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 group of Glu-4 by an isopeptide bond. In addition, NT(2-13) and NT(3-13) together with a fragment (22-38) of dopamine- and adenosine 3 ,5 -monophosphate-regulated phosphoprotein (DARPP-32) were also detected and studied by MALDI MS and Edman degradation. All the NT forms were active in intracellular Ca2+ release assay.

Human brain-specific metallothionein-3 (MT-3) was produced by recombinant technology and studied by ESI MS. The protein was purified as apo-MT-3 by a new three-step procedure. Titration of MT-3 with Zn2+ and Cd2+ ions and the stability of metal complexes were followed by ESI MS and the results were compared with data for common MT-s. We demonstrated that MT-3, in contrast to common MTs, binds divalent metals in a non-cooperative manner and at higher capacity than common MTs.

The combination of bioassays, high performance liquid chromatography, and mass spectrometry has enabled us to detect, purify and characterize novel peptides from biological sources, yielding new knowledge about their localization, structure, processing and functions.

List of scientific papers

I. Bonetto V, Eriste E, Jonsson AP, Efendic S, Jornvall H, Sillard R (2001). Isolation of peptides from porcine intestinal tissue that induce extracellular acidification in CHO cells: identification of the active peptide as IGF-I and characterization of a fragment of calponin H1 processed at a dibasic site. Arch Biochem Biophys. 385(2): 276-82.
https://pubmed.ncbi.nlm.nih.gov/11368008

II. Liu C, Eriste E, Sutton S, Chen J, Roland B, Kuei C, Farmer N, Jornvall H, Sillard R, Lovenberg TW (2003). Identification of relaxin-3/INSL7 as an endogenous ligand for the orphan G-protein-coupled receptor GPCR135. J Biol Chem. 278(50): 50754-64.
https://pubmed.ncbi.nlm.nih.gov/14522968

III. Eriste E, Norberg A, Bonetto V, Nepomuceno D, Lovenberg TW, Sillard R, Jornvall H (1999). A C-terminally elongated form of PHI from porcine intestine. Cell Mol Life Sci. 56(7-8): 709-13.
https://pubmed.ncbi.nlm.nih.gov/11212317

IV. Eriste E, Norberg A, Strupat K, Lovenberg TW, Nepomuceno D, Jornvall H, Sillard R (2003). Purification and structure of a novel form neurotensin posttranslationally modified by arginylation. [Submitted]

V. Eriste E, Kruusel K, Palumaa P, Jornvall H, Sillard R (2003). Purification of recombinant human apometallothionein-3 and reconstitution with zinc. Protein Expr Purif. 31(1): 161-5.
https://pubmed.ncbi.nlm.nih.gov/12963354

VI. Palumaa P, Eriste E, Njunkova O, Pokras L, Jornvall H, Sillard R (2002). Brain-specific metallothionein-3 has higher metal-binding capacity than ubiquitous metallothioneins and binds metals noncooperatively. Biochemistry. 41(19): 6158-63.
https://pubmed.ncbi.nlm.nih.gov/11994011