The Exon 8-Containing Prosaposin Gene Splice Variant Is Dispensable for Mouse Development, Lysosomal Function, and Secretion

Abstract

Prosaposin is a multifunctional protein with diverse functions. Intracellularly, prosaposin is a precursor of four sphingolipid activator proteins, saposins A to D, which are required for hydrolysis of sphingolipids by several lysosomal exohydrolases. Secreted prosaposin has been implicated as a neurotrophic, myelinotrophic, and myotrophic factor as well as a spermatogenic factor. It has also been implicated in fertilization. The human and the mouse prosaposin gene has a 9-bp exon (exon 8) that is alternatively spliced, resulting in an isoform with three extra amino acids, Gln-Asp-Gln, within the saposin B domain. Alternative splicing in the prosaposin gene is conserved from fish to humans, tissue specific, and regulated in the brain during development and nerve regeneration-degeneration processes. To elucidate the physiological role of alternative splicing, we have generated a mouse lacking exon 8 by homologous recombination. The exon 8 prosaposin mutant mice are healthy and fertile with no obvious phenotype. No changes were detected in prosaposin secretion or in accumulation and metabolism of gangliosides, sulfatides, neutral glycosphingolipids, neutral phospholipids, other neutral lipids, and ceramide. These data strongly indicate that the prosaposin variant containing the exon 8-encoded three amino acids is dispensable for normal mouse development and fertility as well as for prosaposin secretion and its lysosomal function, at least in the presence of the prosaposin variant missing the exon 8-encoded three amino acids.

ACKNOWLEDGMENTS

We thank Hassida Orenstein for preparing the slides for immunohistochemical analysis and Tslil Ofir for electrophoresis of the sequencing gels and their quantitative analysis. We thank Jakov Tal (Ben-Gurion University of the Negev, Beer Sheva, Israel) for TM3 and TM4 cells, David Yaffe (The Weizmann Institute of Science, Rehovot, Israel) for the C2 cells, and Daniel Michaelson (Tel Aviv University, Israel) for the N2a cells.

This project was partially supported by grants from The Binational, Israel-USA, Science Foundation (to M.H. and A.L.J.) and Genzyme Inc., USA (to M.H.).