Selective Recognition of Mannose by the Human Eosinophil Charcot-Leyden Crystal Protein (Galectin-10): A Crystallographic Study at 1.8 Å Resolution^,

Abstract

The role(s) of the eosinophil Charcot-Leyden crystal (CLC) protein in eosinophil or basophil function or associated inflammatory processes is yet to be established. Although the CLC protein has been reported to exhibit weak lysophospholipase activity, it shows virtually no sequence homology to any known member of this family of enzymes. The X-ray crystal structure of the CLC protein is very similar to the structure of the galectins, members of a beta-galactoside-specific animal lectin family, including a partially conserved galectin carbohydrate recognition domain (CRD). In the absence of any known natural carbohydrate ligand for this protein, the functional role of the CLC protein (galectin-10) has remained speculative. Here we describe structural studies on the carbohydrate binding properties of the CLC protein and report the first structure of a carbohydrate in complex with the protein. Interestingly, the CLC protein demonstrates no affinity for beta-galactosides and binds mannose in a manner very different from those of other related galectins that have been shown to bind lactosamine. The partial conservation of residues involved in carbohydrate binding led to significant changes in the topology and chemical nature of the CRD, and has implications for carbohydrate recognition by the CLC protein in vivo and its functional role in the biology of inflammation.

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

“…The extremely low specific activity of CLC protein compared with the enzymatic activities of other known mammalian and prokaryotic LPLases (50 -52) and the lack of significant sequence similarities to any other lipolytic enzymes (lipases, phospholipases, or LPLases) (5, 9, 10) have argued against a role for CLC protein as an eosinophil LPLase. These observations, coupled with the significant similarities of CLC protein to members of the galectin superfamily in terms of amino acid sequence (5), three-dimensional structure (8,14), and organization of the CLC gene (10), prompted us to reevaluate the reported LPLase activity of the protein (2-4, 8, 15, 30).…”

“…Our crystal structures showed that both pCMBS and NEM bound to Cys 29 but that only pCMBS bound to Cys 57 near the CLC CRD. In contrast, the interaction of NEM with residues of the CLC CRD was significantly different and of greater interest, since NEM was found to bind in a manner analogous to the binding of mannose in the CLC crystal, involving stacking interactions of the ring portion of NEM against Trp 72 (14), a highly conserved residue in the CRD of the galectin superfamily (53,54).…”

“…However, our recent studies clearly show that these weak carbohydrate binding activities can be attributed to galectin-10 binding to the cross-linked agarose (or Sepharose) matrix and not to the solid-phased lactosamine-containing simple sugars themselves (13). Most recently, using x-ray crystallographic approaches, we have demonstrated that CLC protein does not show any affinity for ␤-galactosides but can bind mannose (in the crystal) in a manner that is unique and different from the binding of lactosamine-containing sugars by other related galectins (14). A detailed analysis of the topology and chemical nature of CLC protein's CRD in complex with mannose provided the first evidence of possible carbohydrate recognition by this unique lectin.…”

Three-Dimensional Structures of Galectins

“…In the X-ray structure of galectin-10 (1QKQ) (18), CRDs related by a crystallographic 2-fold symmetry are associated in the face-to-face orientation with an interface area of 821 2 (Fig. 4C).…”

Members of the Galectin Network with Deviations from the Canonical Sequence Signature. 1. <i>G</i>alectin-<i>R</i>elated <i>I</i>nter-<i>F</i>iber Prote<i>in</i> (GRIFIN)

“…The loss of galactose-binding capacity in mammalian GRIFINs relates these proteins to galectin-10 (Charcot-Leyden crystal protein; 10% of the total protein in eosinophils (67)) and the galectin-related protein (GRP) (22,(68)(69)(70). Of note, human galectin-10 (no orthologue present in rodents to enable to study the role of this eosinophil protein on T cell suppression in animal models (71,72); its ovine orthologue is called galectin-14 (73)) has four deviations in the signature sequence, binds mannose instead of galactose (74) and also associates with a protein, i.e., pancreatic lysophospholipase (75) (for information on GRP, please see accompanying review (35)). In contrast to the variability of lectin activity among GRIFINs, ability of forming homodimers is maintained.…”