Cell surface membranes of motorneurons are enrich glycoconjugates, including gangliosides. Although the physiological functions of these acidic glycosphingolipids are not understood, they have been implicated in cell-cell recognition, regulation of axonal sprouting and maintenance of the neuromuscular junction. They are expressed at some risk to motorneurons, since certain neurotropic viruses and bacterial exotoxins bind specifically to gangliosides to gain entry into these cells. Gangliosides have also been proposed as key antigens in pathologies involving antibody-mediated attack of motorneurons. We reasoned that skeletal muscles may contain endogenous ganglioside-specific binding proteins which serve essential roles in the physiology of the neuromuscular junction. Muscle-derived ganglioside binding proteins released at the nerve terminal may undergo retrograde or transsynaptic retrograde transport within motorneurons comparable to the transport documented for ganglioside-binding pathogenic agents such as tetanus toxin. A search for endogenous ganglioside binding activities from rat skeletal muscle was initiated using povel radioligands consisting of gangliosides covalently linked to a protein carrier--neoganglioproteins. Our preliminary data support the presence of one or more ganglioside- specific binding proteins in rat skeletal muscle cytosol and on muscle cell surface membranes. Radioiodinated neoganglioproteins will be used to further define these ganglioside-specific binding activities with respect to their distribution, binding kinetics and carbohydrate specificity. We will employ chromatographic techniques (with emphasis on affinity chromatography) to purify the ganglioside-specific binding activities from muscle, with the goal of obtaining enough structural information to allow complete molecular characterization via recombinant DNA techniques. Monospecific antisera will be raised against the purified binding protein(s) or specific synthetic polypeptides derived from their induced structure(s). Our ultimate goal is to generate probes to investigate the anatomic, ultrastructural, ontogenetic, and pathological patterns of expression and transport of muscle-derived ganglioside binding proteins. The results promise to reflect on the physiological role of gangliosides and their endogenous binding proteins in neuromuscular junction.
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