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Identification of a vimentin-like function associated molecule (FAM) on rat NK cells: evidence for receptor function
D L Evans1, D T Harris, J H Leary
1Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens 30602.
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.
Insights
Monoclonal antibody 5C6 targets a vimentin-like protein on NK cells, inhibiting cytotoxicity. This protein may function as an antigen-binding receptor initiating target cell lysis.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Monoclonal antibody (MoAb) 5C6 binds to NK cells in fish, rats, and humans, inhibiting their cytotoxic activity.
- The target of MoAb 5C6 is a 50-53 kDa membrane protein on rat leukaemic NK (CRC) cells, termed Function Associated Molecule (FAM).
Purpose of the Study:
- To characterize the FAM and investigate its relationship with vimentin.
- To determine if vimentin antibodies can inhibit NK cell cytotoxicity and bind to the FAM.
Main Methods:
- Partial internal amino acid sequencing of a 42 kDa FAM fragment.
- Homology search against GenBank mouse cDNA sequences for vimentin.
- Amino acid composition analysis.
- Binding and cytotoxicity inhibition assays using anti-vimentin antibodies.
- Biochemical analysis and Western blotting of the FAM.
- Degradation studies of the FAM under different temperature and time conditions.
Main Results:
- Sequenced peptides showed homology to intermediate filament vimentin.
- FAM exhibited a high percentage of non-polar amino acids, similar to vimentin.
- Anti-vimentin MoAbs inhibited rat NK cell cytotoxicity.
- MoAb 5C6 and anti-vimentin MoAb V9 recognized distinct epitopes on the FAM.
- The vimentin-like protein from CRC cells had a molecular weight of 48-50 kDa and cross-reacted with anti-vimentin and anti-intermediate filament antibodies.
- The native 50-53 kDa protein degraded to a 42 kDa fragment over time, an event inhibitable by EGTA.
Conclusions:
- The FAM is a vimentin-like protein expressed on transformed rat NK cells.
- This vimentin-like protein may serve as an antigen-binding receptor crucial for initiating target cell lysis by NK cells.