Biosynthesis of Lipid-Linked Oligosaccharides in Yeast: the ALG3 Gene Encodes the Dol-P-Man:Man5GlcNAc2-PP-Dol Mannosyltransferase
-
Chandra B. Sharma
Abstract
The formation of Nglycosidic linkages of glycoproteins involves the ordered assembly of the common Glc[3]Man[9]GlcNAc[2] coreoligosaccharide on the lipid carrier dolichyl pyrophosphate. Whereas early mannosylation steps occur on the cytoplasmic side of the endoplasmic reticulum with GDPMan as donor, the final reactions from Man[5]GlcNAc[2]PPDol to Man[9]Glc Nac[2]PPDol on the lumenal side use DolPMan. We have investigated these later stages in vitro using a detergentsolubilized enzyme extract from yeast membranes. Mannosyltransfer from DolPMan to [[3]H]Man[5]GlcNAc[2]PPDol with formation of all intermediates up to Man[9]GlcNAc[2]PPDol occured in a rapid, time and proteindependent fashion. We find that the initial reaction from Man[5]GlcNAc[2]PPDol to Man[6]GlcNAc[2]PPDol is independent of metal ions, but further elongations need Mn[2+] that can be partly replaced by Mg[2+] or Ca[2+]. Zn[2+] or Cd[2+] ions were found to inhibit formation of Man[7-9]GlcNAc[2]PPDol, but do not affect synthesis of Man[6]GlcNAc[2]PPDol. Extension did not occur when the acceptor was added as a free Man[5]GlcNAc[2] oligosaccharide or when GDPMan was used as mannosyldonor. The alg3 mutant was described to accumulate Man[5]GlcNAc[2]PPDol. We expressed a functional active HAepitope tagged ALG3 fusion and succeeded to selectively immunoprecipitate the DolPMan:Man[5]GlcNAc[2]PPDol mannosyltransferase activity from the other enzymes of the detergent extract involved in the subsequent mannosylation reactions. This demonstrates that Alg3p represents the mannosyltransferase itself and not an accessory protein involved in the reaction.
Copyright © 2001 by Walter de Gruyter GmbH & Co. KG
Articles in the same Issue
- Highlight: Glycobiology
- O-Glycosylation of the Mucin Type
- Glycoproteins from Insect Cells: Sialylated or Not?
- Congenital Disorders of Glycosylation: Glycosylation Defects in Man and Biological Models for Their Study
- Mitochondrial Single-Stranded DNA-Binding Proteins: in Search for New Functions
- Do Rodent and Human Brains Have Different N-Glycosylation Patterns?
- The Liver Flukes Fasciola gigantica and Fasciola hepatica Express the Leucocyte Cluster of Differentiation Marker CD77 (Globotriaosylceramide) in Their Tegument
- Cloning and Expression of Drosophila melanogaster UDP-GlcNAc:?-3-D-Mannoside ? 1,2-N-Acetylglucosaminyltransferase I
- Pathways of Mucin O-Glycosylation in Normal and Malignant Rat Colonic Epithelial Cells Reveal a Mechanism for Cancer-Associated Sialyl-Tn Antigen Expression
- 6-O-Sulfo De-N-Acetylsialyl Lewis X as a Novel High-Affinity Ligand for Human L-Selectin: Total Synthesis and Structural Characterization
- Segregation of Gangliosides GM1 and GD3 on Cell Membranes, Isolated Membrane Rafts, and Defined Supported Lipid Monolayers
- Structural Characterization of Fucose-Containing Oligosaccharides by High-Performance Liquid Chromatography and Matrix-Assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometry
- Anencephaly: Structural Characterization of Gangliosides in Defined Brain Regions
- Acidic Glycerol Lipids of Trichomonas vaginalis and Tritrichomonas foetus
- Stimulation of Acid Sphingomyelinase Activity by Lysosomal Lipids and Sphingolipid Activator Proteins
- Biosynthesis of N-Acetylneuraminic Acid in Cells Lacking UDP-N-Acetylglucosamine 2-Epimerase/ N-Acetylmannosamine Kinase
- Synthesis of Nucleotide-Activated Oligosaccharides by ?-Galactosidase from Bacillus circulans
- Elucidation of the Role of Functional Amino Acid Residues of the Small Sialidase from Clostridium perfringens by Site-Directed Mutagenesis
- Biosynthesis of Lipid-Linked Oligosaccharides in Yeast: the ALG3 Gene Encodes the Dol-P-Man:Man5GlcNAc2-PP-Dol Mannosyltransferase
- Intra- and Intermolecular Triplex DNA Formation in the Murine c-myb Proto-Oncogene Promoter Are Inhibited by Mithramycin
- Experimental Regulation of STAT Gene Expression Reveals an Involvement of STAT5 in Interleukin-4-Driven Cell Proliferation
Articles in the same Issue
- Highlight: Glycobiology
- O-Glycosylation of the Mucin Type
- Glycoproteins from Insect Cells: Sialylated or Not?
- Congenital Disorders of Glycosylation: Glycosylation Defects in Man and Biological Models for Their Study
- Mitochondrial Single-Stranded DNA-Binding Proteins: in Search for New Functions
- Do Rodent and Human Brains Have Different N-Glycosylation Patterns?
- The Liver Flukes Fasciola gigantica and Fasciola hepatica Express the Leucocyte Cluster of Differentiation Marker CD77 (Globotriaosylceramide) in Their Tegument
- Cloning and Expression of Drosophila melanogaster UDP-GlcNAc:?-3-D-Mannoside ? 1,2-N-Acetylglucosaminyltransferase I
- Pathways of Mucin O-Glycosylation in Normal and Malignant Rat Colonic Epithelial Cells Reveal a Mechanism for Cancer-Associated Sialyl-Tn Antigen Expression
- 6-O-Sulfo De-N-Acetylsialyl Lewis X as a Novel High-Affinity Ligand for Human L-Selectin: Total Synthesis and Structural Characterization
- Segregation of Gangliosides GM1 and GD3 on Cell Membranes, Isolated Membrane Rafts, and Defined Supported Lipid Monolayers
- Structural Characterization of Fucose-Containing Oligosaccharides by High-Performance Liquid Chromatography and Matrix-Assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometry
- Anencephaly: Structural Characterization of Gangliosides in Defined Brain Regions
- Acidic Glycerol Lipids of Trichomonas vaginalis and Tritrichomonas foetus
- Stimulation of Acid Sphingomyelinase Activity by Lysosomal Lipids and Sphingolipid Activator Proteins
- Biosynthesis of N-Acetylneuraminic Acid in Cells Lacking UDP-N-Acetylglucosamine 2-Epimerase/ N-Acetylmannosamine Kinase
- Synthesis of Nucleotide-Activated Oligosaccharides by ?-Galactosidase from Bacillus circulans
- Elucidation of the Role of Functional Amino Acid Residues of the Small Sialidase from Clostridium perfringens by Site-Directed Mutagenesis
- Biosynthesis of Lipid-Linked Oligosaccharides in Yeast: the ALG3 Gene Encodes the Dol-P-Man:Man5GlcNAc2-PP-Dol Mannosyltransferase
- Intra- and Intermolecular Triplex DNA Formation in the Murine c-myb Proto-Oncogene Promoter Are Inhibited by Mithramycin
- Experimental Regulation of STAT Gene Expression Reveals an Involvement of STAT5 in Interleukin-4-Driven Cell Proliferation
