Complexes between CD1 molecules and self or microbial glycolipids represent important immunogenic ligands for specific subsets of T cells. However, the function of one of the CD1 family members, CD1e, has yet to be determined. Here, we show that the mycobacterial antigens hexamannosylated phosphatidyl-myo-inositols (PIM6) stimulate CD1b-restricted T cells only after partial digestion of the oligomannose moiety by lysosomal alpha-mannosidase and that soluble CD1e is required for this processing. Furthermore, recombinant CD1e was able to bind glycolipids and assist in the digestion of PIM6. We propose that, through this form of glycolipid editing, CD1e helps expand the repertoire of glycolipidic T cell antigens to optimize antimicrobial immune responses.
The dihydroxyacetone kinase (DhaK) of Escherichia coli consists of three soluble protein subunits. DhaK (YcgT; 39.5 kDa) and DhaL (YcgS; 22.6 kDa) are similar to the N-and C-terminal halves of the ATPdependent DhaK ubiquitous in bacteria, animals and plants. The homodimeric DhaM (YcgC; 51.6 kDa) consists of three domains. The N-terminal dimerization domain has the same fold as the IIA domain (PDB code 1PDO) of the mannose transporter of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS). The middle domain is similar to HPr and the C-terminus is similar to the N-terminal domain of enzyme I (EI) of the PTS. DhaM is phosphorylated three times by phosphoenolpyruvate in an EI-and HPr-dependent reaction. DhaK and DhaL are not phosphorylated. The IIA domain of DhaM, instead of ATP, is the phosphoryl donor to dihydroxyacetone (Dha). Unlike the carbohydrate-speci®c transporters of the PTS, DhaK, DhaL and DhaM have no transport activity.
CD1 proteins present lipid antigens to T cells. The antigens are acquired in the endosomal compartments. This raises the question of how the large hydrophobic CD1 pockets are preserved between the moment of biosynthesis in the endoplasmic reticulum and arrival to the endosomes. To address this issue, the natural ligands associated with a soluble form of human CD1b have been investigated. Using isoelectric focusing, native mass spectrometry and resolving the crystal structure at 1.8 Å resolution, we found that human CD1b is simultaneously associated with endogenous phosphatidylcholine (PC) and a 41-44 carbon atoms-long spacer molecule. The two lipids appear to work in concert to stabilize the CD1b groove, their combined size slightly exceeding the maximal groove capacity. We propose that the spacer serves to prevent binding of ligands with long lipid tails, whereas short-chain lipids might still displace the PC, which is exposed at the groove entrance. The data presented herein explain how the CD1b groove is preserved, and provide a rationale for the in vivo antigen-binding properties of CD1b.
Dihydroxyacetone kinases are a sequence-conserved family of enzymes, which utilize two different phosphoryldonors, ATP in animals, plants and some bacteria, and a multiphosphoprotein of the phosphoenolpyruvate carbohydrate phosphotransferase system in bacteria. Here we report the 2.5-Å crystal structure of the homodimeric Citrobacter freundii dihydroxyacetone kinase complex with an ATP analogue and dihydroxyacetone. The N-terminal domain consists of two ␣/-folds with a molecule of dihydroxyacetone covalently bound in hemiaminal linkage to the N⑀2 of His-220. The Cterminal domain consists of a regular eight-helix ␣-barrel. The eight helices form a deep pocket, which includes a tightly bound phospholipid. Only the lipid headgroup protrudes from the surface. The nucleotide is bound on the top of the barrel across from the entrance to the lipid pocket. The phosphate groups are coordinated by two Mg 2؉ ions to ␥-carboxyl groups of aspartyl residues. The ATP binding site does not contain positively charged or aromatic groups. Paralogues of dihydroxyacetone kinase also occur in association with transcription regulators and proteins of unknown function pointing to biological roles beyond triose metabolism. Dihydroxyacetone (Dha)1 kinases can utilize either of two sources for high energy phosphate, ATP or a phosphoprotein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) (1). Little is known about the function of this enzyme and the metabolic origin of its substrate, Dha. Dihydroxyacetone phosphate (DhaP) can be formed by aldol cleavage of fructose-1,6-bisphosphate, by isomerization from glyceraldehyde-3-phosphate, and by oxidation of glycerol-3-phosphate in the mitochondrial glycerol phosphate shuttle. DhaP is an obligatory precursor of glyceryl ether phospholipid biosynthesis (2). Free Dha plays a pivotal role in methanol assimilation by methylotrophic yeast and plants where it is produced in the transketolase reaction between xylulose-5-phosphate and formaldehyde catalyzed by dihydroxyacetone synthase (3-6). Bacteria produce free Dha by oxidation of glycerol under anaerobic conditions and aldol cleavage of fructose-6-phosphate (9 -13). Dha is a carbon source for bacteria, and if added to the medium, it is also used as gluconeogenic precursor by mammalian tissues (14 -18). Although the pathways utilizing free Dha appear few and limited in scope, genes for Dha kinases and Dha kinase homologues are widely distributed among plants and animals where their biological function is not obvious (for a survey see accession numbers PF02733 and PF02734 at www.sanger.ac.uk/Software/Pfam/index.shtml). Dha like other triose sugars has an increased propensity to react with proteins in Maillard-type reactions (19,20), because unlike hexoses and pentoses, it cannot be deactivated by formation of cyclic hemiacetals. The chemical reactivity of Dha might be the rationale for its use as a therapeutic tanning agent (21, 22). It has recently been shown that Dha can be toxic to yeast cells and that detoxification is d...
