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Glycobiology Advance Access published online on March 19, 2004
Glycobiology, doi:10.1093/glycob/cwh023
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Submitted on June 19, 2003
Revised on October 21, 2003
Accepted on October 22, 2003

© 2004 Glycobiology © Oxford University Press 2004; all rights reserved.

ORIGINAL ARTICLES

Functional analysis of the ALG3 gene encoding the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase enzyme of P. pastoris

Robert C. Davidson , Byung-Kwon Choi , Stephen R. Hamilton , Huijuan Li , Robert G. Miele , Benton J. Miller , Teresa I. Mitchell , Juergen H. Nett , Eduard Renfer , Terrance A. Stadheim , and Stefan Wildt *

* To whom correspondence should be addressed. E-mail: Swildt{at}glycofi.com.

Abstract

N-glycans are synthesized in both yeast and mammals through the ordered assembly of a lipid-linked core Glc3Man9GlcNAc2 structure that is subsequently transferred to a nascent protein in the endoplasmic reticulum. Once folded, glycoproteins are then shuttled to the Golgi where additional, but divergent processing occurs in mammals and fungi. We have cloned the P. pastoris homolog of the ALG3 gene, which encodes the enzyme that converts Man5GlcNAc2-Dol-PP to Man6GlcNAc2-Dol-PP. Deletion of this gene in an och1 mutant background resulted in the secretion of glycoproteins with a predicted Man5GlcNAc2 structure that could be trimmed to Man3GlcNAc2 by in vitro {alpha}-1,2- mannosidase treatment. However, several larger glycans ranging from Hex6GlcNAc2 to Hex12GlcNAc2 were also observed that were recalcitrant to an array of mannosidase digests. These results contrast the far simpler glycan profile found in S. cerevisiae alg3-1 och1, indicating diverging Golgi processing in these two closely related yeasts. Finally, analysis of the P. pastoris alg3 deletion mutant in the presence and absence of the outer chain initiating Och1p {alpha}-1,6-mannosyltransferase activity suggests that the PpOch1p has a broader substrate specificity compared to its S. cerevisiae counterpart.


ALG3, Mannosyltransferase, N-glycosylation, Lipid-linked glycosylation, Endoplasmic reticulum, Pichia pastoris
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