A synapse-specific carbohydrate at the neuromuscular junction: association with both acetylcholinesterase and a glycolipid

Abstract

With the aim of investigating the roles of carbohydrates in synapse formation, we have characterized a synapse-specific saccharide at the vertebrate neuromuscular junction. Two lectins of similar specificity (Dolichos biflorus agglutinin, DBA, and Vicia villosa-B4 agglutinin, VVA-B4) stain synaptic but not extrasynaptic regions of the rat muscle fiber surface and thus define a synapse-specific carbohydrate. Using these and other probes, we show that the carbohydrate moiety concentrated at the neuromuscular junction resembles N-acetylgalactosamine (GalNAc) linked in the beta-anomeric form to the termini of oligosaccharides. VVA-B4 also selectively stains neuromuscular junctions in human, mouse, rabbit, guinea pig, chick, frog, axolotl, snake, fish, and lamprey muscles, a phylogenetic conservatism that suggests a synapse-related role for GalNAc beta-terminal saccharides. AChE from muscle binds to DBA- and VVA-B4-agarose, and is thereby identified as a glycoconjugate bearing the synapse-specific carbohydrate. Assay of AChE isoforms reveals that asymmetric, collagen-tailed forms, known to be highly concentrated at the rat neuromuscular junction, bind DBA and VVA-B4, while globular forms, which are more widely distributed, do not. A second class of GalNAc-bearing glycoconjugates is demonstrable immunohistochemically with monoclonal antibodies to stage-specific embryonic antigen (SSEA)-3 (Shevinsky et al., 1982) and GM2 (Natoli et al., 1986), which recognize GalNAc-bearing glycolipids. These antibodies selectively stain neuromuscular junctions, where they recognize glycolipid-like molecules that bind VVA-B4 but are distinguishable from AChE. The association of a synapse-specific carbohydrate with at least 2 different synapse-specific molecules raises the possibility that the former plays a role in determining a property that the latter share, such as concentration at the synapse.

Characterization of a monoclonal antibody directed against a sulphoglycolipid that is evolutionarily conserved and developmentally regulated in rat brain

“…This procedure is known to extract lipids and has been used by various authors as a diagnostic test for their presence (e.g., Richardson et al 1982 andScott et al 1988). Chloroform/methanol pretreatment resulted in an overall reduction of the intensity of the staining, but no qualitative differences were observed (results not shown).…”

Postnatal overexpression of the CT GalNAc transferase inhibits muscular dystrophy in mdx mice without altering muscle growth or neuromuscular development: Evidence for a utrophin-independent mechanism

“…In some experiments, CT carbohydrate was visualized with Dolichos biflorus agglutinin (DBA) (Fig 2A). DBA is a GalNAc-binding lectin that can recognize the terminal βGalNAc linkage synthesized by the CT carbohydrate [26,27]. Because DBA normally only stains the neuromuscular junction [28], its e xtrasynaptic expression was easily visualized in infected cells.…”

Developmental regulation of membrane traffic organization during synaptogenesis in mouse diaphragm muscle.

“…The notion of a biochemical differentiation within the subneural GA was already emphasized by synapse-specific glycosylations (Scott et al, 1988;Iglesias et al, 1992) as well as synapse-specific localization of N-acetylgalactosaminyl transferase in skeletal muscle (Scott et al, 1989).…”