CD1b-restricted T lymphocytes recognize a large diversity of mycobacterial lipids, which differ in their hydrophilic heads and the structure of their acyl appendages. Both moieties participate in the antigenicity of lipid Ags, but the structural constraints governing binding to CD1b and generation of antigenic CD1b:lipid Ag complexes are still poorly understood. Here, we investigated the structural requirements conferring antigenicity to Mycobacterium tuberculosis sulfoglycolipid Ags using a combination of CD1b:lipid binding and T cell activation assays with both living dendritic cells and plate-bound recombinant soluble CD1b. Comparison of the antigenicity of a panel of synthetic analogs, sharing the same trehalose-sulfate polar head, but differing in the structure of their acyl tails, shows that the number of C-methyl substituents on the fatty acid, the configuration of the chiral centers, and the respective localization of the two different acyl chains on the sugar moiety govern TCR recognition and T lymphocyte activation. These studies have major implications for the design of sulfoglycolipid analogs with potential use as tuberculosis subunit vaccines.
In methylotrophic yeast, Dha is the primary product of methanol assimilation (1). In bacteria, Dha is formed by oxidation of glycerol or aldol cleavage of fructose-6-phosphate (2, 3). In animal cells, Dha is a gluconeogenic precursor (4-6). The Dha kinase of Escherichia coli was discovered in the proteome as two spots, which were up-regulated in the ptsI mutant, lacking enzyme I of the phosphoenolpyruvate(PEP)-dependent carbohydrate:phosphotransferase system (PTS) (7) and displayed strong amino acid sequence similarities to the N-and C-terminal domains of the ATP-dependent Dha kinase of Citrobacter freundii (7,8). The two subunits, termed DhaK and DhaL (SWISS-PROT entries P76015 and P76014), are encoded in an operon together with a third protein, DhaM (SWISS-PROT entry P37349). DhaM is a multiphosphoryl protein of the PTS with sequence similarity to the IIA domain of the mannose transporter (PDB ID code 1PDO; ref. No similarity with known protein folds could be predicted for DhaK and DhaL. DhaK, DhaL, and DhaM were overexpressed and purified (12). Whereas DhaL precipitates already at low protein concentration, DhaK is soluble. Here we describe the x-ray structure of DhaK with Dha covalently bound to a histidine in the active site. Materials and MethodsProtein Purification and Activity Assay. E. coli DhaK was overproduced in E. coli WA2127⌬HIC(ptsI) as described (12) and purified by ion exchange chromatography over DEAE cellulose (C545, Fluka) and ResourceQ (Amersham Pharmacia) and gel filtration over Superdex 200 (Amersham Pharmacia). DhaK was concentrated to 30 mg⅐ml Ϫ1 in 5 mM Hepes, pH 7.5͞2 mM DTT. The apoform of DhaK was generated between DEAE and ResourceQ chromatography by incubation of the DhaK-Dha complex with PEP and catalytic amounts of the PTS proteins (enzyme I, HPr) DhaM and DhaL. DhaK activity was measured in a coupled assay by reduction of DhaP with glycerol-3-phosphate dehydrogenase. The disappearance of NADH was monitored continuously in a Spectramax 250 plate reader (Molecular Devices) at 30°C (12).Crystallization and Data Collection. The DhaK-Dha complex and the apoprotein were crystallized from 80 mM sodium acetate, pH 5.0͞160 mM (NH 4 ) 2 SO 4 ͞17% (wt͞vol) polyethylene glycol (PEG) 4000͞15% (wt͞vol) 2-methyl-2,4-pentanediol (MPD) using hanging drop vapor diffusion. The crystals had the symmetry of the space group P2 1 2 1 2 (a ϭ 96.5 Å, b ϭ 97.4 Å, c ϭ 86.0 Å, ␣ ϭ  ϭ ␥ ϭ 90°) and they diffracted to 1.75 Å resolution after flash-freezing at 105°K in the native mother liquor. Native and derivative diffraction data were collected on a RAXIS-IV imaging plate detector mounted on a Rigaku RU300 generator equipped with Yale mirrors (Molecular Structure, The Woodlands, TX). All data were processed by using the HKL program package (13).Structure Solution and Refinement. The DhaK structure was determined by the multiple isomorphous replacement (MIR) method. Heavy-atom sites were located with SHELX (14). Phases were calculated by using SHARP (15) and improved by solvent flattening by using the progr...
