Document Detail

Co-expression of two mammalian glycosyltransferases in the yeast cell wall allows synthesis of sLex.
MedLine Citation:
PMID:  15691739     Owner:  NLM     Status:  MEDLINE    
Interactions between selectins and their oligosaccharide-decorated counter-receptors play an important role in the initiation of leukocyte extravasation in inflammation. L-selectin ligands are O-glycosylated with sulphated sialyl Lewis X epitopes (sulpho-sLex). Synthetic sLex oligosaccharides have been shown to inhibit adhesion of lymphocytes to endothelium at sites of inflammation. Thus, they could be used to prevent undesirable inflammatory reactions such as rejection of organ transplants. In vitro synthesis of sLex glycans is dependent on the availability of recombinant glycosyltransferases. Here we expressed the catalytic domain of human alpha-1,3-fucosyltransferase VII in the yeasts Saccharomyces cerevisiae and Pichia pastoris. To promote proper folding and secretion competence of this catalytic domain in yeast, it was fused to the Hsp150 delta carrier, which is an N-terminal fragment of a secretory glycoprotein of S. cerevisiae. In both yeasts, the catalytic domain acquired an active conformation and the fusion protein was externalised, but remained mostly attached to the cell wall in a non-covalent fashion. Incubation of intact S. cerevisiae or P. pastoris cells with GDP-[14C]fucose and sialyl-alpha-2,3-N-acetyllactosamine resulted in synthesis of radioactive sLex, which diffused to the medium. Finally, we constructed an S. cerevisiae strain co-expressing the catalytic domains of alpha-2,3-sialyltransferase and alpha-1,3-fucosyltransferase VII, which were targeted to the cell wall. When these cells were provided with N-acetyllactosamine, CMP-sialic acid and GDP-[14C]fucose, radioactive sLex was produced to the medium. These data imply that yeast cells can provide a self-perpetuating source of fucosyltransferase activity immobilized in the cell wall, useful for the in vitro synthesis of sLex.
Hanna Salo; Eeva Sievi; Taina Suntio; Maria Mecklin; Pirkko Mattila; Risto Renkonen; Marja Makarow
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  FEMS yeast research     Volume:  5     ISSN:  1567-1356     ISO Abbreviation:  FEMS Yeast Res.     Publication Date:  2005 Feb 
Date Detail:
Created Date:  2005-02-04     Completed Date:  2005-05-10     Revised Date:  2009-05-21    
Medline Journal Info:
Nlm Unique ID:  101085384     Medline TA:  FEMS Yeast Res     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  341-50     Citation Subset:  IM    
Program in Cellular Biotechnology, Institute of Biotechnology, University of Helsinki, Viikinkaari 9, 00710 Helsinki, Finland.
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MeSH Terms
Acetylglucosamine / metabolism
Cell Wall / enzymology*,  genetics
Fucosyltransferases / genetics,  metabolism*
Glycoproteins / genetics,  metabolism
Heat-Shock Proteins / genetics,  metabolism
Oligosaccharides / metabolism*
Pichia / enzymology*,  genetics
Recombinant Proteins
Saccharomyces cerevisiae / enzymology*,  genetics
Saccharomyces cerevisiae Proteins / genetics,  metabolism
Sialyltransferases / genetics,  metabolism*
Reg. No./Substance:
0/5-acetylneuraminyl-(2-3)-galactosyl-(1-4)-(fucopyranosyl-(1-3))-N-acetylglucosamine; 0/Glycoproteins; 0/HSP150 protein, S cerevisiae; 0/Heat-Shock Proteins; 0/Oligosaccharides; 0/Recombinant Proteins; 0/Saccharomyces cerevisiae Proteins; 7512-17-6/Acetylglucosamine; EC 2.4.1.-/Fucosyltransferases; EC 3-fucosyltransferase; EC 2.4.99.-/Sialyltransferases; EC alpha-2,3-sialyltransferase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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