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Isolation and partial characterization of concanavalin A receptors on cloned cytotoxic T lymphocytes

Sitkovsky, Michail V. and Pasternack, Mark S. and Lugo, James P. and Klein, John R. and Eisen, Herman N. (1984) Isolation and partial characterization of concanavalin A receptors on cloned cytotoxic T lymphocytes. Proceedings of the National Academy of Sciences of the United States of America, 81 (5). pp. 1519-1523. ISSN 0027-8424. PMCID PMC344868. doi:10.1073/pnas.81.5.1519.

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A small set of concanavalin A (Con A)-binding glycoproteins was isolated from the surface membrane of cloned cytotoxic T lymphocytes (CTL) and partly identified using monoclonal antibodies. The binding of Con A by these glycoproteins on the CTL surface results in the secretion of γ-interferon and in blocking the effector functions of the cells--namely, antigen-specific and lectin-dependent cytotoxicity. The Con A is evidently bound tightly to some surface structures ("Con A-receptors") that are required for the activation and cytotoxic activity of CTL. To isolate and identify these receptors, antibodies to Con A were used. After Con A was allowed to bind to radiolabeled cloned CTL (labeled with 125I or [35S]methionine or 3H-labeled amino acids), the cells were washed thoroughly, lysed in detergents and anti-Con A antibodies were added to bind to the Con A-receptor complexes. The resulting aggregates were adsorbed with protein A-bearing Staphylococci and the receptors were then specifically released from the pelleted bacteria by α-methyl-D-mannoside and analyzed by polyacrylamide gel electrophoresis under reducing conditions. Eight to nine labeled components were seen by autoradiography and with the aid of monoclonal antibodies to known T-cell surface molecules, four were identified as T200, lymphocyte function-associated antigen (LFA)-1, α- and ß-chains, and (on some clones) Lyt-2. Other components with Mr ~= 160,000, 120,000, 46,000, 42,000, and 23,000 have not been identified. The procedures described here may have general application in the studies of the functional properties of other cell surface molecules.

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Additional Information:© 1984 by the National Academy of Sciences. Contributed by Herman N. Eisen, November 17, 1983. We thank Ms. Joan McDowell of the M.I.T. Cell Sorter Laboratory for her help in performing fluorescence-activated cell sorting. The unpublished observations on photoactivated cross-linking of Con A to Con A receptors were carried out with the expert help of Dr. Martin Schwartz. This work was supported by Research Grants CA-15472 and CA-28900, Training Grant 5-T32-CA-09255 and Center Grant CA-14051 to the Center for Cancer Research, Massachusetts Institute of Technology, from the National Cancer Institute. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Funding AgencyGrant Number
NIH Predoctoral Fellowship5-T32-CA-09255
Subject Keywords:cloned T cells; cell-surface antigens; lectins; γ-interferon; glycoproteins
Issue or Number:5
PubMed Central ID:PMC344868
Record Number:CaltechAUTHORS:SITpnas84
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:10043
Deposited By: Tony Diaz
Deposited On:08 Apr 2008
Last Modified:08 Nov 2021 21:04

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