Sugimura et al.

    J. Biol. Chem.

    (2006)
  • Y. Sugimura et al.

    J. Biotechnol.

    (2007)
  • T. Kashiwagi et al.

    J. Biol. Chem.

    (2002)
  • A. Valdivia et al.

    J. Biotechnol.

    (2006)
  • N. Kamiya et al.

    J. Biosci. Bioeng.

    (2007)
  • M. Griffin et al.

    Biochem. J.

    (2002)
  • L. Lorand et al.

    Nat. Rev. Mol. Cell Biol.

    (2003)
  • A. Ichinose

    Thromb. Haemost.

    (2001)
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