Ultrastructural localization of the Charcot-Leyden crystal protein (lysophospholipase) to a distinct crystalloid-free granule population in mature human eosinophils

Abstract

The Charcot-Leyden crystal (CLC) protein is a unique constituent of eosinophils and basophils. This protein forms the hexagonal bipyramidal crystals observed in tissues at sites of eosinophil accumulations, possesses lysophospholipase activity (lysolecithin acylhydrolase E.C.3.1.1.5), and comprises an estimated 7% to 10% of total eosinophil protein. The ultrastructural localization of CLC protein was studied in mature peripheral blood eosinophils from normal donors and from patients with the idiopathic hypereosinophilic syndrome. Subcellular localization was evaluated by immunoelectron microscopy using eosinophils, both from buffy coat and purified cell suspensions, that were fixed by a variety of methods. Immunochemical detection of CLC protein employed rabbit antiserum to eosinophil CLC protein, affinity chromatography-purified monospecific IgG antibodies, and postembedding immunogold techniques. Controls for specificity included (1) omission of the primary antibody to CLC protein and (2) substitution of primary antibody with a nonimmune preimmunization serum, a protein A-purified nonimmune IgG, or a protein A-purified nonreactive IgG prepared from solid-phase CLC protein-Sepharose-absorbed anti-CLC antiserum. CLC protein was localized to a minor (approximately 5%) subpopulation of eosinophil granules. These membrane-bound cytoplasmic granules were large (greater than 0.5 mu), were devoid of crystalloid inclusions, and were morphologically compatible with persisting eosinophil primary granules. The crystalloid-containing, large, specific granules did not stain for CLC protein. Insufficient numbers of small dense granules, lipid bodies, and vesiculotubular structures were present to adequately evaluate their potential as additional sites for the subcellular localization of CLC protein. The cellular specificity of the immunogold localization of CLC protein in the eosinophil was affirmed by the absence of staining in neutrophils and lymphocytes present in the same sections. The ultrastructural immunogold localization of CLC protein (lysophospholipase) to a large, crystalloid-free granule in mature circulating eosinophils supports the persistence of a distinct “primary” granule population that serves as a major intracytoplasmic repository for this enzyme.

A sputum gene expression signature predicts oral corticosteroid response in asthma

“…These findings show that the signature is responsive to change. The CLC protein, also known as galectin-10, comprises up to 10% of the total proteins in eosinophils [20] and is expressed by both basophils [21] and regulatory T-cells [22]. A dominant protein in mast cell granules, CPA3, is present in a subtype of mast cells that also contain tryptase.…”

Charcot-Leyden crystal protein/galectin-10 interacts with cationic ribonucleases and is required for eosinophil granulogenesis

“…Following incubation of cell lysates with crystal-derived CLC/Gal-10 protein, interacting protein bands were detected by a highly specific affinity-purified anti-CLC/Gal-10 antibody. 59 In addition to detecting the expected endogenous CLC/Gal-10 present in blood eosinophils, this approach also identified a 21-kD protein that interacted with CLC/Gal-10 (Fig 1, A). A and B, CLC/Gal-10 was used as a probe to ''ligand-blot'' blood eosinophil lysate, followed by detection with anti-CLC/Gal-10 antibody.…”

The Enigmatic Charcot-Leyden Crystal Protein (Galectin-10): Speculative Role(s) in the Eosinophil Biology and Function

“…Charcot-Leyden crystal protein is a major constituent of eosinophils (and basophils) [9] and a hallmark of eosinophil-associated inflammatory reactions [10][11][12]. The crystals are distinct, colorless, hexagonal and bi-pyramidal; 20µm to 40µm in length, and 2 µm to 4um across [13,14].…”

“…They are unique to primate eosinophils [15] and are frequently observed in human tissues and secretions in association with eosinophilic inflammatory responses, such as asthma, myeloid leukemias, allergic and parasitic diseases [11,16], and various types of cancers [17][18][19] (Figure 1). The protein is among the most abundant of eosinophil constituents [11,20], comprising an estimated 7% to10% of total eosinophil cellular protein [21]-an amount comparable with the eosinophil's content of toxic cationic proteins and enzymes, such as MBP and EPO [10]. The 16.5 kDa hydrophobic protein [20], localized primarily to a small cytoplasmic granule fraction [9] and to the nucleus of eosinophils [10], lacks a secretion signal peptide and transmembrane domain, and is secreted under certain conditions by nonclassical and novel apocrine mechanisms [4].…”