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Isolation and Structure–Bioactivity Characterization of Glycosylated N ‐Pro‐Opiomelanocortin Isoforms
Author(s) -
Lu J.,
Swinnen E.,
Proost P.,
De Vreese B.,
Vankelecom H.,
Denef C.
Publication year - 2002
Publication title -
journal of neuroendocrinology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.062
H-Index - 116
eISSN - 1365-2826
pISSN - 0953-8194
DOI - 10.1046/j.1365-2826.2002.00851.x
Subject(s) - gene isoform , prolactin , microbiology and biotechnology , monoclonal antibody , messenger rna , biochemistry , chemistry , biology , glycosylation , medicine , endocrinology , antibody , hormone , gene , immunology
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.