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

Abstract

Charcot-Leyden crystal (CLC) protein, initially reported to possess weak lysophospholipase activity, is still considered to be the eosinophil's lysophospholipase, but it shows no sequence similarities to any known lysophospholipases. In contrast, CLC protein has moderate sequence similarity, conserved genomic organization, and near structural identity to members of the galectin superfamily, and it has been designated galectin-10. To definitively determine whether or not CLC protein is a lysophospholipase, we reassessed its enzymatic activity in peripheral blood eosinophils and an eosinophil myelocyte cell line (AML14.3D10). Antibody affinity chromatography was used to fully deplete CLC protein from eosinophil lysates. The CLC-depleted lysates retained their full lysophospholipase activity, and this activity could be blocked by sulfhydryl group-reactive inhibitors, N-ethylmaleimide and p-chloromercuribenzenesulfonate, previously reported to inhibit the eosinophil enzyme. In contrast, the affinity-purified CLC protein lacked significant lysophospholipase activity. X-ray crystallographic structures of CLC protein in complex with the inhibitors showed that p-chloromercuribenzenesulfonate bound CLC protein via disulfide bonds with Cys 29 and with Cys 57 near the carbohydrate recognition domain (CRD), whereas N-ethylmaleimide bound to the galectin-10 CRD via ring stacking interactions with Trp 72 , in a manner highly analogous to mannose binding to this CRD. Antibodies to rat pancreatic lysophospholipase identified a protein in eosinophil and AML14.3D10 cell lysates, comparable in size with human pancreatic lysophospholipase, which co-purifies in small quantities with CLC protein. Ligand blotting of human and murine eosinophil lysates with CLC protein as probe showed that it binds proteins also recognized by antibodies to pancreatic lysophospholipase. Our results definitively show that CLC protein is not one of the eosinophil's lysophospholipases but that it does interact with eosinophil lysophospholipases and known inhibitors of this lipolytic activity.

REVIEW ARTICLE: Governing the Invasive Trophoblast: Current Aspects on Intra‐ and Extracellular Regulation

“…Gal-10 was found to be essential for the functional properties of CD25(+) Treg cells. 57,58 Gal-12 is preferentially expressed in peripheral blood leukocytes and adipocytes. Previously, it was shown that Gal-12 is induced by cell cycle block at the G(1) phase and causes G(1) arrest when overexpressed.…”

A Unique β1,3-Galactosyltransferase Is Indispensable for the Biosynthesis of N-Glycans Containing Lewis a Structures in Arabidopsis thaliana

“…In addition to the galactosyltransferase domain, all members of subfamily 1 contain a putative galactoside binding lectin domain with similarity to human galectins (Ackerman et al, 2002). Interestingly, such a lectin domain is not present in mammalian B3GALTs (Hennet, 2002).…”

Proteomic identification of Galectin-11 and 14 ligands from Haemonchus contortus

“…LGALS-14 is thought to be the homologue of human galectin-10, which is also secreted by eosinophils (Ackerman et al 2002). Analysis of H. contortus infected sheep demonstrated release of LGALS-14 into the gastrointestinal mucus, the interface of host and parasite interaction (Dunphy et al 2002).…”