Cloning and characterization of a putative gene encoding serine protease inhibitor (251Hbpi) with antifungal activity against Trichophyton rubrum from Hevea brasiliensis leaves

Abstract

A novel serine protease inhibitor gene was isolated from Hevea brasiliensis leaves, a RRIT251 cultivar and designated RRIT251 H. brasiliensis protease inhibitor (251Hbpi). Reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were used to isolate 251Hbpi. A full-length cDNA of 251Hbpi encoded a 70 amino acid protein. 251HbPI is a member of the potato inhibitor I (PI-I) family of serine protease inhibitors. The amino acid residues at the active site of 251HbPI were predicted as Met 46-Glu 47. Multiple alignments of the homologous PI-I family revealed one motif WPELVG of 251HbPI conserved across the family. 251Hbpi was cloned into expression vector pFLAG-ATS and expressed in Escherichia coli strain BL21. Molecular weight of the recombinant 251HbPI (r251HbPI) was approximately 11 kDa. Protease inhibition analysis revealed that r251HbPI inhibited the activity of chymotrypsin and subtilisin A but did not trypsin protease. Moreover, purified r251HbPI protein inhibited Trichophyton rubrum with a minimum inhibitory concentration of 0.7 mg/ml and a minimum fungicidal concentration of 1.4 mg/ml. The specific T. rubrum protease targets of r251HbPI were analyzed by co-immunoprecipitation. r251HbPI interacted with approximate 27 and 61 kDa T. rubrum proteins, suggesting a role in the inhibition of T. rubrum growth. These results suggest that 251HbPI could be a candidate for the development of a novel drug to treat T. rubrum infection.

Isolation, expression, and characterization of the serine protease inhibitor gene (600Hbpi) from Hevea brasiliensis leaves, RRIM600 cultivar

“…1). Compared with the fulllength 251Hbpi cDNA sequence from a previous study (Chinnapun et al, 2016), the 600HbPI amino acid sequence shows 87% sequence identity with 251HbPI ( Fig. 1).…”

“…Moreover, the conserved sequence across the PI-I family of 600HbPI is different from that of 251HbPI. Multiple sequence alignments of homologous PI-I family proteins from various plant species revealed one motif WPELVG of 251HbPI conserved across the PI-I family (Chinnapun et al, 2016) and one motif WPEL of 600HbPI conserved across the PI-I family (Fig. 1).…”

“…However, the 600HbPI amino acid sequence lacks a signal peptide and has an active site at Met 46 -Glu 47 (Fig. 2) like the 251HbPI amino acid sequence (Chinnapun et al, 2016). The active site of 600HbPI was predicted based on the other known members of PI-I family as Met-Glu (Svendsen et al, 1980), Met-Asp (Richardson, 1977), Leu-Asp (Richardson, 1977), Ala-Asp (Svendsen et al, 1984), and Lys-Asp (Wingate et al, 1989).…”

“…The RRIM600 cultivar has less capacity to resist pathogens than the RRIT251 cultivar. A previous study found that the RRIT251 cultivar of H. brasiliensis leaves produces a serine protease inhibitor designated as RRIT251 H. brasiliensis protease inhibitor (251HbPI) (Chinnapun et al, 2016). In this study, we hypothesize that serine protease inhibitor characteristics of the RRIM600 cultivar of H. brasiliensis leaves are different from those of 251HbPI because the capacity to resist pathogens of the RRIT251 and RRIM600 cultivars is different.…”

“…The PI-I protein is found in many plant species, including potato tubers (Ryan and Balls, 1962), tomato leaves (Lee et al, 1986), tomato fruit (Margossian et al, 1988;Wingate et al, 1989), and squash phloem exudates (Murray and Christeller, 1995). Moreover, the PI-I family of serine protease inhibitor has been found in latex of Hevea brasiliensis, RRIM600 cultivar (Sritanyarat et al, 2006) and leaves of H. brasiliensis, RRIT251 cultivar (Chinnapun et al, 2016).…”