Functional analysis of the ALG3 gene encoding the Dol-P-Man: Man5GlcNAc2-PP-Dol mannosyltransferase enzyme of P. pastoris.
N-glycans are synthesized in both yeast and mammals through the ordered assembly of a lipid-linked core Glc(3)Man(9)GlcNAc(2) 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 Man(5)GlcNAc(2)-Dol-PP to Man(6)GlcNAc(2)-Dol-PP. Deletion of this gene in an och1 mutant background resulted in the secretion of glycoproteins with a predicted Man(5)GlcNAc(2) structure that could be trimmed to Man(3)GlcNAc(2) by in vitro alpha-1,2-mannosidase treatment. However, several larger glycans ranging from Hex(6)GlcNAc(2) to Hex(12)GlcNAc(2) 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.
Davidson
Robert C
RC
Glycofi, Inc., 21 Lafayette Street Suite 200, Lebanon, NH 03766 Velocity 11; 435 Acacia Ave., Palo Alto, CA 94306, USA.
Nett
Juergen H
JH
Renfer
Eduard
E
Li
Huijuan
H
Stadheim
Terrance A
TA
Miller
Benton J
BJ
Miele
Robert G
RG
Hamilton
Stephen R
SR
Choi
Byung-Kwon
BK
Mitchell
Teresa I
TI
Wildt
Stefan
S
D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism; D008361:Mannosidases / Q000378:metabolism; D008364:Mannosyltransferases / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011134:Polysaccharides / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D008565:Membrane Proteins; D009844:Oligosaccharides; D011134:Polysaccharides; D029701:Saccharomyces cerevisiae Proteins; C058642:mannosyl(5)-N-acetyl(2)-glucose; C104466:ALG3 protein, S cerevisiae; D008364:Mannosyltransferases; D008361:Mannosidases; D008358:Mannose