Dr Geoff KrissansenProfessor Jim WatsonPrint, Cristin Gregor2008-12-042008-12-041995Thesis (PhD--Molecular Medicine)--University of Auckland, 1995.http://hdl.handle.net/2292/3194This thesis describes the investigation of three novel leucocyte activation antigens. The investigation began with the characterisation of the αHML-1 integrin subunit. Immunoprecipitation analysis and Western blotting indicated that αHML-1 associates with the β7 integrin subunit to form a typical integrin heterodimer. αHML-1 appears to be post-translationally cleaved into heavy and light chains which remain linked by disulphide bonds. With the aim of isolating the αHML-1 protein, a 45kDa disulphide-linked polypeptide was isolated from leucocytes by two-dimensional non-reducing / reducing gel electrophoresis, and used to raise a polyclonal antiserum. This polyclonal antiserum detected a protein encoded by a cDNA in a λgt11 leucocyte expression library. This cDNA was designated CG-1. It became apparent that CG-1 did not encode the αHML-1 protein, but rather encoded a novel protein which is abundantly expressed in activated leucocytes and reproductive tissues, and weakly expressed in all other tissues examined. The CG-1 gene may be one member of a larger gene family, as two different chicken relatives of CG-1 have been isolated elsewhere. The deduced CG-1 amino acid sequence contains potential transmembrane domains and a potential nuclear localisation signal motif. On the basis of its primary structure the CG-1 protein is expected to form an α helical coiled-coil dimer. A polyclonal antisera raised against a CG-1 - β-galactosidase fusion protein detected a 300kDa protein on the surface of activated human leucocytes. The apparent molecular weight of this protein is halved following the reduction of disulphide bonds, supporting the contention that CG-1 is a disulphide-linked cell-surface dimer. This protein appears to be associated with tyrosine kinase activity in an in vitro kinase assay. While participating in the Fifth International Conference on Leucocyte Differentiation Antigens we noted that the protein detected by the anti-CG-1 antisera was remarkably similar to a novel leucocyte protein designated CD100. CD100 is a T lymphocyte co-stimulatory antigen capable of signalling in concert with the T lymphocyte antigen receptor to produce T lymphocyte proliferation. Like the protein detected by the anti-CG-1 antisera, CD 100 was associated with tyrosine kinase activity in an in vitro kinase assay, and appeared to be a 300kDa disulphide-linked cell-surface dimer. Anti-CD100 antibodies were able to remove the protein detected by the anti-CG-1 antisera from leucocyte lysates in immunodepletion experiments. This suggests that CG-1 and CD100 are immunologically related, or that CD100 and the protein detected by the anti-CG-1 antisera are physically associated. Current research is directed toward determining the relationship between CG-1 and CD100, and investigating the signalling and costimulatory potential of these molecules. The three leucocyte activation antigens investigated in this thesis all appear to mediate leucocyte transmembrane signalling. All three molecules potentially form the basis of therapeutic approaches for immunoregulation of inflammatory and neoplastic disease.Scanned from print thesisenItems in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated.https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htmThesisFields of Research::320000 Medical and Health SciencesCopyright: The authorQ112853691