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Cloning of the murine lymphocyte function-associated molecule-1 alpha-subunit and its expression in COS cells
Y Kaufmann1, E Tseng, T A Springer
1Center for Blood Research, Harvard Medical School, Boston, MA 02115.
Insights
Researchers isolated and expressed the murine alpha subunit cDNA for lymphocyte function-associated molecule 1 (LFA-1). This integrin is crucial for immune cell adhesion and shows high similarity to its human counterpart.
Area of Science:
- Immunology
- Molecular Biology
- Cell Adhesion
Background:
- Lymphocyte function-associated molecule 1 (LFA-1) is an integrin mediating immune cell adhesion.
- LFA-1 interacts with ICAM-1 and ICAM-2, members of the Ig superfamily.
- LFA-1 is composed of alpha (CD11a) and beta (CD18) subunits.
Purpose of the Study:
- To isolate and express the complementary DNA (cDNA) encoding the murine alpha subunit of LFA-1.
- To characterize the sequence and structure of the murine LFA-1 alpha subunit.
- To investigate the expression of the murine LFA-1 alpha subunit in a heterologous cell system.
Main Methods:
- Isolation and sequencing of murine LFA-1 alpha subunit cDNA (GenBank accession no. M60778).
- Bioinformatic analysis to deduce amino acid sequence and structural domains (extracellular, transmembrane, cytoplasmic).
- Co-transfection of COS cells with murine LFA-1 alpha subunit cDNA and beta subunit cDNA (human or murine) for cell surface expression analysis.
Main Results:
- The deduced murine LFA-1 alpha subunit sequence comprises 1061 extracellular, 29 transmembrane, and 50 cytoplasmic amino acids.
- High amino acid identity (72%) was observed between murine and human LFA-1 alpha subunits.
- Significant identity (34%) was found between murine LFA-1 and Mac-1 alpha subunits.
- Successful expression of the murine LFA-1 alpha subunit on COS cell surfaces when co-transfected with appropriate beta subunit cDNA.
Conclusions:
- The murine LFA-1 alpha subunit cDNA has been successfully isolated and characterized.
- The structural features and high homology to the human counterpart suggest conserved function.
- The study demonstrates the feasibility of expressing functional LFA-1 complexes in a heterologous system, aiding further research into immune cell adhesion mechanisms.
Abstract:
The lymphocyte function-associated molecule 1 (LFA-1, CD11a/CD18) is an integrin that mediates adhesion of immune cells by interaction with two members of the Ig superfamily, ICAM-1 and ICAM-2. LFA-1 consists of an alpha subunit (Mr = 180,000) and a beta subunit (Mr = 95,000). We report here the isolation and expression of the murine alpha subunit cDNA (GenBank accession no. M60778). The deduced sequence comprises a 1061 amino acid extracellular domain, a 29 amino acid transmembrane region, and a 50 amino acid cytoplasmic domain. It has a 72% amino acid identity with its human counterpart and 34% identity with the murine Mac-1 alpha subunit. The murine LFA-1 alpha subunit could be expressed on the cell surface of a fibroblastoid cell line, COS, by cotransfection with either the human or murine beta subunit cDNA.