PROUROGUANYLIN AND PROGUANYLIN - PURIFICATION FROM COLON, STRUCTURE, AND MODULATION OF BIOACTIVITY BY PROTEASES

Uroguanylin and guanylin are peptides isolated from urine and intestin al mucosa, which regulate cyclic GMP production in enterocytes by acti vating an apical membrane, receptor-guanylate cyclase. This study exte nded our previous findings, which showed that colonic mucosa of opossu ms contained uroguanylin and guanylin peptides, by purifying prourogua nylin and proguanylin from this tissue. Prouroguanylin and proguanylin coeluted from Sephadex G-75 gelfiltration columns with a similar mole cular size between 6 and 12 kDa. Mass spectrometry indicated that prog uanylin (approximate to 8.7 kDa) had a 10% lower molecular mass than p rouroguanylin (approximate to 9.7 kDa). Isoelectric focusing separated prouroguanylin (pI approximate to 4.5) from proguanylin (pI approxima te to 7.5). N-terminal sequence analysis of reverse phase-HPLC purifie d prohormones revealed 13 amino acids in opossum proguanylin that shar ed 77-85% identity with human and rat proguanylin, but only 23% identi ty with opossum prouroguanylin. The N-terminal 19 residues obtained fo r opossum prouroguanylin shared 32-42% identity with rat and human pro guanylin. Prouroguanylin and proguanylin were both inactive and requir ed pretreatment with proteases to elicit cyclic GMP responses in T84 c ells. V8 protease treatment of proguanylin liberated a bioactive, 16-a mino acid form of guanylin. Chymotrypsin treatment activated prourogua nylin, but inactivated the bioactive peptide domain within proguanylin . In summary, colonic mucosa contains the bioactive peptide and inacti ve prohormone forms of uroguanylin and guanylin. Thus, after proteolyt ic processing of prouroguanylin and proguanylin, bioactive uroguanylin and guanylin could both function to regulate guanylate cyclase activi ty by autocrine and/or paracrine actions on enterocytes. Also, these p eptide hormones are implicated in an intestinal-renal axis for the end ocrine regulation of salt and water homeostasis.