N-linked glycosylation of a proenkephalin A-derived peptide. Evidence for the glycosylation of an NH2-terminally extended Met-enkephalin Arg6-Gly7-Leu8 variant.
To investigate the possibility that the opioid peptide precursor proenkephalin A was glycosylated, we utilized an antiserum raised against the COOH terminus of Met-enkephalin Arg6-Gly7-Leu8 (MERGL) to identify and characterize enkephalin-containing peptides from extracts of bovine adrenal medulla. Sephadex G-50 gel filtration separated two immunoreactive peaks which had apparent masses of 9 and 6 kDa. Anion-exchange chromatography and reverse-phase high pressure liquid chromatography (HPLC) revealed that the 9-kDa material was a heterogenous mixture of immunoreactive peptides, of which one (9K-MERGL Ia) was purified to homogeneity. The 6-kDa material separated into two major immunoreactive peaks (6K-MERGL I and 6K-MERGL II) on anion-exchange chromatography, and these were obtained in an homogenous form after reverse-phase HPLC. Amino acid sequencing, together with immunological characterization, indicated that the three peptides were identical in chain length, and corresponded to proenkephalin A 116-165. They contained the sequence Asn-Ser-Ser which is a potential N-glycosylation site. In 9K-MERGL Ia, but not the others, automated Edman amino acid sequencing was unable to detect the relevant asparagine residue, suggesting that this residue has been chemically modified. Further investigation of the 9K-MERGL material using lectin affinity chromatography provided direct evidence of glycosylation. Verification of this result was obtained using the specific enzyme glycopeptidase F (glycopeptide-N-glycosidase) which demonstrated that 9K-MERGL contained, in part, N-linked oligosaccharide chains. These results show that an NH2 terminally extended Met-enkephalin Arg6-Gly7-Leu8 variant was N-glycosylated, and hence indicate that the precursor polypeptide proenkephalin A can be glycosylated during translation in the rough endoplasmic reticulum.
Watkinson
A
A
Physiological Laboratory, University of Liverpool, Liverpool, United Kingdom.
Dockray
G J
GJ
Young
J
J
D000313:Adrenal Medulla / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004744:Enkephalin, Methionine / Q000031:analogs & derivatives* / Q000235:genetics* / Q000378:metabolism; D004745:Enkephalins / Q000235:genetics*; D014644:Genetic Variation; D006031:Glycosylation; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000235:genetics*; D011499:Protein Processing, Post-Translational
D004745:Enkephalins; D011498:Protein Precursors; C029992:proenkephalin; D004744:Enkephalin, Methionine; C033453:enkephalin-Met, Arg(6)-Gly(7)-Leu(8)-