Walailak Frontier 21, November 2019

Serine protease inhibitor with antifungal activity against Trichophyton rubrum from Para rubber leaves 

Protease inhibitors in organisms are produced for regulating the proteolytic activity of their targets. The serine protease inhibitor is one type of protease inhibitors. It is widespread in plants and has been found in many species such as potato, squash, barley, wheat, and rubber tree. Serine protease inhibitors have been related to the mobilization of storage proteins, regulation of endogenous enzymatic activities, and stabilization of defense proteins against microorganisms, insects, and animals. The serine protease inhibitor gene isolated from Hevea brasiliensis leaves, a RRIT251 cultivar and designated RRIT251 H. brasiliensis protease inhibitor (251Hbpi) encoded a 70 amino acid protein (Figure 1). 251HbPI is a member of the potato inhibitor I (PI-I) family of serine protease inhibitors. Multiple alignments of the homologous PI-I family revealed one motif WPELVG of 251HbPI conserved across the family (Figure 1). Molecular weight of the recombinant 251HbPI was approximately 11 kDa. Protease inhibition analysis revealed that 251HbPI inhibited the activity of chymotrypsin and subtilisin A but did not trypsin protease.

From research revealed that 251HbPI inhibits Trichophyton rubrum (dermatophyte infection in humans) growth, suggesting that 251HbPI inhibits T. rubrum serine protease, which is an important virulence factor secreted during host infection of T. rubrum.  The anti T. rubrum activity of 251HbPI was effective with minimum inhibitory concentration of 0.7 mg/ml and minimum fungicidal concentration of 1.4 mg/ml. Purified 251HbPI protein was tested for co-immunoprecipitation with secreted T. rubrum proteins. The result shows that 251HbPI co-precipitated approximate 27 kDa and 61 kDa of secreted T. rubrum proteins (Figure 2). This result indicates that both approximate 27 kDa and 61 kDa T. rubrum proteins were targets for 251HbPI to inhibit T. rubrum growth. These proteins are suggested as T. rubrum serine protease. These results suggest that 251HbPI could be a candidate for the development of a novel drug to treat T. rubrum infection.

Figure 1. Alignments of the putative 251HbPI amino acid sequence with members of the PI-I family from various plant species. The amino acid sequence of 251HbPI was aligned with Hb-PI (H. brasiliensis RRIM600 latex protease inhibitor), PI1 (H. brasiliensis protease inhibitor protein 1), CI-1A (Subtilisin-chymotrypsin inhibitor-1A of H. vulgare), CI-1B (subtilisin-chymotrypsin inhibitor-1B of Z. mays), CI-2A (Subtilisin-chymotrypsin inhibitor-2A of H. vulgare), and WSCI (Subtilisin-chymotrypsin inhibitor WSCI of T. aestivum). Identical amino acids are highlighted with a gray background. The asterisks indicate amino acid residues of 251HbPI that were conserved across the PI-I family.

Figure 2. Co-immunoprecipitation of 251HbPI with secreted T. rubrum proteins using anti-FLAG M2 resin. SDS-PAGE gels were stained with Coomassie Brilliant Blue. Lane M represents the protein standard marker. Lane 1 represents the secreted T. rubrum proteins alone. Lane 2 represents the mixture of secreted T. rubrum proteins incubated with 251HbPI.  Lane 3 represents the purified 251HbPI.

Sources:

• Dutsadee Chinnapun, Sarawoot Palipoch and Hatairat Hongphruk. 2016. Cloning and characterization of a putative gene encoding serine protease inhibitor (251Hbpi) with antifungal activity against Trichophyton rubrum from Hevea brasiliensis leaves. Plant Omics Journal. 9(2), 142-148

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