Premium
Saccharomyces cerevisiae α1,6‐mannosyltransferase has a catalytic potential to transfer a second mannose molecule
Author(s) -
Kitajima Toshihiko,
Chiba Yasunori,
Jigami Yoshifumi
Publication year - 2006
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2006.05505.x
Subject(s) - mannose , pichia pastoris , biochemistry , chemistry , oligosaccharide , saccharomyces cerevisiae , glycosylation , yeast , recombinant dna , gene
In yeast, the N ‐linked oligosaccharide modification in the Golgi apparatus is initiated by α1,6‐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 α1,2‐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 α1,3‐linked mannose branching from the α1,6‐linked mannose that is attached to β1,4‐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.