Isolation and structural characterization of novel Rugosin A-like insulinotropic peptide from the skin secretions of Rana saharica frog

Abstract

Skin secretions of Rana saharica were evaluated for the isolation and characterization of novel insulinotropic peptides. Crude secretions obtained from young adult frogs by mild electrical stimulation of the dorsal skin surface were purified by reversed-phase HPLC yielding 80 fractions. In acute incubations with glucose-responsive BRIN-BD11cells, fractions 36–43, 46–54 and 57–63 showed the significant 2–8-fold increase in insulin-releasing activity ( P < 0.001) compared with 5.6 mM of glucose alone. A pool of fractions 36–43 was subsequently rechromatographed to 28 homogenous peaks out of which 7 were capable of subsequent 1.5–3-fold increase in insulin release ( P < 0.001). Structural analysis of the non-toxic peptides with greatest insulin-releasing activity was performed by mass spectrometry and Edman degradation. Mass spectrometry analysis of two peaks indicated the molecular masses of 1892.6 and 2930.8 Da. The sequence of the 1892.6-Da peptide was determined as KGAAKGLLEVASCKLSKSC, which has 68% homology with Rugosin A originally isolated from the skin secretion of Rana rugosa. A partial N-terminal sequence was determined for the 2930.8-Da peptide as AVITGACERDVQCGGGTCCAVSLI…. These data indicate that the skin secretions of Rana saharica frogs contain novel peptides with insulin-releasing activity.