Student Theses and Dissertations
Date of Award
1980
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Christian de Duve
Keywords
lymphocyte mitogenesis, oxidative agents, sialoglycoproteins, protease treatment, monoclonal antibody, blast transformation
Abstract
The objectives of this thesis work involved the identification and characterization of rat lymphocyte surface components involved in mitogenic stimulation by the oxidative agents periodate and neuraminidase plus galactose oxidase (NGO). Lymph node cell blastogenesis induced by these oxidative agents was inhibited by reduction with borohydride or reaction with cysteine methyl ester (CME) without affecting the ability of lymph node cells (LNC) to respond to concanavalin A (Con A). 35S-CME was synthesized, and oxidized LNC were reacted with either the radioactive CME or 3H-borohydride. The radioactively-tagged LNC were then solubilized in sodium dodecyl sulfate (SDS) and electrophoresced on SDS-polyacrylamide gradient slab gels. Fluorography of these gels demonstrated that 35S-CME- and 3H- borohydride-Iabeled LNC had identical radioactive banding patterns. Differential centrifugation and density gradient isopycnic centrifugation indicated that the radioactive components distributed with the plasma membrane markers: 5'-nucleotidase, γ-glutamyl transpeptidase, and alkaline phosphodiesterase. The labeling was shown to be specific and uniform for all oxidized components; radioactive Schiff base adducts could not be detected following 3H-borohydride reduction. Similar sets of molecules were labeled after periodate or NGO reaction. However, some molecules showed a differential sensitivity to periodate or NGO oxidation, or migrated somewhat differently in SDS-polyacrylamide gradient slab gels depending on the oxidizing agent. A heavily-labeled low molecular weight component was shown to be glycolipid. A comparison of the labeling of thoracic duct lymphocytes, thymocytes, cortisone-resistant thymocytes, and LNC indicated that lymphoid populations capable of responding to periodate or NGO stimulation (LNC, thoracic duct lymphocytes, and cortisoneresistant thymocytes) had similar radioactive banding patterns. Non-responsive populations (thymocytes) had somewhat different surface labeling. Rat erythrocytes had labeled glycoproteins and overall banding patterns distinct from those seen with any of the lymphoid preparations examined. Periodate and NGO have been shown by other workers to be T-cell mitogens, and the induction of the stimulation by these agents has been shown to require the interaction of two distinct cell types: the oxidized, responding lymphocyte, and an accessory cell that need not be mitogen-treated. Analysis of nylon wool-purified T-cells and Ficoll gradient-separated responder lymphocytes (both from LNC) showed that these cells had the same labeling patterns as intact LNC populations. Papain was found to inhibit the stimulation by periodate or NGO in a dose-dependent fashion without affecting the kinetics or magnitude of a Con A response. Treatment with the protease either before or after oxidation had the same result. Heat inactivation of the papain abrogated its inhibitory effect. Trypsin, chymotrypsin, and thermolysin were also found to inhibit oxidative mitogenesis (stimulation by periodate or NGO) in a dosedependent fashion without reducing the magnitude of the Con A stimulation. However, the Con A response was delayed by 12-24 hours. None of the proteases affected the immediate or long-term viability of LNC in culture, and none was directly mitogenic for LNC. Protease treatment of Ficoll gradient-separated responder lymphocytes caused an inhibition of oxidative mitogenesis comparable to protease treatment of the entire LNC population. Radioactive labeling of protease-treated, oxidized LNC gave rise to banding patterns which were unique for each enzyme and distinct from non-digested LNC. Papain-treated responder lymphocytes had the same banding pattern as papain-treated whole LNC populations. Membrane-bound, radio-labeled fragments and protease-resistant molecules differed for each of the four enzymes; glycolipid labeling was not affected. Despite the variable effects of the enzymes on the labeling patterns, levels of the proteases which were inhibitory for stimulation by periodate or NGO consistently caused the loss of four labeled components representing sialoglycoproteins of molecular weights 175,000, 170,000, 160,000, and 155,000 daltons (designated t1-t4). A monoclonal mouse immunoglobulin (IgG class) originally raised against a protease fragment of the 155,000 dalton molecule on thymocytes, was found to cross-react with all four of the high molecular weight components on LNC as assessed by immunoprecipitation and gel analysis. Moreover, the monoclonal antibody, designated MRC OX1, was also found to be stimulatory for LNC. Both immunoprecipitation and blast transformation required the use of second antibody specific for mouse immunoglobulin; either F(ab')2 or intact immunoglobulin (Ig) second antibody were found to work. In LNC depleted of B-cells by anti-Ig plus complement lysis, neither MRC OX1 nor anti-Ig second antibody were stimulatory. Combined treatment with optimal concentrations of both antisera (generally in a 1:5 to 1:10 ratio of first:second antibody) gave rise to a blast response with kinetics identical to a periodate response and with a magnitude approximately 1/5 to 1/10 as strong. Efforts to increase the magnitude of the response by using protein A, neuraminidase treatment, heat-aggregated MRC OX1, additional accessory cells, or adding non-mitogenic lectins were not successful. It is not currently clear whether the relatively weak response is caused by the known low affinity of the monoclonal antiserum, or is due to the stimulation of only a sub-population of LNC with a high density of the relevant t1-t4 molecules. Taken together, the results strongly implicate a role for the high molecular weight t1-t4 tetrad of sialoglycoproteins in oxidative mitogenesis, and indicate that direct cross-linking of these molecules results in blast transformation. The mechanism of stimulation via this cross-linking, however, remains at best speculative.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Mitchell, Richard Neal, "Rat Lymphocyte Surface Glycoproteins Mediating Mitogenesis by Periodate or Neuraminidase Plus Galactose Oxidase" (1980). Student Theses and Dissertations. 475.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/475
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy