Abstract
N-Linked glycosylation involves the ordered, stepwise synthesis of the unique lipid-linked oligosaccharide precursor Glc(3)Man(9) GlcNAc(2)-PP-Dol on the endoplasmic reticulum (ER), catalyzed by a series of glycosyltransferases. Here we characterize Alg2 as a bifunctional enzyme that is required for both the transfer of the alpha1,3- and the alpha1,6-mannose-linked residue from GDP-mannose to Man(1)GlcNAc(2)-PP-Dol forming the Man(3)GlcNAc(2)-PP-Dol intermediate on the cytosolic side of the ER. Alg2 has a calculated mass of 58 kDa and is predicted to contain four transmembrane-spanning helices, two at the N terminus and two at the C terminus. Contradictory to topology predictions, we prove that only the two N-terminal domains fulfill this criterion, whereas the C-terminal hydrophobic sequences contribute to ER localization in a nontransmembrane manner. Surprisingly, none of the four domains is essential for transferase activity because truncated Alg2 variants can exert their function as long as Alg2 is associated with the ER by either its N- or C-terminal hydrophobic regions. By site-directed mutagenesis we demonstrate that an EX(7)E motif, conserved in a variety of glycosyltransferases, is not important for Alg2 function in vivo and in vitro. Instead, we identify a conserved lysine residue, Lys(230), as being essential for activity, which could be involved in the binding of the phosphate of the glycosyl donor.
Highlights
11900 JOURNAL OF BIOLOGICAL CHEMISTRY syl donors
Because a bioinformatic approach of the yeast data base did not reveal an unknown open reading frame that might encode an additional putative mannosyltransferase being involved in linked oligosaccharide (LLO) synthesis, we reasoned that ALG2 may have a dual function, i.e. synthesizing both Man2GlcNAc2-PP-Dol and Man3GlcNAc2-PP-Dol
The results presented in this paper demonstrate that ALG2 encodes a bifunctional glycosyltransferase, catalyzing both the ␣1,3- and the ␣1,6-mannose linkage onto Man1GlcNAc2-PPDol with formation of the trimannosyl core Man3GlcNAc2-PPDol (Fig. 8)
Summary
11900 JOURNAL OF BIOLOGICAL CHEMISTRY syl donors. Upon translocation of the heptasaccharide to the luminal site, which is facilitated by Rft1 [11] and another not yet identified protein [12], it is extended by four mannose and three glucose residues deriving from Man-P-Dol and Glc-P-Dol. One of the temperature-sensitive alg mutants, alg, was shown to accumulate lipid-linked Man2GlcNAc2 at the restrictive temperature [15], indicating that alg might have a defect in the glycosyltransferase catalyzing the transfer of the third, ␣1,6-linked mannose, i.e. in the formation of the branched pentasaccharide Man3GlcNAc2-PP-Dol (see Fig. 8). Biochemical studies in human fibroblasts from a patient with a defect in the human ALG2 ortholog, causing congenital disorder of glycosylation type CDG1i, pointed to a role in the transfer of the second, ␣1,3-linked mannose residue, because no elongation of Man[1,6]ManGlcNAc2-PP-Dol occurred [21]. We perform a mutational analysis of Alg and identify amino acids required for its activity
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