Identification of O-Linked N-Acetylglucosamine Modification of AnkyrinG Isoforms Targeted to Nodes of Ranvier

doi:10.1074/jbc.271.49.31391

Identification of O-Linked N-Acetylglucosamine Modification of Ankyrin_G Isoforms Targeted to Nodes of Ranvier*

Xu Zhang^‡ and
Vann Bennett

From the Departments of Cell Biology and Biochemistry and the Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710

^‡ To whom correspondence should be addressed. Tel.: 919-684-3538; Fax: 919-684-3590.

Abstract

Ankyrin_Gs of 270 and 480 kDa are localized at nodes of Ranvier and are candidates to couple the voltage-dependent sodium channel and neurofascin to the spectrin/actin network. This study presents evidence that these ankyrins contain O-linked GlcNAc residues and identifies as the site of glycosylation a serine-rich domain that distinguishes them from other ankyrin isoforms. The 480-kDa ankyrin_G, extracted from brain membranes associated with wheat germ agglutinin-affinity columns, was [³H]galactose-labeled with UDP-[³H] galactose and galactosyltransferase, and cross-reacted with an antibody against O-GlcNAc monosaccharides. Ankyrin_G-associated sugars are O-linked monosaccharides based on resistance to peptide-N-glycosidase F and analysis of saccharides released by β-elimination. The serine-rich domain is the site of glycosylation based on wheat germ agglutinin binding activity of polypeptides produced by in vitro translation in reticulocyte lysates. Immunofluorescence revealed co-localization of ankyrin_G and O-GlcNAc immunoreactivity at nodes of Ranvier. These observations suggest that ankyrin at the node of Ranvier is O-GlcNAc-glycosylated and are the first demonstration of a post-translational modification that is concentrated at the node of Ranvier and not in adjacent areas of myelinated axons.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PAGE

polyacrylamide gel electrophoresis

WGA

wheat germ agglutinin

HA

hemagglutinin

GFP

green fluorescent protein

PNGase F

peptide-N-glycosidase F

PVDF

polyvinylidene difluoride.
↵² X. Zhang and V. Bennett, unpublished data.
- Received June 26, 1996.
- Revision received September 4, 1996.