Conformational and amino acid residue requirements for the saposin C neuritogenic effect

Prosaposin is the precursor of four activator proteins, termed saposins A, B, C, and D, that are required for much of glycosphingolipid hydrolysis. Th e intact precursor also has neurite outgrowth activity ex vivo and in vivo that is localized to amino acid residues 22-31 of saposin C. Across species , this saposin C region has a high degree of identity and similarity with a mino acids in the analogous region of saposin A. Wild-type and mutant sapos ins C and A from human and mouse were expressed in E. coli. Pure proteins, synthetic peptide analogues, conformation-specific antibodies, and CD spect roscopy were used to evaluate the basis of the ex vivo neuritogenic effect. Wild-type saposin A had no neuritopenic activity whereas reduced and alkyl ated saposin A did. Introduction of the conserved saposin A Tyr 30 (Y30) in to saposin C at the analogous position 31, a conserved Ala(A)/Gly(G)31, dim inished neuritogenic activity by 50-60%. Nondenatured saposin A with an int roduced A30 acquired substantial neuritogenic activity. Polyclonal antibodi es directed against the NH2-terminus of saposin C cross-reacted well with r educed and alkylated saposins C and A, wild-type saposin C, and saposin A [ Y30A], poorly with saposin C [A31Y], and not at all with wild-type saposin A. CD spectra of wild-type and mutant saposins C and A, the corresponding n euritogenic region of saposin C, and the analogous region of saposin A show ed that more "saposin C-like" molecules had neuritogenic properties. Those with more "saposin A-like" spectra did not. These studies show that the neu ritogenic activity of saposin C requires specific placement of amino acids, and that Y30 of saposin A significantly alters local conformation in this critical region and suppresses neuritogenic activity.