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Glycobiology Advance Access published online on April 14, 2004
Glycobiology, doi:10.1093/glycob/cwh062
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Received October 4, 2003
Revised February 13, 2004
Accepted February 13, 2004

ORIGINAL ARTICLES

Analysis of the site-specific N-glycosylation of {beta}1,6 N-acetylglucosaminyltransferase V

Maria Kamar 1, Gerardo Alvarez-Manilla 2, Trina Abney 1, Parastoo Azadi 1, V. S. Kumar Kolli 1, Ron Orlando 1, Michael Pierce 1*

1 Complex Carbohydrate Research Center, Athens, Georgia 30602
2 Complex Carbohydrate Research Center, Athens, Georgia 30602; Centro de Investigacion en Alimentacion y Desarrollo Ac. Km 0.6. carr a La Victoria, Hermosillo, Sonora, Mexico, 83000

* To whom correspondence should be addressed. E-mail: hawkeye{at}uga.edu.


  Abstract

N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of a {beta}1,6-linked GlcNAc to the {alpha}1,6 mannose of the trimannosyl core to form tri- and tetra-antennary 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 biantennary 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.

Key words: glycoforms, MALDI MS, N-acetylglucosaminyltransferase V, N-glycosylation, LC-MS


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