Oligosaccharide heterogeneity of insulin receptors. Comparison of N-linked glycosylation of insulin receptors in adipocytes and brain.
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.
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*
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