Five novel lectin isoforms, Volvariella volvacea (VV) lectins, designated VVA, VVB, VVC, VVD & VVE, were isolated and purified from the fruiting bodies of an edible mushroom, Volvariella volvacea , by ion-exchange chromatographies in a FPLC system. Their molecular masses are very close, as measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS); they are 12740, 12737, 12709, 12708 & 12707 Da for VVA, VVB, VVC, VVD & VVE, respectively, but the pI values between VVA and the others show distinct differences; the pI value of VVA is around 6.7 and the others are much closer to each other (higher than pI 9.3). Their sugar-binding specificities are thyroglobulin, N-acetylneuramic acid and galacturonic acid. The mitogenic activities of VVA and VVE with distinct pI values were measured using [methyl -3H]thymidine (3H-TdR) incorporation assays, nucleic acid sequence by rapid amplification of cDNA ends analysis, amino acid sequencing and molecular masses by MALDI-TOF/MS and gel electrophoresis, respectively. / VVA and VVE share 98.2% amino acid sequence similarities. Both VVA and VVE are potent mitogens toward mouse CD3+ & CD4 + T-cells, which were mediated through a calcium-dependent activation signaling pathway (Sze et al., 2004). VVA is slightly more effective than VVE in the induction of T cell activation and proliferation, as demonstrated by 3H-TdR incorporation assays, cell flow cytometry for calcium ion mobilization, immunoblotting blot analysis for tyrosine phosphorylation of Lck proteins and Lck shift (p60lck protein), and two dimensional gel electrophoresis for up-regulated proteins. The gene encoding VV lectin was cloned and characterized. The recombinant protein possessed hemagglutinating activity and mitogenic activity, as demonstrated by hemagglutination assays and 3H-TdR incorporation assays respectively. The endoproteinase Arg-C-digested VVA retained the mitogenic activity but lost the hemagglutinating activity, indicating that the mitogenic activity of VVA is not only dependent on the dimerization and tertiary structure of the protein (Paaventhan et al., 2003; Lin et al., 1997), but also on the primary structure of unique amino acid sequences. These endoproteinase fragments have also been used for study of structure-function relationship. / Sze Cho Wing. / "July 2004." / Adviser: Ken W. K. Liu. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0171. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 156-189). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_343622 |
| Date | January 2004 |
| Contributors | Sze, Cho Wing., Chinese University of Hong Kong Graduate School. Division of Anatomy. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, theses |
| Format | electronic resource, microform, microfiche, 1 online resource (xii, 189 p. : ill.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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