The gene for human eosinophil Charcot-Leyden crystal protein directs expression of lysophospholipase activity and spontaneous crystallization in transiently transfected COS cells.
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.
Zhou
Z
Z
Department of Medicine, Beth Israel Hospital, Boston, Massachusetts 02215.
Tenen
D G
DG
Dvorak
A M
AM
Ackerman
S J
SJ
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004804:Eosinophils / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D015971:Gene Expression Regulation, Enzymologic; D006023:Glycoproteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008245:Lysophospholipase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000032:analysis; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure; D014162:Transfection
D006023:Glycoproteins; D011994:Recombinant Proteins; D004247:DNA; D008245:Lysophospholipase; C011711:lysolecithin acylhydrolase