Purification, Characterization And Molecular Cloning Of Zephyranthes Candida Herb Agglutinin

The study on Zephyranthes Candida Herb Agglutinin(ZCA) included two parts.The first part was the study on purification and characterization of ZCA, the second included the study on molecular cloning and sequence analysis of ZCA.ZCA was isolated from bulbs of Zephyranthes Candida Herb by extraction,fraction with (NHOaSO-t, and followed by combination of ion-exchange chromatography on DEAE-Sepharose Fast Flow and gel filtration on Sephacryl S-100. The purified ZCA gave one band on SDS-acrylamide gel electrophoresis. The molecular weight was 48 KD determined by gel filtration. The subunit molecular weight was 12.5KD on SDS-PAGE. The results included that there were four same subunits in ZCA molecule.There were 12.2 Trp residues in a ZCA molecule , determinded by colorimetry and all the Trp residues are on the surface of ZCA.ZCA could agglutinate rabbit erythrocytes at 0.95 g/mK E.coli cell at 1.95 g/ml and some sorts of Yeast cells . The result of carbohydrate inhibition assayed that Mannan and Thyroglobulin could inhibit the hemagglutination activity of ZCA. The study on the fluorescence spectra and the agglutinate activity of ZCA under different temperature and pH showed that ZCA was stable in various temperature and pH between pH 4-9.5 and below 80 , but the molecular structure and biological activity changed much under pH 2, pH 4, pH 12 and 80 , and theactivity of ZC A became weak.The study on fluorescence quenching showed that 71% Trp could be quenched by CsCl. Chemical modification indicated that the Arginine and Tryptophan are essential for the hemagglutination activity of ZCA. The modification of Arginine and Tryptophan resulted the loss of rabbit hemaggulutinating activity. The modification of Ser/Thr didn't effect the hemagglutinating activity of ZCA. ZCA could inhibited the infection of the vero cells by Herpes simplex Virus type n(HSV-II) at IC50 values of 5~ 10 ug/ml and it showed no cytotoxic activity to vero cell proliferation at 500 g/ml.The N-terminal amino acids were D-S-I-L-Y-S-G-E-T-L-S-A-G-Q-S; the C-terminal amino acids were G-T-A-W-K-A-P determined by amino acid sequence analysis. The sequence shared high homology with other Amaryllidaceae mannose-binding lectins.Degenerate primers were designed in accordance with the N-terminal partial sequence of purified ZCA. The full-length cDNA was cloned by RT-PCR, 3'-RACE and 5'-RACE, and sequenced (Genbank AF503622). The full-length cDNAhad 661 bp, and the sequence encoded an open reading frame of 168 amino acids..The start codon was at 37-39 bp and the stop codon was at 544-546 bp. The result showed that ZCA gene encoded a protein precursor with a signal peptide, mature protein and C-terminal cleavage amino acids sequence by the study of N-terminal and C-terminal partial amino acids sequence of ZCA. The mature protein included 106 amino acids residues and the molecular weight is 11.7KD . The mature protein sequence showed the identity to those of Galanthus nivalis agglutinin, Narcissus hybrid cultivar agglutinin , Lycoris radiate agglutinin, Clivia miniata agglutinin respectively are 75.2%, 72.4%, 76.2%, 76.2% , Blocks'analysis revealed that the deduced amino acid sequence of ZCA had three functional domains specific for lectin and three sugar-binding boxes(QDNY).In the mature protein,there were 38.7% hydrophobic amino acids, 43.4% hydrophilic amino acids, 10.4% basic amino acids and 7.5% acidic amino acids. The signal peptide of ZCA had 24 aminoacids,included a hydrophobia core of 9 ammo acids. There was no intron in the ZCA gene by the DNA PCR. The cloning of this gene established a important base to the study of ZCA gene structure, the cleavage mechanism, the mechanism of expression and regulation, the relationship of structure and function of ZCA...