Post-translational incorporation of [35S]sulfate into oligosaccharide side chains of pro-opiomelanocortin in rat intermediate lobe cells.
Pro-opiomelanocortin (POMC), the common precursor to beta-endorphin and alpha-melanocyte-stimulating hormone in rat neurointermediate lobe cells, exhibits both charge and size heterogeneity on two-dimensional gel electrophoretograms. Short term [3H]phenylalanine pulse-labeling, and pulse-chase studies, revealed that this heterogeneity is acquired either co-translationally, through the addition of mannose-rich oligosaccharide chains to the nascent protein, or post-translationally, probably during the period of oligosaccharide processing from the high mannose to the complex forms. In this process, radioactive sulfate is incorporated into different glycoprotein variants of POMC. In the presence of tunicamycin, an inhibitor of the N-glycosylation process, [35S]sulfate incorporation does not occur in any of the major variant forms of POMC, thereby preventing the appearance of the most acidic forms on two-dimensional gels. POMC tryptic fragments were separated by high-pressure liquid chromatography. Sulfate incorporation occurred in only two peptides that were also labeled with [3H]glucosamine. Extensive alkaline digestion of these peptides in the presence of sodium borohydride released the sulfate-containing moieties which were separated from free amino acids by gel filtration. Sulfate bearing moieties could also be released by almond emulsin peptide:N-glycosidase digestion. All these results unambiguously show that sulfate moieties preferentially enter asparagine-linked carbohydrate side chains and not amino acid residues of the POMC polypeptide. It is also likely that differential sulfation, conferring unequal amounts of negative charge upon various glycoprotein variants of POMC, is responsible for much of the charge heterogeneity displayed by the prohormone.
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004590:Electrophoresis, Paper; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D009844:Oligosaccharides / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D010904:Pituitary Gland, Posterior / Q000378:metabolism*; D011333:Pro-Opiomelanocortin / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D013431:Sulfates / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
D009844:Oligosaccharides; D010446:Peptide Fragments; D013431:Sulfates; D014415:Tunicamycin; D011333:Pro-Opiomelanocortin; D014357:Trypsin