Highly oriented recombinant glycosyltransferases: Site-specific immobilization of unstable membrane proteins by using staphylococcus aureus sortase a

Takaomi Ito, Reiko Sadamoto, Kentaro Naruchi, Hiroko Togame, Hiroshi Takemoto, Hirosato Kondo, Shin Ichiro Nishimura

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

Recombinant glycosyltransferases are potential biocatalysts for the construction of a compound library of oligosaccharides, glycosphingolipids, glycopeptides, and various artificial glycoconjugates on the basis of combined chemical and enzymatic synthetic procedures. The structurally defined glycan-related compound, library is a key resource both in the basic studies of their functional roles in various biological processes and in the discovery research of new diagnostic biomarkers and therapeutic reagents. Therefore, it is clear that the immobilization of extremely unstable membrane-bound glycosyltransferases on some suitable supporting materials should enhance the operational stability and activity of recombinant enzymes and makes facile separation of products and recycling use of enzymes possible. Until now, however, it seems that no standardized protocol preventing a significant loss of enzyme activity is available due to the lack of a general method of site-selective anchoring between glycosyltransferases and scaffold materials through a stable covalent bond. Here we communicate a versatile and efficient method for the immobilization of recombinant glycosyltransferases onto commercially available solid supports by means of transpeptidase reaction by Staphylococcus aureus sortase A. This protocol allowed for the first time highly specific conjugation at the designated C-terminal signal peptide moiety of recombinant human β1,4-galaetosyltranseferase or recombinant Helicobacter pylori α1,3-fucosyltransferase with simple aliphatic amino groups displayed on the surface of solid materials. Site-specifically immobilized enzymes exhibited the desired sugar transfer activity, an improved, stability, and a practical reusability required for rapid and large-scale synthesis of glycoconjugates. Considering that most mammalian enzymes responsible for the posttranslational modifications, including the protein kinase family, as well as glycosyltransferases are unstable and highly oriented, membrane proteins, the merit of our strategy based on "site-specific" transpeptidation is evident because the reaction proceeds only at an engineered C-terminus without any conformational influence around the active sites of both enzymes as well as heptad repeats of rHFucT required to maintain native secondary and quaternary structures during the dimerization on cell surfaces.

Original languageEnglish
Pages (from-to)2604-2614
Number of pages11
JournalBiochemistry
Volume49
Issue number11
DOIs
Publication statusPublished - Mar 23 2010
Externally publishedYes

Fingerprint

Glycosyltransferases
Immobilization
Staphylococcus aureus
Membrane Proteins
Enzymes
Glycoconjugates
galactoside 3-fucosyltransferase
Equipment Reuse
Peptidyl Transferases
Glycosphingolipids
Immobilized Enzymes
Covalent bonds
Dimerization
Glycopeptides
Biological Phenomena
Reusability
Enzyme activity
Biomarkers
Protein Sorting Signals
Oligosaccharides

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Highly oriented recombinant glycosyltransferases : Site-specific immobilization of unstable membrane proteins by using staphylococcus aureus sortase a. / Ito, Takaomi; Sadamoto, Reiko; Naruchi, Kentaro; Togame, Hiroko; Takemoto, Hiroshi; Kondo, Hirosato; Nishimura, Shin Ichiro.

In: Biochemistry, Vol. 49, No. 11, 23.03.2010, p. 2604-2614.

Research output: Contribution to journalArticle

Ito, Takaomi ; Sadamoto, Reiko ; Naruchi, Kentaro ; Togame, Hiroko ; Takemoto, Hiroshi ; Kondo, Hirosato ; Nishimura, Shin Ichiro. / Highly oriented recombinant glycosyltransferases : Site-specific immobilization of unstable membrane proteins by using staphylococcus aureus sortase a. In: Biochemistry. 2010 ; Vol. 49, No. 11. pp. 2604-2614.
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