Colocalization and Complex Formation Between Prosaposin and Monosialoganglioside GM3 in Neural Cells

Abstract

Prosaposin, the precursor of saposins A, B, C, and D, was recently identified as a neurotrophic factor in vitro as well as in vivo. Its neurotrophic activity has been localized to a linear 12‐amino acid sequence located in the NH2‐terminal portion of the saposin C domain. In this study, we show the colocalization of prosaposin and ganglioside GM3 on NS20Y cell plasma membrane by scanning confocal microscopy. Also, TLC and western blot analyses showed that GM3 was specifically associated with prosaposin in immunoprecipitates; this binding was Ca2+‐independent and not disassociated during sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The association of prosaposin‐GM3 complexes on the cell surface appeared to be functionally important, as determined by differentiation assays. Neurite sprouting, induced by GM3, was inhibited by antibodies raised against a 22‐mer peptide, prosaptide 769, containing the neurotrophic sequence of prosaposin. In addition, pertussis toxin inhibited prosaptide‐induced neurite outgrowth, as well as prosaptide‐enhanced ganglioside concentrations in NS20Y cells, suggesting that prosaposin acted via a G protein‐mediated pathway, affecting both ganglioside content and neuronal differentiation. Our findings revealed a direct and right GM3‐prosaposin association on NS20Y plasma membranes. We suggest that ganglioside‐protein complexes are structural components of the prosaposin receptor involved in cell differentiation.

Shedding and uptake of gangliosides and glycosylphosphatidylinositol-anchored proteins

“…In a similar way, saposin C itself was shown to prevent apoptosis in prostate cancer cells [128]. These effects are consistent with the observations that prosaposin addition rescues neuroblastoma cells, primary hippocampal neurons [129], Schwann cells [125], and PC12 pheochromocytoma cells [119] from apoptosis induced by various agens. Neurotrophic, neuroregenerative and anti-apoptotic effects of prosaposin are apparently mediated by modification of signaling pathways and prosaposin was shown to be involved in ERK phosphorylation [130].…”

“…It has been suggested that its association with the cell membrane proceeds through the interaction with membrane gangliosides [118,119]. Prosaposin can also be found in many biological fluids such as seminal plasma, human milk, and cerebrospinal fluid (reviewed in [120]).…”

2. Involvement of ganglioside GM3 in G_2/M cell cycle arrest of human monocytic cells induced by Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin

“…Recently, it has been reported that the HA1, HA3, and HA3b subcomponents of hemagglutinin, which is a component of the Clostridium botulinum progenitor toxin, use GM3 as a receptor (8,14). Misasi et al suggested that GM3-protein complexes are structural components of the prosaposin receptor involved in cell differentiation (25). We used flow cytometry with a monoclonal antibody to confirm that GM3 was expressed in U937 cells (data not shown).…”

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

“…3A, Western blot analysis of testis revealed several prosaposin isoforms: one of 65 kDa, two isoforms higher than 70 kDa, and one of 98 kDa in size. The different forms may correspond to different glycosylation states of prosaposin or to a prosaposin-lipid complex (10,21,39). Lower prosaposin levels were obvious in the testis of the Pro ⌬8neo homozygotes, while the Pro ⌬8 homozygotes had levels comparable to that of wild-type mice.…”