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.