Isolation and characterization of a novel proopiomelanocortin-derived peptide from hemofiltrate of chronic renal failure patients.
We report the isolation of a novel human circulating proopiomelanocortin-derived peptide, VA-beta-MSH, from hemofiltrate and its pharmacological characterization. Screening for lipolytic activity in differentiated 3T3-L1 adipocytes led to the isolation from a hemofiltrate peptide library by alternating reverse phase and cation exchange chromatography. In the course of this isolation, we also identified human beta-MSH-(1-22). We synthesized VA-beta-MSH by the N-(9-fluorenyl)-methoxycarbonyl (F-moc) solid phase method and used synthetic beta-MSH-(1-22) to confirm that both isolated peptides are lipolytically active in a dose-dependent manner in differentiated 3T3-L1 adipocytes in the nanomolar range. Using cAMP ELISA, we demonstrate that stimulation with both peptides caused a strong cAMP elevation in this cell system. Furthermore, we show that the selective inhibitors of cAMP-dependent protein kinase, 8-(4-Chlorophenylthio)adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-CPT-cAMPS); N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), significantly reduce VA-beta-MSH- and beta-MSH-(1-22)-mediated lipolysis. Although isolated after its lipolytic activity on 3T3-L1 cells, this newly identified circulating human melanocortin may serve other functions in human physiology. Moreover, the fact that these peptides have been identified after a functional assay, but have been overseen in large proteomic approaches, underscores the importance of such approaches in identifying previously undescribed circulating bioactive molecules.
Fricke
Katrin
K
IPF PharmaCeuticals GmbH, Feodor Lynen Strasse 31, D-30625 Hannover, Germany.
Schulz
Axel
A
John
Harald
H
Forssmann
Wolf-Georg
WG
Maronde
Erik
E
D041721:3T3-L1 Cells; D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D006440:Hemofiltration; D006801:Humans; D007676:Kidney Failure, Chronic / Q000378:metabolism*; D008066:Lipolysis; D051379:Mice; D008969:Molecular Sequence Data; D044103:Receptor, Melanocortin, Type 2 / Q000032:analysis; D018043:Receptors, Corticotropin / Q000032:analysis; D044101:Receptors, Melanocortin; D019824:beta-MSH / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology
D044103:Receptor, Melanocortin, Type 2; D018043:Receptors, Corticotropin; D044101:Receptors, Melanocortin; D019824:beta-MSH; C086971:melanocortin 5 receptor; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases