Functional and biosynthetic studies on granulysin, a cytotoxic and antimicrobial protein produced by cytolytic lymphocytes

Cytolytic T lymphocytes (CTL) and natural killer (NK) cells destroy cells infected with viruses and bacteria, and tumor cells. The directional release of preformed granules containing effector molecules constitutes one mechanism by which they mediate this cytolytic activity. Granulysin is a recently identified molecule that is selectively expressed by CTL and NK cells and localizes to cytolytic granules. This dissertation characterizes the cytotoxic activity, antimicrobial properties, and biosynthesis of granulysin.;CTL express two granulysin proteins of 15 and 9 kDa. Recombinant 9 kDa granulysin possessed a dose-dependent cytotoxic activity, lysing the T cell tumor Jurkat with hallmarks of apoptotic cell death. The cytotoxic activity was resistant to boiling for 10 minutes. Reduction of intramolecular disulfide bonds with dithiothreitol quantitatively increased granulysin's specific activity and qualitatively altered the cytotoxic activity, illustrating the importance of structure to the nature of the cytotoxicity induced.;Recombinant 9 kDa granulysin also displayed broad antimicrobial activity, killing bacteria, yeasts, and a parasite. A granule-dependent killing activity of intracellular Mycobacterium tuberculosis displayed by CD8+ T cells correlated with granulysin expression, implicating it in this activity. In support of this hypothesis, recombinant 9 kDa granulysin killed extracellular Mycobacterium tuberculosis. However it did not kill intracellular Mycobacterium tuberculosis. Granulysin co-incubated with purified perforin, with which it co-localized inside T cells, lysed over 90% of intracellular Mycobacterium tuberculosis, further implicating granulysin as an antimicrobial effector molecule of T cells.;CTL express three granulysin messages (519, 520, and 522) but overexpression of the predominate 520 message in a NK cell tumor was sufficient for the production of both granulysin proteins. 15 kDa granulysin is post-translationally processed to 9 kDa granulysin in the acidic environment of cytolytic granules. An altered structure of the 15 kDa granulysin in comparison to 9 kDa granulysin is proposed to render the 15 kDa proprotein non-lytic. 9 kDa granulysin's lytic activity is inhibited by a decrease in pH, precisely the condition necessary for it's liberation from 15 kDa granulysin. Thus the details of granulysin's biosynthesis illuminate a strategy by which expressing cells can produce and store high levels of a lytic protein without causing autolysis.