Purification and characterization of the lymphocyte function-associated-2 (LFA-2) molecule

Area of Science:

• Immunology
• Cell Biology
• Protein Biochemistry

Background:

• The lymphocyte function-associated-2 (LFA-2) molecule, equivalent to CD2 and the E rosette receptor, plays a role in T lymphocyte interactions.
• Jurkat T lymphoma cells express high levels of LFA-2 (1.0 X 10(5) sites/cell), making them a suitable source for purification.

Purpose of the Study:

• To purify the LFA-2 molecule from Jurkat T lymphoma cells.
• To characterize the biochemical properties of purified LFA-2.
• To investigate the direct binding of LFA-2 to sheep erythrocytes (E).

Main Methods:

• Monoclonal antibody (MAb) affinity chromatography was used for LFA-2 purification.
• A two-site radioimmunometric assay was developed to monitor purification progress.
• Techniques including immunoblotting, immunoprecipitation, 2D IEF-SDS-PAGE, Cleveland peptide mapping, and gel filtration were employed for characterization.

Main Results:

• Purification yielded 230 micrograms of LFA-2 from 50g of packed cells with a 65% yield and a 13,000-fold purification factor.
• The major purified components had molecular masses of 58,000 and 54,000, corresponding to LFA-2.
• Purified LFA-2 inhibited T lymphocyte rosetting with sheep E and disrupted preformed rosettes, demonstrating direct binding to sheep E.

Conclusions:

• The CD2/LFA-2 molecule directly binds to sheep erythrocytes.
• The purified LFA-2 molecule is suitable for further biochemical and functional studies.
• This study provides the first evidence for the direct interaction between CD2/LFA-2 and sheep E.