Cloning and characterization of a viral α2-3-sialyltransferase (vST3Gal-I) for the synthesis of sialyl Lewisx

Go Sugiarto, Kam Lau, Hai Yu, Stephanie Vuong, Vireak Thon, Yanhong Li, Shengshu Huang, Xi Chen

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

Sialyl Lewisx (SLex, Siaα2-3Galβ1- 4(Fucα1-3)GlcNAcβOR) is an important sialic acid-containing carbohydrate epitope involved in many biological processes such as inflammation and cancer metastasis. In the biosynthetic process of SLex, α2-3-sialyltransferase-catalyzed sialylation generally proceeds prior to β1-3-fucosyltransferase-catalyzed fucosylation. For the chemoenzymatic synthesis of SLex containing different sialic acid forms, however, it would be more effi- cient if diverse sialic acid forms are transferred in the last step to the fucosylated substrate Lewisx (Lex). An α2-3- sialyltransferase obtained from myxoma virus-infected European rabbit kidney RK13 cells (viral α2-3-sialyltransferase (vST3Gal-I)) was reported to be able to tolerate fucosylated substrate Lex. Nevertheless, the substrate specificity of the enzyme was only determined using partially purified protein from extracts of cells infected with myxoma virus. Herein we demonstrate that a previously reported multifunctional bacterial enzyme Pasteurella multocida sialyltransferase 1 (PmST1) can also use Lex as an acceptor substrate, although at a much lower efficiency compared to nonfucosylated acceptor. In addition, N-terminal 30-aminoacid truncated vST3Gal-I has been successfully cloned and expressed in Escherichia coli Origami™ B(DE3) cells as a fusion protein with an N-terminal maltose binding protein (MBP) and a C-terminal His6-tag (MBP-δ30vST3Gal-IHis6). The viral protein has been purified to homogeneity and characterized biochemically. The enzyme is active in a broad pH range varying from 5.0 to 9.0. It does not require a divalent metal for its α2-3-sialyltransferase activity. It has been used in one-pot multienzyme sialylation of Lex for the synthesis of SLex containing different sialic acid forms with good yields.

Original languageEnglish (US)
Pages (from-to)387-396
Number of pages10
JournalGlycobiology
Volume21
Issue number3
DOIs
StatePublished - Mar 2011

Keywords

  • cloning
  • Lewis
  • sialic acid
  • sialyl Lewis
  • sialyltransferase

ASJC Scopus subject areas

  • Biochemistry

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