Expression And Functional Analysis Of The Two Domains Of Pinellia Ternata F. Angustata Agglutinin (PTA)

Pinellia ternata agglutinin (PTA) from P. ternata f. angustata is a two-domain GNA-related lectin. The current study indicates that the PTA gene encodes a precursor consisting of two tandemly arrayed domains, N-terminal domain (PTA-DOM1) and C-terminal domain (PTA-DOM2). Previous studies have indicated that each subunit of two-domain GNA-related lectins contains zero to three active mannose-binding sites, which may be the carbohydrate binding sites that perform the carbohydrate recognition function. By contrast, the full-length amino acid sequence of the PTA precursor contains three active mannose-binding sites (QDNVY), one belongs to PTA-D0M1, and the other two belong to PTA-D0M2.According to the assay for lectin activity, the obtained fusion proteins demonstrated excellent hemagglutinating activity, indicating that the procedures of expression and purification of the proteins were effective. Inhibition of agglutination by sugars and oligosaccharides showed that the fusion proteins have similar specificity to native agglutinin, even though the latter is a mixture of isoforms. The fusion proteins expressed as PTA domains were effective against a variety of fungal species, including A. alternata, B. sorokiniana, and C. lunata. The PTA-D0M2 protein was more potent against these fungal species than that of the PTA-D0M1, which may be a consequence of its greater number of active carbohydrate-binding sites. Analyses of the three fusion proteins, PTA-D0M1, PTA-D0M2 and PTA-P, expressed in Escherichia coli revealed that one mannose-binding site the agglutination activity while the additional sites do not possess such activity. However, the number of carbohydrate-binding sites suggests some significant properties on the antifungal effectiveness. In addition, each of the PTA domains has the same function when compared with the natural PTA (N-PTA).Using semi-quantitative RT-PCR analysis, the PTA in P. ternata f. angustata was expressed in all tested tissues with slight differences. The failure to detect the fragments of PTA in the vegetative tissues may be attributed to further proteolysis and/or transport to the storage tissues, such as bulbils and tubers, since it is a storage protein to help plants survive periods of adverse conditions or to survive between growing seasons and provide nutrients to support the growth of new plants as seedlings or shoots once it is produced in P. ternata cells.In conclusion, the two domains of PTA including the precursor form were effectively expressed in E. coli, and the fusions proteins all have the same activity, similar to the native protein. To our knowledge, this method was the first time to study lectin domains separately. Furthermore, we determined that one carbohydrate-binding site of PTA is sufficient for agglutinin activity and that the presence of additional sites may have no further effect on this activity. These conclusions could help researchers to produce large amounts of PTA by expressing either one of its two domains in E. coli. The information on PTA gene obtained in this study will served as baseline information in developing this protein as a form of transgenic plant protection.