Phylogenetic and structural insights into the origin of C-type lectin Mincle in vertebrates

Abstract

Our bodies are continuously exposed to injurious insults by infection and tissue damage, which are primarily sensed by innate immune receptors to maintain homeostasis. Among such receptors is macrophage-inducible C-type lectin (Mincle, gene symbol CLEC4E), a member of the C-type lectin receptor (CLR) family, which functions as an immune sensor for both pathogens and damaged self. To monitor these injurious stimuli, Mincle recognizes disaccharide-based pathogen-derived glycolipids and monosaccharide-based intracellular metabolites, such as β-glucosylceramide. Mincle is well-conserved among mammals; however, there are questions that remain unclear, such as from which lower vertebrate did it arise and whether the original ligand was self or non-self. Here, we found homologues of Mincle and its signaling subunit Fc receptor γ chain (FcRγ) in lower vertebrates, such as reptiles, amphibians, and fishes. The crystal structure of a Mincle homologue revealed that fish Mincle possesses a narrower sugar-binding pocket than that of mammalian Mincle, and accommodates only monosaccharide moieties. These results suggest that Mincle may have evolved from a self-recognizing receptor, and its sugar-binding pocket widened during evolution, presumably to adapt to disaccharide-based glycolipids derived from life-threatening pathogens.

Keywords: C-type lectin receptors; Crystal structure; FcRγ; Ligand specificity; Molecular phylogenetics; Vertebrates.

Fig. 1

Mincle homologue in Takifugu rubripes. A Amino acid sequence alignment of full-length Mincle and FcRγ from human and T. rubripes. The transmembrane region and the CRD are shaded in gray and yellow, respectively. Amino acid residues that interact with a calcium ion are shown in red, hydrophobic groove-forming residues are shown in blue, and the characteristics of the mammalian Mincle are indicated in bold. The immunoreceptor tyrosine-based activation motif (ITAM) is labeled below the amino acid sequence. B Amino acid sequence alignment of full-length huMincle and Mincle homologues in P. spathula and S. kaupii. The transmembrane region and the CRD are shaded in gray and yellow, respectively. Amino acid residues that interact with a calcium ion are shown in red, hydrophobic groove-forming residues are shown in blue, and residues that are defined as the characteristics of the mammalian Mincle are indicated in bold. C Flow cytometry analysis showing the cell surface expression levels of HA-tagged Mincle and FLAG-tagged FcRγ on HEK293 transfectants. The species are indicated above. Data are representative of three independent experiments

Fig. 2

trMincle is structurally similar to boMincle. A Schematic representation of full-length trMincle (upper) and soluble trMincle (lower). TM, transmembrane helix; CRD, carbohydrate recognition domain. B Overall structure of the trMincle CRD homodimer in complex with glycerol (PDB ID: 9KS7). Protein, glycerol, and calcium ions are shown in ribbon, stick, and sphere models, respectively. C Close-up view of the glycerol binding site in trMincle. The hydroxyl groups 1 and 2 of the glycerol are labeled in gray. Coordination bonds are indicated by brown dotted lines, and hydrogen bonds are indicated by black dotted lines. D List of the top ten PDB structures that are structurally similar to trMincle obtained from a full PDB search of Dali server. PDB IDs and chain names are indicated in the chain column. Descriptions of each structure are indicated in the PDB Description column. Mincle in the list is highlighted in red. E Surface representation of overall ligand binding region of boMincle (PDB ID: 4KZV) (left panel) and trMincle (PDB ID: 9KS7) (right panel). Schematic drawings of the receptor showing representative grooves are given above the panel. Asterisks indicate the corresponding grooves in the structure. F Representative state of trMincle ligand binding region inferred from molecular dynamics simulation. The left panel is the initial state, while the right panel shows the final state of the simulation. These images are depicted from the same view angle. G Comparison of the position of the hydrophobic groove on boMincle (left panel) and the putative hydrophobic groove on trMincle in the final state (right panel). Calcium ion is represented as a gray sphere

Fig. 3

Structural differences in the sugar-binding pocket. A Overall structure of the trMincle CRD homodimer in complex with glucose (PDB ID: 9KPL). Proteins, glucose, and calcium ions are shown in ribbon, stick, and sphere models, respectively. B Close-up view of the glucose binding site in trMincle. The hydroxyl groups 3 and 4 of the glucose molecule are labeled in gray. Coordination bonds are indicated by brown dotted lines, and hydrogen bonds are indicated by black dotted lines. C Distances of amino acid residues shaping the sugar-binding pocket. The measured distances are indicated near the dotted lines. D Overlayed structure of trMincle and boMincle-trehalose complex. Structures were superposed according to the position of the calcium ions in their sugar-binding pockets. Surface of the boMincle and trMincle is shown in orange and blue, respectively. The calcium ion is shown as a gray sphere

Fig. 4

Evolution of ligand binding region of Mincle CRD. A Maximum likelihood tree for amino acid sequences inferred from the BLAST top 10 hits for searching Mincle homologues in amphibians and reptiles. The numbers beside each node indicate bootstrap values calculated with 1000 replications. Amphibian-derived molecules and reptile-derived molecules are highlighted in cyan and green, respectively. Molecules in the same clade with trMincle are indicated as the trMincle group, and those in the same clade with huMincle and boMincle are indicated as the mammalian Mincle group. Human Dectin-1 (huDecin-1) was used as an outgroup. Dali Z-score distributions of B amphibian Mincle homologues and C reptilian Mincle homologues in Dali search. Dali Z-scores were calculated by comparing the structures of the AlphaFold3-predicted CRD structure of Mincle homologues and the crystal structure of huMincle or boMincle. D Superposition of boMincle (PDB ID: 4KZV) (brown) and amphibian Mincle homologues (cyan). The structures were superposed according to the positions of calcium ions in their putative sugar-binding pockets. Structures in trMincle groups and mammalian Mincle groups are shown in the left and right panels, respectively. Individual structures viewed from the same angle are shown in Supplementary Fig. 2C. Trehalose bound in the sugar-binding pocket of boMincle is shown in a stick model. Calcium ion is shown in a sphere model. E Superposition of boMincle (PDB ID: 4KZV) (brown) and reptilian Mincle homologues (green). The structures are superposed according to the positions of calcium ions in their putative sugar-binding pockets. Structures that have steric clashes with trehalose (clash (+)) and those without steric clash (clash (–)) are shown in the left and right panels, respectively. The arrow in the clash (+) panel indicates the location of the clash observed in the structure. Structures of the sugar-binding pocket in individual reptilian protein viewed from the same angle are shown in Supplementary Fig. 2D. Trehalose and calcium ions are shown in a stick model and in a sphere model, respectively. F Scheme that shows the widening of the sugar-binding pocket and the broadened capacity of expected ligands during evolution