Novel carboxylated N-glycans contain oligosaccharide-linked glutamic acid.
We previously reported that N-glycans from bovine lung contain novel carboxylate groups. Here, we provide evidence that the carboxylated glycans contain glutamic acid. We labeled HeLa cells with [2,3-(3)H]glutamate and used a carboxylate-specific monoclonal antibody to enrich for the desired proteins. PNGaseF digestion of these proteins released labeled N-glycans with a free amino group and 1-3 carboxylates. Mild acid hydrolysis had no effect, but strong acid hydrolysis of the glycans released >80% of the (3)H as glutamate. Reducing the carboxylates to alcohols prior to hydrolysis eliminated the [(3)H]glutamate and generated [(3)H]4-amino 5-hydroxy pentanoic acid, suggesting that [(3)H]glutamate was linked to the glycan through its gamma-carboxyl. The glutamate-containing N-glycans resisted exoglycosidase digestion and oligosaccharide processing inhibitors greatly reduced [(3)H]glutamate incorporation. These results demonstrate that mammalian cells synthesize complex-type N-glycans with glutamate linked to their antennae, further expanding their potential for covalent or ionic interactions.
Srikrishna
Geetha
G
Glycobiology Program, The Burnham Institute, La Jolla, CA, USA.
Brive
Lena
L
Freeze
Hudson H
HH
D000143:Acids / Q000494:pharmacology; D000596:Amino Acids / Q000494:pharmacology; D002264:Carboxylic Acids / Q000378:metabolism; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003208:Concanavalin A / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D018698:Glutamic Acid / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D006847:Hydrocarbons, Iodinated / Q000737:chemistry; D006868:Hydrolysis; D047468:Immunoprecipitation; D007477:Ions; D009844:Oligosaccharides / Q000737:chemistry*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000494:pharmacology; D011134:Polysaccharides / Q000737:chemistry*; D014974:Xanthomonas / Q000378:metabolism
D000143:Acids; D000596:Amino Acids; D002264:Carboxylic Acids; D006847:Hydrocarbons, Iodinated; D007477:Ions; D009844:Oligosaccharides; D011134:Polysaccharides; D003208:Concanavalin A; D018698:Glutamic Acid; C004387:2-amino-5-hydroxyvaleric acid; C014055:methyl iodide; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase