Saccharomyces cerevisiae alpha1,6-mannosyltransferase has a catalytic potential to transfer a second mannose molecule.
In yeast, the N-linked oligosaccharide modification in the Golgi apparatus is initiated by alpha1,6-mannosyltransferase (encoded by the OCH1 gene) with the addition of mannose to the Man(8)GlcNAc(2) or Man(9)GlcNAc(2) endoplasmic reticulum intermediates. In order to characterize its enzymatic properties, the soluble form of the recombinant Och1p was expressed in the methylotrophic yeast Pichia pastoris as a secreted protein, after truncation of its transmembrane region and fusion with myc and histidine tags at the C-terminus, and purified using a metal chelating column. The enzymatic reaction was performed using various kinds of pyridylaminated (PA) sugar chains as acceptor, and the products were separated by high performance liquid chromatography. The recombinant Och1p efficiently transferred a mannose to Man(8)GlcNAc(2)-PA and Man(9)GlcNAc(2)-PA acceptors, while Man(5)GlcNAc(2)-PA, which completely lacks alpha1,2-linked mannose residues, was not used as an acceptor. At high enzyme concentrations, a novel product was detected by HPLC. Analysis of the product revealed that a second mannose was attached at the 6-O-position of alpha1,3-linked mannose branching from the alpha1,6-linked mannose that is attached to beta1,4-linked mannose of Man(10)GlcNAc(2)-PA produced by the original activity of Och1p. Our results indicate that Och1p has the potential to transfer two mannoses from GDP-mannose, and strictly recognizes the overall structure of high mannose type oligosaccharide.
Kitajima
Toshihiko
T
Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 6, Tsukuba-shi, Ibaraki, Japan.
Chiba
Yasunori
Y
Jigami
Yoshifumi
Y
D050260:Carbohydrate Metabolism; D002384:Catalysis; D002478:Cells, Cultured; D006031:Glycosylation; D008358:Mannose / Q000378:metabolism*; D008364:Mannosyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D010843:Pichia / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D008562:Membrane Glycoproteins; C075636:OCH1 protein, S cerevisiae; D009844:Oligosaccharides; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D008364:Mannosyltransferases; C058478:alpha 1,6-mannosyltransferase; D008358:Mannose