Substrate specificity of α‐1,6‐mannosyltransferase that initiates N ‐linked mannose outer chain elongation in Saccharomyces cerevisiae
1997; Wiley; Volume: 412; Issue: 3 Linguagem: Inglês
10.1016/s0014-5793(97)00634-0
ISSN1873-3468
AutoresKen-ichi Nakayama, Y. Nakanishi-Shindo, Atsushi Tanaka, Yoshiko Haga-Toda, Yoshifumi Jigami,
Tópico(s)Fermentation and Sensory Analysis
ResumoYeast Saccharomyces cerevisiae OCH1 gene encodes the mannosyltransferase that is essential for the outer chain elongation of N ‐linked oligosaccharides. Mannosyltransferase activity of OCH1 gene product (Och1p) was measured on HPLC by using pyridylaminated Man 8 GlcNAc 2 (Man 8 GlcNAc 2 ‐PA) as an acceptor and the reaction product was observed at the retention time corresponding to Man 9 GlcNAc 2 ‐PA. 1 H‐NMR and fast atom bombardment mass spectrometry (FAB‐MS) fragmentation analysis of Man 9 GlcNAc 2 ‐PA showed that the additional mannose was attached with an α‐1,6 linkage at the site where mannose outer chain elongation initiates. Substrate specificity of Och1p was investigated by using various high mannose‐type oligosaccharides as acceptors. Man 8 GlcNAc 2 was the best acceptor for Och1p. The loss of one or two α‐1,2‐mannoses from Man 8 GlcNAc 2 reduced the mannosyltransferase activity and the Man 5 GlcNAc 2 completely lacking α‐1,2‐mannose residues did not serve as an acceptor. Man 8 GlcNAcOH that involves an open sugar ring by reduction of reducing terminal GlcNAc residue did not serve as an acceptor for Och1p. The loss of three mannoses at the α‐1,6‐branch also reduced the Och1p activity. These results suggest that Och1p is an initiation specific α‐1,6‐mannosyltransferase that requires the intact structure of Man 8 GlcNAc for efficient mannose outer chain initiation.
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