Publications

• LEW3, encoding a putative alpha-1,2-mannosyltransferase (ALG11) in N-linked glycoprotein, plays vital roles in cell-wall biosynthesis and the abiotic stress response in Arabidopsis thaliana.

  Zhang M., Henquet M., Chen Z., Zhang H., Zhang Y., Ren X., van der Krol S., Gonneau M., Bosch D., Gong Z.

  N-linked glycosylation is an essential protein modification that helps protein folding, trafficking and translocation in eukaryotic systems. The initial process for N-linked glycosylation shares a common pathway with assembly of a dolichol-linked core oligosaccharide. Here we characterize a new Ar ... >> More

  N-linked glycosylation is an essential protein modification that helps protein folding, trafficking and translocation in eukaryotic systems. The initial process for N-linked glycosylation shares a common pathway with assembly of a dolichol-linked core oligosaccharide. Here we characterize a new Arabidopsis thaliana mutant lew3 (leaf wilting 3), which has a defect in an alpha-1,2-mannosyltransferase, a homolog of ALG11 in yeast, that transfers mannose to the dolichol-linked core oligosaccharide in the last two steps on the cytosolic face of the ER in N-glycan precursor synthesis. LEW3 is localized to the ER membrane and expressed throughout the plant. Mutation of LEW3 caused low-level accumulation of Man(3)GlcNAc(2) and Man(4)GlcNAc(2) glycans, structures that are seldom detected in wild-type plants. In addition, the lew3 mutant has low levels of normal high-mannose-type glycans, but increased levels of complex-type glycans. The lew3 mutant showed abnormal developmental phenotypes, reduced fertility, impaired cellulose synthesis, abnormal primary cell walls, and xylem collapse due to disturbance of the secondary cell walls. lew3 mutants were more sensitive to osmotic stress and abscisic acid (ABA) treatment. Protein N-glycosylation was reduced and the unfolded protein response was more activated by osmotic stress and ABA treatment in the lew3 mutant than in the wild-type. These results demonstrate that protein N-glycosylation plays crucial roles in plant development and the response to abiotic stresses. << Less

  Plant J. 60:983-999(2009) [PubMed] [EuropePMC]

• The biosynthesis of oligosaccharide-lipids. Formation of an alpha-1,2-mannosyl-mannose linkage.

  Schutzbach J.S., Springfield J.D., Jensen J.W.

  An enzyme present in rabbit liver microsomes has been found to catalyze mannosyltransfer from GDP-mannose to exogenously added oligosaccharide-lipid acceptor resulting in the formation of an alpha-1,2-mannosyl-mannose linkage. Several lines of evidence suggest that the product is a heptasaccharide ... >> More

  An enzyme present in rabbit liver microsomes has been found to catalyze mannosyltransfer from GDP-mannose to exogenously added oligosaccharide-lipid acceptor resulting in the formation of an alpha-1,2-mannosyl-mannose linkage. Several lines of evidence suggest that the product is a heptasaccharide containing a single 1,6-linked branched glycosyl unit with 65% of the newly added mannosyl units in a terminal nonreducing position. The enzyme has been solubilized with non-ionic detergents and was partially purified on DEAE-cellulose and hydroxylapatite. The partially purified enzyme no longer catalyzed the formation of dolichol-P-mannose indicating a direct transfer from GDP-mannose to oligosaccharide-lipid acceptor. << Less

  J Biol Chem 255:4170-4175(1980) [PubMed] [EuropePMC]

• A severe human metabolic disease caused by deficiency of the endoplasmatic mannosyltransferase hALG11 leads to congenital disorder of glycosylation-Ip.

  Rind N., Schmeiser V., Thiel C., Absmanner B., Lubbehusen J., Hocks J., Apeshiotis N., Wilichowski E., Lehle L., Korner C.

  A new type of congenital disorders of glycosylation, designated CDG-Ip, is caused by the deficiency of GDP-Man:Man3GlcNAc2-PP-dolichol-alpha1,2-mannosyltransferase, encoded by the human ortholog of ALG11 from yeast. The patient presented with a multisystemic disorder characterized by muscular hypo ... >> More

  A new type of congenital disorders of glycosylation, designated CDG-Ip, is caused by the deficiency of GDP-Man:Man3GlcNAc2-PP-dolichol-alpha1,2-mannosyltransferase, encoded by the human ortholog of ALG11 from yeast. The patient presented with a multisystemic disorder characterized by muscular hypotonia, seizures, developmental retardation and death at the age of 2 years. The isoelectric focusing pattern of the patient's serum transferrin showed the partial loss of complete N-glycan side chains, which is a characteristic sign for CDG-I. Analysis of dolichol-linked oligosaccharides in patient-derived fibroblasts revealed an accumulation of Man3GlcNAc2-PP-dolichol and Man4GlcNAc2-PP-dolichol. Determination of mannosyltransferase activities of early steps of lipid-linked oligosaccharide biosynthesis in fibroblasts indicated that the patient was deficient in elongating Man3GlcNAc2-PP-dolichol. These findings gave rise to genetic analysis of the hALG11 cDNA, in which homozygosity for mutation c.T257C (p.L86S) was identified. Verification of the mutation as a primary cause for the genetic defect was proved by retroviral expression of human wild-type and mutated ALG11 cDNA in patient-derived fibroblasts as well as using a yeast alg11 deletion strain as a heterologous expression system for hALG11 variants. Immunofluorescence examinations combined with western blotting showed no differences of intracellular localization or expression of ALG11 between control and patient fibroblasts, respectively, indicating no mislocalization or degradation of the mutated transferase. << Less

  Hum. Mol. Genet. 19:1413-1424(2010) [PubMed] [EuropePMC]

• In vitro evidence for the dual function of Alg2 and Alg11: essential mannosyltransferases in N-linked glycoprotein biosynthesis.

