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

JORGE BENAVIDES; AVILA RODRÍGUEZ, MARÍA ISABELA; 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 multistep process in which matrix metalloproteases (MMP) and serine proteases (SP) are strongly involved. When dysregulation of these components occur, the healing process is precluded. This type of proteases has been also described within fish epithelial mucus, acting as innate immune response against pathogenic agents. Empirical evidence has shown that epithelial mucus of the Iridescent shark (Pangasius hypophthalmus) promotes skin regeneration. Under this evidence, the characterization of the proteases within P. hypophthalmus epithelial mucus was performed, as to identify new MMP and SP with potential use for wound care. Epithelial mucus was extracted, pooled and clarified through centrifugation. Supernatant was then concentrated by vacuum evaporation (EME). Casein and gelatin are considered universal substrates for protease characterization, by which protease activity of mucus extract was measured by caseinolytic activity and zymography (gelatin and casein). Different inhibitors (PMSF, benzamidine, EDTA, O-phenanthroline, and iodoacetamide) and reported activating ions (Zn2+, Ca2+, K+, Na+ and no ion) for specific protease families were evaluated through zymography. The protease activity of EME was of 5.33 ± 0.37 U/mg at 25 ºC and 1.67 ± 0.31 U/mg at 37 ºC. As for zymography analysis, 10 active bands within 130-15 kDa were identified in gelatin substrate, while only one active band of 63 kDa was identified in casein substrate. As well treatment with presence and absence of ions revealed that compared to control treatment (Zn2+), K+ and Na+ ions enhanced the activity of they high molecular weight bands (63, 58 and 48 kDa), while Ca2+ depleted the activity of most of the protease profile in both substrates. On the other hand, PMSF and iodoacetamine had similar inhibition profiles by reducing the activity of 63 kDa and inhibiting 58, 56, 30 kDa bands. MMP inhibitors (EDTA and O-phenanthroline) exerted light inhibition over superior weight bands (114, 90 and 71 kDa). Benzamidine only depleated 45 kDa band. The present analysis proves the presence of the MMPs and SPs within P. hypopthalmus epithelial mucus. This positive result opens the possibility for further protease characterization and isolation for its evaluation as feasible agents for wound healing treatments.