Identification of a GM1-binding protein on the surface of murine neuroblastoma cells.
S20Y murine neuroblastoma cells appear to express a protein component(s) able to adhere specifically to the oligosaccharide portion of GM1 (oligo-GM1). To identify proteins with which the oligo-GM1 becomes closely associated, a radiolabeled (125I), photoactivatable derivative of oligo-GM1 was prepared. This was accomplished by reductive amination of the glucosyl moiety of oligo-GM1 to 1-deoxy-1-aminoglucitol, followed by reaction of the amine with sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD). Crosslinking studies using the photoactivatable probe indicated that it came in close proximity to a protein with an apparent molecular mass of approximately 71 kDa. In competition experiments, as little as a 10-fold molar excess of oligo-GM1 resulted in a selective reduction in labeling of this protein; preincubation with a 200-fold molar excess of siayllactose was necessary to observe the same change in the labeling pattern, lending additional support to the hypothesis that the approximately 71-kDa protein specifically associates with oligo-GM1. Cell surface location of the oligo-GM1 binding protein was confirmed using subcellular fractionation and morphological analyses.
Fueshko
S M
SM
Department of Biological Chemistry, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
Schengrund
C L
CL
D000818:Animals; D002458:Cell Fractionation; D002478:Cells, Cultured; D005347:Fibroblasts / Q000737:chemistry / Q000166:cytology / Q000648:ultrastructure; D005677:G(M1) Ganglioside / Q000378:metabolism*; D007457:Iodine Radioisotopes; D051379:Mice; D008807:Mice, Inbred BALB C; D009447:Neuroblastoma / Q000737:chemistry* / Q000473:pathology* / Q000648:ultrastructure; D009844:Oligosaccharides / Q000378:metabolism; D011485:Protein Binding; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000473:pathology / Q000648:ultrastructure
D007457:Iodine Radioisotopes; D009844:Oligosaccharides; D005677:G(M1) Ganglioside