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Glycobiology Advance Access originally published online on March 19, 2004
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Glycobiology vol 14 no 5 pp. 399-407, 2004
Glycobiology vol. 14 no. 5 © Oxford University Press 2004; all rights reserved.

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

Robert C. Davidson2, Juergen H. Nett2, Eduard Renfer2, Huijuan Li2, Terrance A. Stadheim2, Benton J. Miller3, Robert G. Miele2, Stephen R. Hamilton2, Byung-Kwon Choi2, Teresa I. Mitchell2 and Stefan Wildt1,2

2 Glycofi, Inc., 21 Lafayette Street Suite 200, Lebanon, NH 03766 3 Velocity 11; 435 Acacia Ave., Palo Alto, CA 94306

Received on June 19, 2003; revised on October 21, 2003; accepted on October 22, 2003

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 cloned the Pichia 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 Saccharomyces 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.

1 To whom correspondence should be addressed; e-mail: swildt{at}glycofi.com


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