title: Expression of soluble isoforms of rat CD45. Analysis by electron microscopy and use in epitope mapping of anti-CD45R monoclonal antibodies.
abstract: The CD45 or leucocyte-common antigens are encoded by a single gene but can be found in various forms due to alternative splicing of three exons near the 5' end of the gene. The CD45 antigens are major glycoproteins of all types of leucocytes. Monoclonal antibodies recognizing restricted epitopes of CD45 have been used to distinguish phenotypic and functional subsets of lymphocytes. To facilitate epitope mapping and biochemical studies, we have expressed the extracellular portions for four different isoforms of rat CD45 in Chinese hamster ovary cells. Constructs were prepared to give four soluble CD45 isoforms, with sequence incorporating either all three alternative exons (sCD45.ABC), the B exon (sCD45.B), the C exon (sCD45.C), or no alternative exons (sCD45.O). These were expressed at approximately 5 mg/l of spent tissue culture supernatant and were antigenically active with monoclonal antibodies (mAb) that recognize all CD45 isoforms. The MRC OX22 and OX32 mAb have been used to split rat CD4+ T cells into functionally distinct subpopulations and the epitopes for these were mapped to the product of exon C. The epitope for MRC OX33, a marker for B cells, requires expression of either the A exon or the A/B exon junction. Electron microscopy showed that the extra segments contributed to an extended structure as has been predicted from the sequence. The shape of the molecule is discussed with regard to other molecules at the leucocyte cell surface.
authors.affiliation: MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford.
authors.forename: M N
authors.initials: MN
authors.lastname: McCall
authors.forename: D M
authors.initials: DM
authors.lastname: Shotton
authors.forename: A N
authors.initials: AN
authors.lastname: Barclay
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D000939:Epitopes / Q000235:genetics*; D006649:Histocompatibility Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D017493:Leukocyte Common Antigens; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000939:Epitopes; D006649:Histocompatibility Antigens; D021382:Protein Sorting Signals; D017493:Leukocyte Common Antigens