The gene for human eosinophil Charcot-Leyden crystal protein directs expression of lysophospholipase activity and spontaneous crystallization in transiently transfected COS cells

Abstract

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.

Charcot-Leyden Crystal Protein (Galectin-10) Is Not a Dual Function Galectin with Lysophospholipase Activity but Binds a Lysophospholipase Inhibitor in a Novel Structural Fashion

“…Although CLC protein is clearly not one of the eosinophil's LPLases, our results suggest that it may interact with eosinophil LPLases in vitro, perhaps due to an artifactual interaction that occurs during cell lysis procedures used to purify the enzyme activity (15) or CLC protein itself (8,30). Our ligand blotting results indicate that CLC protein does have the capacity to bind the pancreatic-like LPLase expressed by both human and murine eosinophils.…”

“…One unit of enzyme activity represents 1 mol of fatty acid released/h at 37°C and pH 7.5. The identity of the released reaction product was confirmed by thin layer chromatography (15,30). All LPLase assays included a positive control enzyme, Vibrio sp.…”

“…Lysophospholipase Activity Assay-Lysophospholipase activity was measured using a radioenzyme assay as described previously without any modifications (15,30). LPLase activity was measured in 50 -200-l samples of whole cell lysates, the pooled and concentrated CLC-depleted flow-through, and the eluted affinity-purified CLC protein or bacterially expressed recombinant CLC protein in a final volume of 1 ml of buffer containing 100 mM Tris-HCl (pH 7.5), 0.01% Tween 20, and 2 mM EDTA.…”

“…We reassessed the contribution of CLC protein/galectin-10 to eosinophil LPLase activity using antibody affinity chromatography to entirely deplete eosinophil lysates of the protein, a maneuver confirmed by highly sensitive and specific Western blotting (30) and radioimmunoassay (29). We followed this with the demonstration that CLC-depleted eosinophil lysates retained essentially 100% of their LPLase activity.…”

A role for vesicles in human basophil secretion

“…This protein is also present in human eosinophils (Ackerman et al 1982). It exhibits lysophospholipase activity (Weller et al 1984;Zhou et al 1992), has recently been cloned and expressed in COS and Chinese hamster ovary cells (Ackerman et al 1993;Zhou et al 1992), and has had its three-dimensional structure resolved by X-ray crystallography (Leonidas et al 1995). The latter studies reveal that the CLC protein is a unique protein with a dual functional role, based on structural topology similar to galectins-1 and -2 and the identification of a putative lysophospholipase active site (Leonidas et al 1995).…”