  O'Reilly M.K., Zhang G., Imperiali B.

  The biosynthesis of asparagine-linked glycoproteins utilizes a dolichylpyrophosphate-linked glycosyl donor (Dol-PP-GlcNAc(2)Man(9)Glc(3)), which is assembled by the series of membrane-bound glycosyltransferases that comprise the dolichol pathway. This biosynthetic pathway is highly conserved throu ... >> More

  The biosynthesis of asparagine-linked glycoproteins utilizes a dolichylpyrophosphate-linked glycosyl donor (Dol-PP-GlcNAc(2)Man(9)Glc(3)), which is assembled by the series of membrane-bound glycosyltransferases that comprise the dolichol pathway. This biosynthetic pathway is highly conserved throughout eukaryotic evolution. While complementary genetic and bioinformatic approaches have enabled identification of most of the dolichol pathway enzymes in Saccharomyces cerevisiae, the roles of two of the mannosyltransferases in the pathway, Alg2 and Alg11, have remained ambiguous because these enzymes appear to catalyze only two of the remaining four unannotated transformations. To address this issue, a biochemical approach was taken using recombinant Alg2 and Alg11 from S. cerevisiae and defined dolichylpyrophosphate-linked substrates. A cell-membrane fraction isolated from Escherichia coli overexpressing thioredoxin-tagged Alg2 was used to demonstrate that this enzyme actually carries out an alpha1,3-mannosylation, followed by an alpha1,6-mannosylation, to form the first branched pentasaccharide intermediate of the pathway. Then, using thioredoxin-tagged Alg2 for the chemoenzymatic synthesis of the dolichylpyrophosphate pentasaccharide, it was thus possible to define the biochemical function of Alg11, which is to catalyze the next two sequential alpha1,2-mannosylations. The elucidation of the dual function of each of these enzymes thus completes the identification of the entire ensemble of glycosyltransferases that comprise the dolichol pathway. << Less

  Biochemistry 45:9593-9603(2006) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Biochemical characterization, membrane association and identification of amino acids essential for the function of Alg11 from Saccharomyces cerevisiae, an alpha1,2-mannosyltransferase catalysing two sequential glycosylation steps in the formation of the lipid-linked core oligosaccharide.

  Absmanner B., Schmeiser V., Kaempf M., Lehle L.

  The biosynthesis of asparagine-linked glycans occurs in an evolutionarily conserved manner with the assembly of the unique lipid-linked oligosaccharide precursor Glc3Man9GlcNAc2-PP-Dol at the ER (endoplasmic reticulum). In the present study we characterize Alg11 from yeast as a mannosyltransferase ... >> More

  The biosynthesis of asparagine-linked glycans occurs in an evolutionarily conserved manner with the assembly of the unique lipid-linked oligosaccharide precursor Glc3Man9GlcNAc2-PP-Dol at the ER (endoplasmic reticulum). In the present study we characterize Alg11 from yeast as a mannosyltransferase catalysing the sequential transfer of two alpha1,2-linked mannose residues from GDP-mannose to Man3GlcNAc2-PP-Dol and subsequently to Man4GlcNAc2-PP-Dol forming the Man5GlcNAc2-PP-Dol intermediate at the cytosolic side of the ER before flipping to the luminal side. Alg11 is predicted to contain three hydrophobic transmembrane-spanning helices. Using Alg11 topology reporter fusion constructs, we show that only the N-terminal domain fulfils this criterion. Surprisingly, this domain can be deleted without disturbing glycosyltransferase function and membrane association, indicating also that the other two hydrophobic domains contribute to ER localization, but in a non-transmembrane manner. By site-directed mutagenesis we investigated amino acids important for transferase activity. We demonstrate that the first glutamate residue in the EX7E motif, conserved in a variety of glycosyltransferases, is more critical than the second, and loss of Alg11 function occurs only when both glutamate residues are exchanged, or when the mutation of the first glutamate residue is combined with replacement of another amino acid in the motif. This indicates that perturbations in EX7E are not restricted to the second glutamate residue. Moreover, Gly85 and Gly87, within a glycine-rich domain as part of a potential flexible loop, were found to be required for Alg11 function. Similarly, a conserved lysine residue, Lys319, was identified as being important for activity, which could be involved in the binding of the phosphate of the glycosyl donor. << Less

  Biochem. J. 426:205-217(2010) [PubMed] [EuropePMC]