Identification of protein-bound oligosaccharides on the surface of growth cones that bind to muscle cells

In the accompanying paper (Gabel, Den, and Ambron, in press) it was shown that eight populations of glycopeptides are synthesized by single neurons of Aplysia californica. To see which glycopeptides might mediate interactions with target cells, we first identified glycopeptides that are transported selectively to synapses and growth cones. The giant neuron R2 was injected intrasomatically with 3H-glucosamine. Twenty-four hours later, 3H-glycopeptides in the axon and cell body were isolated and resolved by serial lectin affinity chromatography. Of the eight populations, the biantennary-type glycopeptides (GPbi) and those that bind to WGA (GPwga) were preferentially associated with rapidly transported glycoproteins. In contrast, the glycopeptide that consists of N-acetylglucosamine O-linked to ser/thr was mostly retained in the cell body. GPbi and GPwga were also preferentially transported to growth cones. Analyses of RUQ cells, exposed to 3H-glucosamine in vitro for 36 h showed an enrichment of GPbi and GPwga at the growth cone relative to the cell body. The disposition of the various glycopeptides in growing neurons was also examined using FITC lectins. FITC-coupled WGA, Vicia vellosa, and lentil lectin showed extensive staining of the cell body, but only WGA stained the growth cones. To investigate if GPwga interacts specifically with target cells, these glycopeptides were isolated from the neurons of 180 abdominal ganglia. GPwga, other Aplysia glycopeptides, and glycopeptides prepared from ovalbumin were coupled separately to fluorescent spheres. The spheres were then added to muscle cells isolated from the auricle of the heart, which is innervated by many neurons from the ganglion. While spheres coupled to GPwga bound to the muscle cell surface, the other glycopeptides did not. These results indicate that glycopeptides class GPwga, found among rapidly transported glycoproteins and on the growth cone surface, is able to bind to muscle cells and may therefore play some role in neuron-target interactions.