Modulation of human saposin B sphingolipid-binding specificity by alternative splicing. A study with saposin B-derived synthetic peptides.

The saposins A, B, C, and D, produced by proteolytic maturation of the same precursor protein, prosaposin, are sphingolipid-binding proteins which function as activators for lysosomal enzymes involved in sphingolipid hydrolysis. The alternative splicing of the prosaposin gene results in the inclusion or exclusion of exon 8 into transcribed prosaposin mRNA through the use of alternative acceptor sites. The relative abundance of each alternatively spliced mRNA was determined by reverse transcription-polymerase chain reaction in various human tissues and cell lines. Exon 8 codes for only three amino acid residues, Gln-Asp-Gln, in the saposin B domain of prosaposin. The prosaposin mRNA containing exon 8 is the major species in cultured skin fibroblasts, brain, and pituitary glands together with a smaller amount of mRNA devoid of exon 8, whereas the prosaposin mRNA detected in liver and lymphoblasts was devoid of exon 8 insertion. Previous structural modeling studies on saposin B have suggested that the Gln-Asp-Gln insertion occurs in an amphipathic alpha-helix region of the protein which is implicated in the binding of GM1-ganglioside. We report that synthetic peptides containing the alpha-helix, with and without the Gln-Asp-Gln insertion, have different binding affinities for GM1-ganglioside, sulfatide, and sphingomyelin. The insertion of the Gln-Asp-Gln sequence completely abolishes the capacity of the peptide to bind GM1-ganglioside, whereas its affinity for sulfatide and sphingomyelin is increased about 4-fold and almost 2-fold, respectively. No significant binding of glucosylceramide was observed with both peptides. These results suggest that alternative splicing of prosaposin mRNA may change binding specificity of saposin B presumably to adapt to the variable sphingolipid composition of tissues.