title: Identification of a vimentin-like function associated molecule (FAM) on rat NK cells: evidence for receptor function.
abstract: Monoclonal antibody (MoAb) 5C6 specifically binds to fish, rat and human NK cells and inhibits cytotoxicity. The molecule recognized by this MoAb is a 50-53-kDa membrane protein on rat leukaemic NK (CRC) cells. In the present study, we have obtained a partial internal amino acid sequence from a purified 42-kDa fragment of the CRC-function associated molecule (FAM). Three tryptic peptide fragments were sequenced and each showed homology to intermediate filament vimentin sequences as deduced from (GenBank) mouse cDNA sequences. Amino acid composition analysis indicated that similar to cytoskeletal vimentin, the FAM contained a high percentage of non-polar amino acids. To further assess the similarities between this protein and vimentin, two commercially available anti-vimentin MoAbs and one anti-vimentin polyclonal antibody were tested for binding and inhibition of NK cytotoxicity. All anti-vimentin MoAbs inhibited killing by rat NWNA cells of appropriate targets. Anti-vimentin MoAb 13.2 bound to 41% of NWNA cells compared with approximately 58% binding for MoAb 5C6. Capping and sequential binding experiments showed that MoAb 5C6 effectively removed, from CRC-cell membranes, the protein recognized by MoAb V9. Sequential addition of these two MoAbs (MoAb 13.2 followed by MoAb V9) to CRC cells did not produce competitive binding. Biochemical and Western blot analysis of the vimentin-like protein obtained from CRC cells indicated that this protein has a molecular weight of 48-50 kDa, with an isoelectric point of pH 6.1-6.3. This protein is cross-reactive by Western blot analysis with anti-vimentin and anti-intermediate filament (IFA) antigen MoAbs but not with anti-desmin or anti-actin MoAbs. The molecular weight heterogeneity (43 versus 48-50 kDa) of the CRC protein was also examined. Western blot analysis of the CRC extract after different in vitro incubation times at 37 degrees C and 4 degrees C demonstrated that the 50-53-kDa 'native' protein degraded to a 42-kDa protein by 24 and 48 h respectively. This degradation was inhibitable by 10 mM EGTA. Evidence is presented which indicates that a vimentin-like protein on transformed rat NK cells may be an antigen binding receptor which initiates target cell lysis.
authors.affiliation: Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens 30602.
authors.forename: D L
authors.initials: DL
authors.lastname: Evans
authors.forename: D T
authors.initials: DT
authors.lastname: Harris
authors.forename: J H
authors.initials: JH
authors.lastname: Leary
authors.forename: A L
authors.initials: AL
authors.lastname: St John
authors.forename: L
authors.initials: L
authors.lastname: Jaso-Friedman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000954:Antigens, Surface / Q000737:chemistry; D001667:Binding, Competitive; D006224:Cricetinae; D003429:Cross Reactions; D003602:Cytotoxicity, Immunologic / Q000276:immunology; D005399:Fishes; D005434:Flow Cytometry; D007152:Immunologic Capping; D007694:Killer Cells, Natural / Q000737:chemistry* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D051381:Rats; D011955:Receptors, Drug / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured / Q000473:pathology; D014746:Vimentin / Q000378:metabolism / Q000502:physiology*
chemical_list: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D008565:Membrane Proteins; D010446:Peptide Fragments; D011955:Receptors, Drug; D014746:Vimentin