Laminin‐like immunoreactivity in the snail Helisoma: Involvement of a ∼300 kD extracellular matrix protein in promoting outgrowth from identified neurons

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Abstract
Polyclonal antibodies directed against laminin (LM), and against the A and B chains of reduced LM were used to identify antigenically related proteins in the extracellular matrix (ECM) of the snail Helisoma trivolvis Immunofluorescence of snail central ganglionic rings using either the anti-LM or anti-B chain antibodies labeled the ECM within ganglionic sheaths as well as basal laminae surrounding the ganglia. Both the anti-LM and anti-B chain antibodies recognized a prominent, ∼300-kD protein on immunoblots of a snail central ganglion preparation enriched in ECM components. The anti-A chain antibody failed to label any structures in sections of snail ganglia or to recognize any proteins on immunoblots of ganglionic ECM. A polyclonal antibody was raised against the ∼300-kD snail protein. Immunofluorescence of snail ganglia with the anti-∼300-kD antibody gave a distribution of labeled structures comparable to that obtained with the anti-LM antibody. Immunofluorescent labeling of sections of snail muscle and salivary gland with the anti-∼300-kD antibody revealed a distribution of reactive protein characteristic of an ECM component. Probing immunoblots of ganglionic ECM with the anti- ∼300-kD antibody revealed the recognition of the same ∼ 300-kD protein as identified by the anti-LM antibodies. Media conditioned by Helisoma central ganglionic rings (CM) contains an unidentified neurite outgrowth promoting factor (NOPF). Immunoblots of CM probed with the anti-B chain and anti- ∼300-kD antibodies reveal the recognition of a soluble ∼300-kD protein similar to the ∼300-kD protein identified in snail ECM. The ganglionic ECM preparation containing the ∼300-kD protein supported outgrowth from cultured snail buccal neurons B5, and addition of anti- ∼300-kD Fab fragments to CM abolished its outgrowth promoting activity. These results suggest that the ∼300-kD ECM protein may be the NOPF in CM and /or functions in promoting neurite outgrowth.