Characterization and tissue distribution of opioid-binding cell adhesion molecule (OBCAM) using monoclonal antibodies.
Monoclonal antibodies to opioid-binding cell adhesion molecule (OBCAM) were produced against a synthetic OBCAM peptide. Immunoblotting analysis revealed that the antibodies reacted with 58 and/or 51 kDa proteins in P2 membranes from bovine, rat, mouse, guinea pig and rabbit brains. In bovine brain, the 58 and 51 kDa proteins were present in the striatum and cerebral cortex at high levels, but not in the pituitary. OBCAM was also detected in the cerebellum mainly in the 51 kDa form. In other tissues, the proteins were found in the spleen at very low levels, but not at all in the liver or kidney of the rat. OBCAM was effectively solubilized from bovine P2 membranes by bacterial phosphatidylinositol specific-phospholipase C (PI-PLC), indicating that OBCAM is a glycosylphosphatidylinositol (GPI)-anchored protein. PI-PLC treatment, however, had little effect on the opioid binding activity of the residual P2 membranes. The molecular weight of the proteins (58 and 51 kDa) was reduced to 36 kDa following treatment with N-glycanase but not further reduced after subsequent treatment with neuraminidase and O-glycanase, suggesting that OBCAM has N-glycosylated carbohydrate chains and that its two isoforms are different, at least, in the degree of N-glycosylation. Taken together, these results suggest that OBCAM consists of 58/51 kDa GPI-anchored glycoproteins which are highly N-glycosylated and are expressed mainly in the nervous system.
Hachisuka
A
A
Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, Tokyo, Japan.
Yamazaki
T
T
Sawada
J
J
Terao
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols / Q000032:analysis*; D006168:Guinea Pigs; D006596:Hexosaminidases; D015151:Immunoblotting; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000032:analysis; D009439:Neuraminidase; D009928:Organ Specificity; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases; D010902:Pituitary Gland / Q000737:chemistry; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011957:Receptors, Opioid / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols; D009419:Nerve Tissue Proteins; C489214:Opcml protein, mouse; C489215:Opcml protein, rat; D010446:Peptide Fragments; D011957:Receptors, Opioid; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D006596:Hexosaminidases; D009439:Neuraminidase; C060638:glycopeptide alpha-N-acetylgalactosaminidase; D043265:Phosphatidylinositol Diacylglycerol-Lyase