Removal of lymphocyte surface molecules with phosphatidylinositol-specific phospholipase C: effects on mitogen responses and evidence that ThB and certain Qa antigens are membrane-anchored via phosphatidylinositol.
We reported previously that the Thy-1 antigen was released from murine thymocytes and thymoma cells by S. aureus-derived phosphatidylinositol-specific phospholipase C (PI-PLC). It is therefore part of a small group of proteins known to use a unique form of membrane attachment. This finding has now been extended in studies with peripheral lymphocytes and additional leukocyte markers. Retention of viability and responsiveness to LPS were excellent in PI-PLC-treated spleen cells and there was no appreciable effect on lectin-binding surface glycoproteins. Thy-1 regeneration was insignificant on unstimulated spleen cells within 24 hr of treatment, but nearly complete at this time with a continuously dividing cell line. In contrast to the result with LPS, responses to the mitogens Con A, PHA, and PWM were virtually eliminated. Of more than 40 monoclonal antibodies tested, only staining with ThB and particular Qa specificities were diminished by PI-PLC treatment. The latter included Qa-2, Qa-4, Qa-5, and possibly also Qa-6, whereas Qa-1, TLa, and other class I and class II histocompatibility antigens were unaffected. Although the validity of the Qa results seems assured by the total PI-PLC resistance of many other lymphocyte antigens, the pattern of release was notably different from that observed with Thy-1 and ThB. That is, the density of Qa-2 was usually unchanged on a subpopulation of Qa-2-positive cells. This raises interesting questions about lymphocyte heterogeneity and flexibility in the use of this form of surface protein anchoring. Glycosyl-phosphatidylinositol-linked proteins may be functionally significant in immunological responses, and this experimental approach should continue to be valuable for their identification and characterization.
D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000378:metabolism*; D006017:Glycolipids / Q000502:physiology*; D017261:Glycosylphosphatidylinositols; D015395:Histocompatibility Antigens Class I; D008213:Lymphocyte Activation / Q000187:drug effects; D008214:Lymphocytes / Q000276:immunology / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008934:Mitogens / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000502:physiology*; D011975:Receptors, Mitogen / Q000378:metabolism; D010738:Type C Phospholipases
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins; D008934:Mitogens; D010716:Phosphatidylinositols; C032907:Q surface antigens; D011975:Receptors, Mitogen; D010738:Type C Phospholipases