Isolation and structure-bioactivity characterization of glycosylated N-pro-opiomelanocortin isoforms.
The N-terminal fragment of mouse pro-opiomelanocortin (N-POMC) was isolated from AtT-20 cell-conditioned medium on the basis of immunoreactivity to an anti-POMC1-50 monoclonal antibody by a concentration step, a cation exchange step, reversed phase high-performance liquid chromatography (HPLC) and size exclusion HPLC. Two groups of N-POMC isoforms with a molecular weight (MW) of approximately 11 kDa and 13 kDa, respectively, were identified by mass spectrometry and N-terminal amino acid sequencing. C-terminal sequencing indicated that 11 kDa isoforms correspond to POMC1-74 and 13 kDa isoforms to POMC1-95. Isoforms from both groups enhanced the prolactin mRNA content (measured by means of TaqMan real-time reverse transcription-polymerase chain reaction) in cultured rat pituitary cell aggregates in a dose-dependent manner, but not all of them showed this activity. POMC1-74 compounds were significantly more potent than POMC1-95 isoforms. The observed effects were abolished by coincubation with the monoclonal anti-POMC1-50 antibody, showing the specificity of this biological action. Incorporation of bromodeoxyuridine into DNA of immunostained lactotrophs was enhanced by only a minor part of the isoforms. Some of these had no effect on prolactin mRNA expression. The N-POMC isoforms appeared to be N- and at least in part O-glycosylated. After enzymatic N-deglycosylation of selected N-POMC isoforms, the stimulatory effect on the prolactin mRNA level was depressed (in case of the POMC1-95 isoforms) or totally abolished (in case of the POMC1-74 isoforms). The present findings show that N-POMC is a mixture of differentially glycosylated isoforms, that the isoforms of POMC1-74 are the biologically more effective forms and that different isoforms induce different biological responses in the same cell population. The data also show the essential role of N-glycosylation in the biological response.
Lu
J
J
Laboratory of Cell Pharmacology, University of Leuven (KU Leuven), Medical School, Gasthuisberg O&N, Belgium.
Swinnen
E
E
Proost
P
P
De Vreese
B
B
Vankelecom
H
H
Denef
C
C
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001973:Bromodeoxyuridine / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D006031:Glycosylation; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism; D011333:Pro-Opiomelanocortin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D011388:Prolactin / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
D000906:Antibodies; D010446:Peptide Fragments; D020033:Protein Isoforms; D012333:RNA, Messenger; D011333:Pro-Opiomelanocortin; D011388:Prolactin; D001973:Bromodeoxyuridine