Expression of the gene encoding human
eosinophil lysophospholipase, the Charcot-Leyden crystal (CLC)
protein, was studied in transiently transfected COS cells. Recombinant CLC (rCLC)
protein expression was demonstrated both by Western blot and radioimmunoassay inhibition analyses of transfected COS cell extracts and by immunofluorescent staining and ultrastructural immunogold analyses of intact cells. The rCLC
protein was immunochemically indistinguishable from native eosinophil-derived CLC
protein, and each transfected COS cell expressed approximately 11 pg of rCLC
protein as determined by radioimmunoassay and assessment of transfection efficiency. Immunofluorescent microscopy and ultrastructural immunogold analyses localized rCLC
protein to the nucleus, cytoplasm, and plasma membrane of COS cells. Lysates from transfected COS cells producing CLC
protein expressed significant
lysophospholipase activity. Furthermore, rCLC
protein expressed in COS cells spontaneously formed the distinctive intracytoplasmic and intranuclear hexagonal bipyramidal crystals characteristic of the native eosinophil and basophil-derived
protein. Expression of the CLC gene confirms the authenticity of the CLC
cDNA, the expression of
lysophospholipase activity by this unique eosinophil and basophil constituent, and will facilitate the routine purification of the active
enzyme for in vitro and animal model studies of its role (or roles) in eosinophil and basophil associated allergic
inflammation and eosinophil-parasite interactions.