Cloning, expression and characterization of antisecretory factor

Abstract

Antisecretory factor (AF) is a protein known to inhibit pathological intestinal fluid secretion induced by enterotoxins. The aim of this thesis was to perform a genetic and chemical characterization of AF and to evaluate the biological activity of the recombinant protein and its peptide derivative. The human AF-gene was cloned by screening a cDNA-library with antiserum raised against purified AF from the porcine pituitary gland. The entire AF-gene and fragments of the gene were cloned by PCR technique. The nucleotide sequence was, at that time, coding for an unknown 382-residue protein that was expressed in pGEX/E.coli and purified. The recombinant AF manifested similar immunoreactivity, migration in SDS-PAGE and biological activity with the previously characterized AF. Antiserum against recombinant AF showed an intracellular staining in cells of the adenohypophysis. The recombinant AF inhibited hypersecretion induced by cholera toxin, toxin A from Clostridium difficile, or by okadaic acid from phytoplankton. The inflammatory reaction in the gut caused by toxin A was abolished after treatment with AF. The recombinant AF had an increased effect after treatment with trypsin, indicating a peptide activity. A N-terminal peptide, 35IVCHSKTR42, was determined to be the smallest active site of the AF molecule by testing truncated PCR-amplificated AF-fragments that were expressed in E.coli and peptides made by the solid phase synthesis. Human probes detected AF in DNA in all eukaryotic species examined. The coding region, including the antisecretory activity, was cloned in porcine, bovine and rat DNA. The identities at nucleotide level with human AF were 91.8%-93.5% whereas the amino acid identities were 100%. AF-like sequences from Drosophila, Arabidopsis and Saccharomyces were compared and showed the highest homology in the N-terminal part with human AF. The AF-gene is evolutionary conserved, but the protein's antisecretory effect in non-mammalian species is debatable. Because there are functional similarities to neuropeptides, the effect of AF is probably mediated by the enteric nerve system. This recombinant protein and its peptide derivatives might be therapeutically useful to regulate intestinal pathological fluid secretion.