Purification, structure and function of bioactive peptides

Peptides are vitally important molecules and many evoke cellular responses. The completion of several genome sequencing projects has revealed a number of new genes. However, as functional peptides often contain posttranslational modifications and/or occur at various lengths, it is of great importance to detect, purify and characterize novel bioactive peptides. To achieve these goals, new methods for peptide detection, isolation and functional characterization have to be developed. In the present thesis, a broad range of different techniques for detection, purification, structural and functional characterization of new peptides, the production of recombinant peptides, their reconstitution with metals and functional characterization was utilized. A detection method was developed to screen for receptor ligands from tissue extracts using Chinese hamster ovary (CHO) cells with a Cytosensor. As a proof-of-concept, an active component in tissue extracts was purified by a seven-step protocol. The structure was determined by mass spectrometry (MS), which identified it as insulin-like growth factor-I (IGF-I). A novel ligand for an orphan G-protein coupled receptor, GPCR135, was detected in porcine brain extracts by guanosine 5`-O-(3-thiotriphosphate) (GTPγS) incorporation assay. The ligand was purified by a six-step procedure. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry analyses demonstrated that the ligand is relaxin 3, which is composed of two peptide chains linked together with disulfide bridges similarly to insulin-relaxin family of peptides. Pharmacological experiments showed that relaxin-3 is the only ligand that activates GPCR135. An elongated form of peptide histidine isoleucine amide (PHI), PHI-42, was detected in porcine intestinal extracts by cAMP assays on IMR-32 neuroblastoma cells. PHI-42 was purified from the mixture by a four-step procedure. The structure was determined by MALDI MS and Edman degradation, which showed that the 12-residue sequence of porcine PHI-42 is unique and links together PHI-27 and vasoactive intestinal polypeptide (VIP) in the precursor. A novel posttranslationally modified form of neurotensin (NT) was identified from porcine intestine and purified by a five-step procedure. Linear ion trap-Fourier transform, MALDI, and ESI quadropole time-of-flight (Q-TOF) mass spectrometries identified the posttranslational modification as an Arg-residue coupled to the gamma-carboxyl g