title: Epitope mapping of anti-rat CD53 monoclonal antibodies. Implications for the membrane orientation of the Transmembrane 4 Superfamily.
abstract: CD53 is a pan-leukocyte glycoprotein which is a member of the recently described Transmembrane 4 Superfamily (TM4SF) of membrane proteins that are predicted to span the lipid bilayer four times. The major hydrophilic region of murine CD53 was expressed as a glutathione-S-transferase fusion protein, and the epitopes of four mouse anti-rat CD53 monoclonal antibodies (mAb) (OX-44, 2D1, 6E2 and 7D2) were mapped to this region using mouse/rat chimeric fusion proteins. The epitopes of OX-44, 6E2 and 7D2 are restored by the substitution of a single isoleucine residue for threonine at position 154 in the mouse protein. The 2D1 epitope is non-linear and appears to require the juxtaposition of isoleucine at position 154 with one or more of the amino acids arginine (132), methionine (133) and serine (140). All of these epitopes are shown to be sensitive to reduction, thus indicating the importance of disulfide bonding in the correct folding of the CD53 hydrophilic domain. Moreover, as these four mAb recognize CD53 at the cell surface, the data provide direct molecular evidence for the proposed membrane orientation of the TM4SF.
authors.affiliation: MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, GB.
authors.forename: M G
authors.initials: MG
authors.lastname: Tomlinson
authors.forename: A F
authors.initials: AF
authors.lastname: Williams
authors.forename: M D
authors.initials: MD
authors.lastname: Wright
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015703:Antigens, CD / Q000276:immunology*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology*; D001483:Base Sequence; D000939:Epitopes / Q000032:analysis*; D005982:Glutathione Transferase / Q000276:immunology; D008565:Membrane Proteins / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000276:immunology; D060265:Tetraspanin 25
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; C554895:Cd53 protein, mouse; C554896:Cd53 protein, rat; D000939:Epitopes; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D060265:Tetraspanin 25; D005982:Glutathione Transferase