Colocalization and complex formation between prosaposin and monosialoganglioside GM3 in neural cells.
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 tight GM3-prosaposin association on NS20Y plasma membranes. We suggest that ganglioside-protein complexes are structural components of the prosaposin receptor involved in cell differentiation.
Misasi
R
R
Dipartimento di Medicina Sperimentale e Patologia, Università La Sapienza, Roma, Italy.
Sorice
M
M
Garofalo
T
T
Griggi
T
T
Campana
W M
WM
Giammatteo
M
M
Pavan
A
A
Hiraiwa
M
M
Pontieri
G M
GM
O'Brien
J S
JS
D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D005679:G(M3) Ganglioside / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D018613:Microscopy, Confocal; D016501:Neurites / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D037342:Pertussis Toxin; D011233:Precipitin Tests; D049231:Saposins; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D005679:G(M3) Ganglioside; D006023:Glycoproteins; C483344:Psap protein, mouse; D049231:Saposins; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin