Sequence, Structure, and Binding Site Analysis of Kirkiin in Comparison with Ricin and Other Type 2 RIPs.

Kirkiin is a new type 2 ribosome-inactivating protein (RIP) purified from the caudex of Adenia kirkii with a cytotoxicity compared to that of stenodactylin. The high toxicity of RIPs from Adenia genus plants makes them interesting tools for biotechnology and therapeutic applications, particularly in cancer therapy. The complete amino acid sequence and 3D structure prediction of kirkiin are here reported. Gene sequence analysis revealed that kirkiin is encoded by a 1572 bp open reading frame, corresponding to 524 amino acid residues, without introns. The amino acid sequence analysis showed a high degree of identity with other Adenia RIPs. The 3D structure of kirkiin preserves the overall folding of type 2 RIPs. The key amino acids of the active site, described for ricin and other RIPs, are also conserved in the kirkiin A chain. Sugar affinity studies and docking experiments revealed that both the 1α and 2γ sites of the kirkiin B chain exhibit binding activity toward lactose and D-galactose, being lower than ricin. The replacement of His246 in the kirkiin 2γ site instead of Tyr248 in ricin causes a different structure arrangement that could explain the lower sugar affinity of kirkiin with respect to ricin.

A Polylactosamine Specific Lectin from Adenia hondala Induces Apoptosis and Necrosis in Human Epithelial Colon Cancer HT-29 Cells.

BACKGROUND: Altered expression of N-glycans such as polylactosamine is observed in colon cancer. AHL, a polylactosamine specific lectin from Adenia hondala from a medicinal plant from the Passifloraceae family has been reported earlier. OBJECTIVE: The aim of the present study is to study the interaction of AHL with human colon cancer epithelial HT-29 cells and colon cancer tissues. METHODS: Cell viability was determined by MTT [3-[4, 5- dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide] assay, while cell surface binding, apoptosis by Annexin-V-PI assay and ROS production using DCFDA [2',7' - dichlorofluorescindiacetate] kit method were analysed by flowcytometry, immunohistochemistry was performed using biotinylated AHL, protein purification by affinity chromatography using asialofetuin-coupled Sepharose -4B column. RESULTS: AHL strongly binds to HT-29 cells with a Mean Fluorescence Intensity of 12.4, which could be blocked by competing glycoprotein asialofetuin. AHL inhibits HT-29 cell growth in a dose and time-dependent manner with IC50 of 2.5 µg/mL and differentially binds to human normal and cancerous tissues. AHL induces apoptosis and slight necrosis in HT-29 cells with an increase in the early apoptotic population of 25.1 and 36% for 24 h and 48 h respectively and necrotic population of 1.5 and 4.6% at 24 h and 48 h respectively as revealed by Annexin-V-PI assay. AHL induces the release of Reactive Oxygen Species in HT-29 cells in a dose-dependent manner. CONCLUSION: To the best of knowledge, this is the first report on lectin from Adenia hondala which is not a RIP with apoptotic and necrotic effects. These findings support the promising potential of AHL in cancer research.

Purification, characterization and fine sugar specificity of a N-Acetylgalactosamine specific lectin from Adenia hondala.

Plant lectins are gaining interest because of their interesting biological properties. Several Adenia species, that are being used in traditional medicine to treat many health ailments have shown presence of lectins or carbohydrate binding proteins. Here, we report the purification, characterization and biological significance of N-Acetyl galactosamine specific lectin from Adenia hondala (AHL) from Passifloraceae family. AHL was purified in a single step by affinity chromatography on asialofetuin Sepharose 4B column, characterized and its fine sugar specificity determined by glycan array analysis. AHL is human blood group non specific and also agglutinates rabbit erythrocytes. AHL is a glycoprotein with 12.5% of the carbohydrate, SDS-PAGE, MALDI-TOF-MS and ESI-MS analysis showed that AHL is a monomer of 31.6 kDa. AHL is devoid of DNase activity unlike other Ribosome inactivating proteins (RIPs). Glycan array analysis of AHL revealed its highest affinity for terminal lactosamine or polylactosamine of N- glycans, known to be over expressed in hepatocellular carcinoma and colon cancer. AHL showed strong binding to human hepatocellular carcinoma HepG2 cells with MFI of 59.1 expressing these glycans which was effectively blocked by 93.1% by asialofetuin. AHL showed dose and time dependent growth inhibitory effects on HepG2 cells with IC50 of 4.8 µg/ml. AHL can be explored for its clinical potential.

Respuesta in vitro de diferentes biotipos y explantos de Passiflora caerulea L / In vitro response of different biotypes and explants of Passiflora caerulea L

Passiflora caerulea L., al igual que otras especies de la familia Passifloraceae, es utilizada en la medicina popular por sus propiedades antiespasmódicas y para el tratamiento de la ansiedad, el insomnio y el nerviosismo. La belleza de sus flores les otorga valor ornamental, mientras que sus frutos son apreciados por su importancia alimenticia. Se evaluó la respuesta in vitro de diferentes explantos y tres biotipos de P. caerulea: Corral de Bustos (provincia de Córdoba), Zavalla (provincia de Santa Fe) y Neuquén (provincia de Neuquén). Se utilizaron dos tipos de explantos: entrenudos y segmentos nodales, y como medio de cultivo Murashige y Skoog (1962) (MS), suplementado con vitaminas de Gamborg (1976) y 1 mg/L-1 de benciladenina (BA). Las respuestas fueron diferentes según el genotipo y el explanto. Los entrenudos ubicados tanto horizontal como verticalmente en medio de cultivo generaron callos como única respuesta. El biotipo de Neuquén mostró los mayores porcentajes de segmentos nodales con brotes. A través de estudios histológicos se determinó que en medio de cultivo MS con 1 mg/L-1 de BA, los segmentos nodales de P. caerulea originan brotes a partir de las yemas axilares preformadas y raíces que parten de callos en la base de los mismos. En iguales condiciones, los entrenudos originan callo como única respuesta.

As other species of the Passifloraceae family, Passiflora caerulea L. is used in popular medicine for its antispasmodic properties and as a remedy for anxiety, insomnia and nervousness. It is also highly prized for the ornamental value of its beautiful flowers, as well as for the nutritional importance of its fruits. The in vitro response of different explants and three biotypes of P. caerulea: the Corral de Bustos (Province of Córdoba), the Zavalla (Province of Santa Fe) and the Neuquén (Province of Neuquén) genotypes, was evaluated using two types of explants: internodes and nodal segments on Murashige and Skoog (1962) (MS) culture medium supplemented with Gamborg’s vitamins (1976) and 1 mg.L-1 of benzyladenine (BA). There were different responses depending on the genotype and the explant. The internodes placed both horizontally and vertically in the culture medium produced callus as sole response. The Neuquén biotype showed the highest percentages of nodal segments with shoots. Histological tests allowed to establish that in MS culture medium with 1 mg.L-1 of BA, the nodal segments of P. caerulea produce shoots from the preformed axillary buds and roots that develop from the callus situated on its base. Under similar conditions, the internodes produce callus as sole response.