Abstract
The VLA-4 (alpha 4 beta 1) integrin is involved in the adhesion of cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In order to study alpha 4 structure-function relationships, we have expressed mutated alpha 4 subunit by transfection into VLA-4-negative K562 cells. Substitutions at alpha 4 residues Arg89-Asp90, which show the highest surface probability indexes inside the N-terminal alpha 4/80 fragment, resulted in a reduction in the reactivity of all anti-alpha 4 epitope A monoclonal antibodies (mAbs) tested, compared with the reactivity with anti-alpha 4 epitopes B1, B2, and C mAb, both by transfectant flow cytometry, and by immunoprecipitation and SDS-polyacrylamide gel electrophoresis analysis of transfectant surface-iodinated proteins. In contrast, substitutions at nearby residues, Gln101, Pro102, and Ile105 did not affect the reactivity of any anti-alpha 4 mAb representing the known alpha 4 epitopes. Homotypic cell aggregation triggered by anti-alpha 4 epitope A mAb was prevented in the transfectants expressing mutated alpha 4 Arg89-Asp90Asp residues, while cell aggregation was fully achieved with either anti-alpha 4 epitope B2 or anti-beta 1 mAb. Mutations at alpha 4 residues Gln101, Pro102, and Ile108 did not affect the homotypic cell aggregation of the transfectants expressing these mutations. In addition, the adhesion of mutant Arg89-Asp90 alpha 4 transfectants to the connecting segment-1-containing fibronectin-40 (FN-40) fragment of fibronectin was diminished compared to wild type alpha 4 transfectants, as well as to other mutant alpha 4 transfectants. This adhesion to FN-40 was restored when the activating anti-beta 1 TS2/16 mAb was present in the adhesion assays. In contrast, adhesion to VCAM-1 was not affected by mutations at Arg89-Asp90, nor at Gln101, Pro102, and Ile108 alpha 4 residues. Altogether, these results indicate that alpha 4 residues Arg89 and Asp90 are included in a region involved in homotypic cell aggregation, as well as in adhesion to FN-40, but not to VCAM-1.
Highlights
The ␣4 integrins play an important role in leukocyte extravasation during inflammation, lymphocyte traffic to lymphoid organs during a normal immune response, and hematopoietic progenitor cell adhesion to bone marrow stroma
In the present study we show that residues Arg89 and Asp90 form part of the integrin ␣4 subunit epitope A
Substitutions at these residues resulted in a diminished capability of three anti-␣4 epitope A monoclonal antibodies (mAbs) (HP1/1, HP1/3, and HP1/7) to recognize the ␣4 subunit expressed by RD(89 –90)SA transfectants, which correlated with a decrease in cell adhesion to the FN-40 fragment of fibronectin and lack of homotypic cell aggregation of these transfectants
Summary
The ␣4 integrins play an important role in leukocyte extravasation during inflammation, lymphocyte traffic to lymphoid organs during a normal immune response, and hematopoietic progenitor cell adhesion to bone marrow stroma (reviewed in Refs. 1– 4). Interaction with other sequences on fibronectin [11,12,13], as well as with thrombospondin [14], requires VLA-4 to be activated. ␣47 known ligands include the fibronectin CS-1 region, VCAM-1, and mucosal addressin cell adhesion molecule (MadCAM) (16 –19). The inhibitory effect of the anti-␣4 mAb in these processes comes from the blockade of VLA-4/ligand interactions, resulting in an inhibition of leukocyte recruitment. Epitope A anti-␣4 mAb were able to induce homotypic aggregation, blocked partially adhesion to the CS-1-containing FN-40 fibronectin fragment and did not inhibit adhesion to VCAM-1. Epitope B mAb were subdivided into B1 and B2, both blocking adhesion to FN-40 and VCAM-1, with the difference that B2 mAb triggered homotypic cell aggregation, while B1 mAb did not [39]. The only effect so far described for epitope C mAb is the blocking of homotypic aggregation [39]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
