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An exoglycosidase-sensitive triggering site on NK cells which is coupled to transmethylation of membrane
Abstract:
Glycosidic enzymes were used as probes to analyze the mechanism of NK cell-mediated cytotoxicity. Pretreatment of nylon wool-enriched CBA/J spleen cells, a murine NK clone, or human peripheral blood lymphocytes (PBL) with alpha-mannosidase, an exoglycosidase, led to a marked dose-dependent inhibition of NK lytic activity against YAC-1.2 or K562 tumor cells. Maximal inhibition occurred after a 60-min pretreatment of murine effectors at 37 degrees C, and the kinetics of NK inhibition by alpha-mannosidase was similar to the reported kinetics for enzymatic activity. Released hexose was detected chemically in the supernatant of mouse spleen cells treated with NK inhibitory dose of alpha-mannosidase, and inactivation of enzymatic function with EDTA reversed the NK inhibitory effect. These results suggest that alpha-mannosidase inhibited NK function by virtue of its enzymatic action. Culture of human PBL for 20-hr after treatment with this enzyme led to a greater than 70% recovery in NK lytic function. Recovery was blocked by incorporating tunicamycin, a glycosylation inhibitor of asparagine-linked glycoproteins, into the culture medium. These results suggest that the alpha-mannosidase-sensitive site may be de novo synthesized glycoprotein. Neuraminidase, beta-galactosidase, endo-beta-N-acetylglucosaminidase-D and H, and peptide-N-glycosidase treatments did not inhibit human NK cell lysis of K562 cells. Pretreatment of nylon wool-enriched CBA/J spleen cells or Percoll-enriched human LGL with alpha-mannosidase did not influence their capacity to bind YAC 1.2 target cells or K562 target cells, respectively, Ca++ pulse experiments revealed that the alpha-mannosidase-sensitive site on the NK cells was involved after target-effector binding but before the Ca++ influx. Pretreatment of effector cells with this enzyme which normally occurs after effector-target cell interaction. These results suggest that the phospholipid methylation reaction is coupled to the alpha-mannosidase-sensitive site on the NK cells. By analogy to other physiologic systems, such as histamine release in mast cells, the triggering of phospholipid methylation in the NK cells may serve as a mechanism for signal transduction across the plasma membrane.
Insights
Alpha-mannosidase enzyme treatment inhibits natural killer (NK) cell cytotoxicity by targeting a specific glycoprotein site involved in signal transduction. This inhibition is reversible and suggests a role for phospholipid methylation in NK cell activation.
Area of Science:
- Immunology
- Cellular Biology
- Biochemistry
Background:
- Natural killer (NK) cells are crucial for innate immunity, mediating cytotoxicity against tumor cells.
- The precise molecular mechanisms underlying NK cell activation and function remain an active area of research.
- Glycosidic enzymes offer potential as probes to dissect cellular processes, including immune cell function.
Purpose of the Study:
- To investigate the role of glycosidic enzymes, specifically alpha-mannosidase, in modulating NK cell-mediated cytotoxicity.
- To elucidate the mechanism by which alpha-mannosidase affects NK cell function and identify the sensitive molecular targets.
- To explore the potential involvement of signal transduction pathways, such as phospholipid methylation, in NK cell activation.
Main Methods:
- Treatment of murine and human NK cells with alpha-mannosidase and other glycosidases.
- Assays for NK cell lytic activity against target tumor cells (YAC-1.2 and K562).
- Detection of released hexose, use of EDTA to assess enzyme activity, tunicamycin to inhibit glycosylation, and Ca++ pulse experiments.
- Analysis of target-effector cell binding and phospholipid methylation assays.
Main Results:
- Alpha-mannosidase treatment caused a dose-dependent inhibition of NK cell cytotoxicity, suggesting enzymatic action is responsible.
- The alpha-mannosidase-sensitive site appears to be a de novo synthesized glycoprotein, as recovery of function was blocked by tunicamycin.
- Inhibition occurred after target-effector binding but before Ca++ influx, implicating a role in signal transduction potentially linked to phospholipid methylation.
Conclusions:
- Alpha-mannosidase acts as a probe to identify a critical glycoprotein on NK cells involved in cytotoxicity.
- This glycoprotein site is essential for signal transduction, likely preceding Ca++ influx and potentially involving phospholipid methylation.
- The findings provide insights into the molecular mechanisms of NK cell activation and cytotoxic function.