Analysis of the site-specific N-glycosylation of beta1,6 N-acetylglucosaminyltransferase V.
N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of a beta1,6-linked GlcNAc to the alpha1,6 mannose of the trimannosyl core to form tri- and tetraantennary N-glycans and contains six putative N-linked sites. We used mass spectrometry techniques combined with exoglycosidase digestions of recombinant human GnT-V expressed in CHO cells, to identify its N-glycan structures and their sites of expression. Release of N-glycans by PNGase F treatment, followed by analysis of the permethylated glycans using MALDI-TOF MS, indicated a range of complex glycans from bi- to tetraantennary species. Mapping of the glycosylation sites was performed by enriching for trypsin-digested glycopeptides, followed by analysis of each fraction with Q-TOF MS. Predicted tryptic glycopeptides were identified by comparisons of theoretical masses of peptides with various glycan masses to the masses of the glycopeptides determined experimentally. Of the three putative glycosylation sites in the catalytic region, peptides containing sites Asn 334, 433, and 447 were identified as being N-glycosylated. Asn 334 is glycosylated with only a biantennary structure with one or two terminating sialic acids. Sites Asn 433 and 447 both contain structures that range from biantennary with two sialic acids to tetraantennary terminating with four sialic acids. The predominant glycan species found on both of these sites is a triantennary with three sialic acids. The appearance of only biantennary glycans at site Asn 433, coupled with the appearance of more highly branched structures at Asn 334 and 447, demonstrates that biantennary acceptors present at different sites on the same protein during biosynthesis can differ in their accessibility for branching by GnT-V.
Kamar
Maria
M
Complex Carbohydrate Research Center, 315 Riverbend Road, Athens, GA 30602, USA.
Alvarez-Manilla
Gerardo
G
Abney
Trina
T
Azadi
Parastoo
P
Kumar Kolli
V S
VS
Orlando
Ron
R
Pierce
Michael
M
D000818:Animals; D001216:Asparagine / Q000737:chemistry*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle; D006224:Cricetinae; D006026:Glycoside Hydrolases / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry*; D019158:N-Acetylneuraminic Acid / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011134:Polysaccharides; D011994:Recombinant Proteins; D001216:Asparagine; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase; D006026:Glycoside Hydrolases; D019158:N-Acetylneuraminic Acid