In vitro evidence for the dual function of Alg2 and Alg11: essential mannosyltransferases in N-linked glycoprotein biosynthesis.
The biosynthesis of asparagine-linked glycoproteins utilizes a dolichylpyrophosphate-linked glycosyl donor (Dol-PP-GlcNAc(2)Man(9)Glc(3)), which is assembled by the series of membrane-bound glycosyltransferases that comprise the dolichol pathway. This biosynthetic pathway is highly conserved throughout eukaryotic evolution. While complementary genetic and bioinformatic approaches have enabled identification of most of the dolichol pathway enzymes in Saccharomyces cerevisiae, the roles of two of the mannosyltransferases in the pathway, Alg2 and Alg11, have remained ambiguous because these enzymes appear to catalyze only two of the remaining four unannotated transformations. To address this issue, a biochemical approach was taken using recombinant Alg2 and Alg11 from S. cerevisiae and defined dolichylpyrophosphate-linked substrates. A cell-membrane fraction isolated from Escherichia coli overexpressing thioredoxin-tagged Alg2 was used to demonstrate that this enzyme actually carries out an alpha1,3-mannosylation, followed by an alpha1,6-mannosylation, to form the first branched pentasaccharide intermediate of the pathway. Then, using thioredoxin-tagged Alg2 for the chemoenzymatic synthesis of the dolichylpyrophosphate pentasaccharide, it was thus possible to define the biochemical function of Alg11, which is to catalyze the next two sequential alpha1,2-mannosylations. The elucidation of the dual function of each of these enzymes thus completes the identification of the entire ensemble of glycosyltransferases that comprise the dolichol pathway.
O'Reilly
Mary K
MK
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Zhang
Guofeng
G
Imperiali
Barbara
B
D001216:Asparagine / Q000378:metabolism*; D002384:Catalysis; D004286:Dolichols / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis*; D008358:Mannose / Q000378:metabolism; D008364:Mannosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011105:Polyisoprenyl Phosphate Sugars / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014312:Trisaccharides / Q000737:chemistry
D004286:Dolichols; C066723:Glc(3)Man(9)(GlcNAc)(2)-diphosphate-dolichol; D006023:Glycoproteins; D011105:Polyisoprenyl Phosphate Sugars; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014312:Trisaccharides; D001216:Asparagine; C083733:ALG2 protein, S cerevisiae; D008364:Mannosyltransferases; C035777:chitobiosyldiphosphodolichol beta-mannosyltransferase; D008358:Mannose