title: Oligosaccharide heterogeneity of insulin receptors. Comparison of N-linked glycosylation of insulin receptors in adipocytes and brain.
abstract: We tested the hypothesis that the molecular weight discrepancy between insulin receptors in brain and adipocytes is due to differences in glycosylation by treating photoaffinity-labeled insulin receptors from both tissues with endo-beta-N-acetylglucosaminidase F (Endo F) and analyzing the products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Endo F removed glycans from the adipocyte 125-kilodalton (kDa) subunit and the brain 115-kDa subunit in a manner dependent upon the concentration of enzyme and time of incubation. At a maximally effective concentration of Endo F, the adipocyte alpha-subunit was reduced from 125-kDa to 100-kDa and the brain alpha-subunit from 115-kDa to 100-kDa. We also examined the type of oligosaccharides present in both alpha-subunits by treating the proteins with endo-beta-N-acetylglucosaminidase H (Endo H), which selectively removes high mannose residues, and neuraminidase. Endo H treatment reduced the apparent molecular weight of both the adipocyte and brain alpha-subunits. In both receptors, the deglycosylated product obtained with Endo H was larger than that generated by Endo F. The adipocyte alpha-subunit demonstrated a shift in mobility on sodium dodecyl sulfate gels after neuraminidase treatment, whereas the brain alpha-subunit did not. We conclude from these studies that 1) The discrepancy in apparent molecular weight of alpha-subunits in brain and adipocytes is due to differences in N-linked glycosylation; 2) high mannose and complex type oligosaccharides are present in both receptor types; and 3) the complex oligosaccharides in the adipocyte alpha-subunit are terminated in a manner different from the complex glycans of the brain alpha-subunit.
mesh_terms: D000118:Acetylglucosaminidase / Q000378:metabolism; D000273:Adipose Tissue / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism; D001921:Brain / Q000378:metabolism*; D002236:Carbohydrate Conformation; D007328:Insulin / Q000031:analogs & derivatives / Q000378:metabolism; D008297:Male; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008970:Molecular Weight; D009439:Neuraminidase / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism*; D010777:Photochemistry; D011499:Protein Processing, Post-Translational; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; D001386:Azides; D007328:Insulin; D009844:Oligosaccharides; C035459:insulin, (2-nitro-4-azidophenylacetyl)(B2)-des-Phe(B1)-; D011972:Receptor, Insulin; D009439:Neuraminidase; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase