Biosynthesis of HNK-1 glycans on O-linked oligosaccharides attached to the neural cell adhesion molecule (NCAM): the requirement for core 2 beta 1,6-N-acetylglucosaminyltransferase and the muscle-specific domain in NCAM.
The HNK-1 glycan, sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->R, is highly expressed in neuronal cells and apparently plays critical roles in neuronal cell migration and axonal extension. The HNK-1 glycan synthesis is initiated by the addition of beta1,3-linked GlcA to N-acetyllactosamine followed by sulfation of the C-3 position of GlcA. The cDNAs encoding beta1,3-glucuronyltransferase (GlcAT-P) and HNK-1 sulfotransferase (HNK-1ST) have been recently cloned. Among various adhesion molecules, the neural cell adhesion molecule (NCAM) was shown to contain HNK-1 glycan on N-glycans. In the present study, we first demonstrated that NCAM also bears HNK-1 glycan attached to O-glycans when NCAM contains the O-glycan attachment scaffold, muscle-specific domain, and is synthesized in the presence of core 2 beta1,6-N-acetylglucosaminyltransferase, GlcAT-P, and HNK-1ST. Structural analysis of the HNK-1 glycan revealed that the HNK-1 glycan is attached on core 2 branched O-glycans, sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->3)GalNAc. Using synthetic oligosaccharides as acceptors, we found that GlcAT-P and HNK-1ST almost equally act on oligosaccharides, mimicking N- and O-glycans. By contrast, HNK-1 glycan was much more efficiently added to N-glycans than O-glycans when NCAM was used as an acceptor. These results are consistent with our results showing that HNK-1 glycan is minimally attached to O-glycans of NCAM in fetal brain, heart, and the myoblast cell line, C2C12. These results combined together indicate that HNK-1 glycan can be synthesized on core 2 branched O-glycans but that the HNK-1 glycan is preferentially added on N-glycans over O-glycans of NCAM, probably because N-glycans are extended further than O-glycans attached to NCAM containing the muscle-specific domain.
Ong
Edgar
E
Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, California 92037, USA.
Suzuki
Misa
M
Belot
Frederic
F
Yeh
Jiunn-Chern
JC
Franceschini
Isabelle
I
Angata
Kiyohiko
K
Hindsgaul
Ole
O
Fukuda
Minoru
M
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D001923:Brain Chemistry; D018959:CD57 Antigens / Q000096:biosynthesis*; D002240:Carbohydrate Sequence; D002852:Chromatography, Ion Exchange; D006321:Heart / Q000196:embryology; D051379:Mice; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009077:Mucins / Q000378:metabolism; D009132:Muscles / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D019006:Neural Cell Adhesion Molecules / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*
D018959:CD57 Antigens; D009077:Mucins; D019006:Neural Cell Adhesion Molecules; D009844:Oligosaccharides; D017351:N-Acetylglucosaminyltransferases