Characterization of proteases from Pangasius hypophthalmus epithelial mucus and their potential use as wound healing agent

AVILA RODRÍGUEZ, MARÍA ISABELA; JORGE BENAVIDES; MIRNA LORENA SANCHEZ

Simposio; "8th Symposium of the Mexican Proteomics Society 3rd PanAmerican-Human Proteome Organization (Pan-HUPO) Meeting 2nd Ibero-American Symposium on Mass Spectrometry"; 2019

Wound healing is a process in which matrix metalloproteases (MMP)1 and serine proteases (SP) 2 are strongly involved. When dysregulation of these components occurs, the healing process is precluded. These proteases have also been described as innate immune response agents in epithelial mucus of several 3 fish species . Empirical evidence has shown that, the epithelial mucus of Iridescent shark (Pangasius hypophthalmus) promotes skin regeneration. Under this evidence, the characterization of the proteases present in P. hypophthalmus epithelial mucus was held, to identify new MMP and SP with potential use for wound care. Epithelial mucus was extracted by rinsing specimens with extraction buffer (NaCl 50mM pH 7.4) in polyethylene bags and held back to tank. Then, mucus extracts were pooled and centrifuged. Supernatant was concentrated through vacuum evaporation and desalted with PD-10 columns. Protease activity of DME was evaluated through caseinolytic activity and zymography (gelatin and casein), as casein and gelatin are considered universal protease substrates. Within zymography, different inhibitors (PMSF, benzamidine, EDT A, O- phenanthroline, and iodoacetamide) and reported activating ions (Zn 2+, Ca 2+ , K +, Na + and no ion) for specific protease families were evaluated. Caseinolytic activity for DME was detected (5.33 ± 0.37 U/mg at 25 oC). As for zymography analysis, active bands within 130-15 kDa were identified for gelatin, while only one active band of 63 kDa was identified for casein. Compared to control treatment (Zn2+), K+ and Na+ enhanced gelatinolytic activity of medium weight bands (63, 58 and 48 kDa), while Ca2+ depleted most protease activity. Serine and cysteine protease inhibitors, PMSF and iodoacetamide, excerpted similar inhibition by reducing 63 kDa and inhibiting 58, 56, 30 kDa activity. MMP inhibitors exerted slight inhibition to superior weight bands (114, 90 and 71 kDa). Benzamidine only depleted 45 kDa activity. The present analysis proves the presence of the MMPs and SPs within P. hypophthalmus epithelial mucus. This positive result opens the possibility for further protease characterization and isolation for their evaluation as feasible agents for wound healing treatments.