Cord factor, also called trehalose-6,6'-dimycolate (TDM), is a potent mycobacterial adjuvant. We herein report that the C-type lectin MCL (also called Clec4d) is a TDM receptor that is likely to arise from gene duplication of Mincle (also called Clec4e). Mincle is known to be an inducible receptor recognizing TDM, whereas MCL was constitutively expressed in myeloid cells. To examine the contribution of MCL in response to TDM adjuvant, we generated MCL-deficient mice. TDM promoted innate immune responses, such as granuloma formation, which was severely impaired in MCL-deficient mice. TDM-induced acquired immune responses, such as experimental autoimmune encephalomyelitis (EAE), was almost completely dependent on MCL, but not Mincle. Furthermore, by generating Clec4e(gfp) reporter mice, we found that MCL was also crucial for driving Mincle induction upon TDM stimulation. These results suggest that MCL is an FcRγ-coupled activating receptor that mediates the adjuvanticity of TDM.

Mincle [macrophage inducible Ca2+ -dependent (C-type) lectin; CLEC4E] and MCL (macrophage C-type lectin; CLEC4D) are receptors for the cord factor TDM (trehalose-6,6′-dimycolate), a unique glycolipid of mycobacterial cell-surface components, and activate immune cells to confer adjuvant activity. Although it is known that receptor-TDM interactions require both sugar and lipid moieties of TDM, the mechanisms of glycolipid recognition by Mincle and MCL remain unclear. We here report the crystal structures of Mincle, MCL, and the Mincle-citric acid complex. The structures revealed that these receptors are capable of interacting with sugar in a Ca 2+ -dependent manner, as observed in other C-type lectins. However, Mincle and MCL uniquely possess shallow hydrophobic regions found adjacent to their putative sugar binding sites, which reasonably locate for recognition of fatty acid moieties of glycolipids. Functional studies using mutant receptors as well as glycolipid ligands support this deduced binding mode. These results give insight into the molecular mechanism of glycolipid recognition through C-type lectin receptors, which may provide clues to rational design for effective adjuvants.X-ray crystallography | innate immunity | mycobacteria | pattern-recognition receptors | myeloid cells

New dinucleating ligands having two metal-binding sites bridged by an imidazolate moiety, Hbdpi and HMe4bdpi (Hbdpi = 4,5-bis(di(2-pyridylmethyl)aminomethyl)imidazole, HMe4bdpi = 4,5-bis(di(6-methyl-2-pyridylmethyl)aminomethyl)imidazole), have been designed and synthesized as model ligands for copper−zinc superoxide dismutase (Cu,Zn-SOD). The corresponding mononucleating ligand, MeIm(Py)2 (MeIm(Py)2 = ((1-methyl-4-imidazolyl)methyl)bis(2-pyridylmethyl)amine), has also been synthesized for comparison. The imidazolate-bridged Cu(II)−Cu(II) homodinuclear complexes represented as [Cu2(bdpi)(CH3CN)2](ClO4)3·CH3CN·3H2O (1), [Cu2(Me4bdpi)(H2O)2](ClO4)3·4H2O (2), a Cu(II)−Zn(II) heterodinuclear complex of the type of [CuZn(bdpi)(CH3CN)2](ClO4)3·2CH3CN (3), and a Cu(II) mononuclear complex of [Cu(MeIm(Py)2(CH3CN)](ClO4)2·CH3CN (4) have been synthesized, and the structures of complexes 1−4 were determined by X-ray crystallography. The Cu(II)−Zn(II) distance of 6.197(2) Å in 3 agrees well with that of native Cu,Zn-SOD (6.2 Å). All the metals in 1−4 have pentacoordinate geometries with the imidazolate or 1-methylimidazole nitrogen, two pyridine nitrogens, the tertiary amine nitrogen, and a solvent (CH3CN or H2O). The coordination site occupied by a solvent can be susceptible to ligand substitution, providing a binding site for substrate superoxide. Magnetic measurements of the Cu(II)−Cu(II) homodinuclear complexes 1 and 2 have shown an antiferromagnetic exchange interaction with a coupling constant of −2J = 73.4 and 145.9 cm-1, respectively. The ESR spectra of 1 and 2 exhibited broad signals centered at g ≅ 2.13 due to the spin−spin interaction between two copper ions, while the ESR spectrum of the Cu(II)−Zn(II) heterodinuclear complex 3 showed a signal which is characteristic of mononuclear trigonal-bipyramidal Cu(II) complexes (g ∥ = 2.10, g ⊥ = 2.24, |A ∥| = 11.7 mT, and |A ⊥| = 12.4 mT). The cyclic voltammograms of homodinuclear complexes (1 and 2) in CH3CN gave two reversible waves which correspond to the Cu(I,I)/Cu(I,II) and Cu(I,II)/Cu(II,II) redox processes: E 1/2 = −0.31 and −0.03 V vs Ag/AgCl for 1 and E 1/2 = −0.29 and +0.12 V vs Ag/AgCl for 2, respectively. On the other hand, the Cu(II)−Zn(II) heterodinuclear complex 3 exhibited one Cu(I)/(II) reversible wave, E 1/2 = −0.03 V vs Ag/AgCl, which is shifted in a positive direction (0.21 and 0.19 V) as compared to those of the corresponding Cu(II) mononuclear complexes. All the examined complexes catalyzed the dismutation of superoxide at biological pH; the SOD activity increased in the order 2 < 1 < 3.