The mechanisms permitting nonpolymorphic CD1 molecules to present lipid antigens that differ considerably in polar head and aliphatic tails remain elusive. It is also unclear why hydrophobic motifs in the aliphatic tails of some antigens, which presumably embed inside CD1 pockets, contribute to determinants for T-cell recognition. The 1.9-Å crystal structure of an active complex of CD1b and a mycobacterial diacylsulfoglycolipid presented here provides some clues. Upon antigen binding, endogenous spacers of CD1b, which consist of a mixture of diradylglycerols, moved considerably within the lipid-binding groove. Spacer displacement was accompanied by F' pocket closure and an extensive rearrangement of residues exposed to T-cell receptors. Such structural reorganization resulted in reduction of the A' pocket capacity and led to incomplete embedding of the methyl-ramified portion of the phthioceranoyl chain of the antigen, explaining why such hydrophobic motifs are critical for T-cell receptor recognition. Mutagenesis experiments supported the functional importance of the observed structural alterations for T-cell stimulation. Overall, our data delineate a complex molecular mechanism combining spacer repositioning and ligandinduced conformational changes that, together with pocket intricacy, endows CD1b with the required molecular plasticity to present a broad range of structurally diverse antigens.three-dimensional structure | groove shrinking | diacylglycerol endogenous ligand | T lymphocyte activation | CD1b mutant transfectant T lymphocytes have developed the capacity to recognize as antigens a large variety of molecules including peptides, (glyco)lipids, and phosphorylated metabolites (1). Specific recognition of peptides or lipids by T-cell receptors (TCR) occurs when these molecules form antigenic complexes with dedicated antigen-presenting molecules belonging to MHC or CD1 families, respectively. Diversity has forced the immune system to develop appropriate strategies to present antigens in immunogenic form. Polymorphic MHC molecules cope with the peptide repertoire by constraining the ligand conformational space (2). Less clear is how the immune system adapts to the large glycolipid antigenic range and forms antigenic complexes using the functionally nonpolymorphic CD1 molecules.Human antigen-presenting cells (APC) display the CD1a, CD1b, CD1c, and CD1d proteins on their plasma membranes (1, 3). CD1 ectodomains consist of a heavy chain, which folds into three extracellular domains (α1-α3) noncovalently associated with β2-microglobulin (4). Antigen-binding grooves nestle between the α1 and α2 domains and are mostly lined by hydrophobic residues. This allows the antigenic lipids to be anchored via their hydrophobic chains, so that polar motifs protrude toward the aqueous milieu. Consequently, polar heads but not hydrophobic tails are assumed to establish stimulatory contacts with TCRs. Nevertheless, modifications in the lipid chains may also indirectly impact on TCR recognition (5).The number, shape, and conn...
CD1e is the only human CD1 protein existing in soluble form in the late endosomes of dendritic cells, where it facilitates the processing of glycolipid antigens that are ultimately recognized by CD1b-restricted T cells. The precise function of CD1e remains undefined, thus impeding efforts to predict the participation of this protein in the presentation of other antigens. To gain insight into its function, we determined the crystal structure of recombinant CD1e expressed in human cells at 2.90-Å resolution. The structure revealed a groove less intricate than in other CD1 proteins, with a significantly wider portal characterized by a 2 Å-larger spacing between the α1 and α2 helices. No electron density corresponding to endogenous ligands was detected within the groove, despite the presence of ligands unequivocally established by native mass spectrometry in recombinant CD1e. Our structural data indicate that the water-exposed CD1e groove could ensure the establishment of loose contacts with lipids. In agreement with this possibility, lipid association and dissociation processes were found to be considerably faster with CD1e than with CD1b. Moreover, CD1e was found to mediate in vitro the transfer of lipids to CD1b and the displacement of lipids from stable CD1b-antigen complexes. Altogether, these data support that CD1e could have evolved to mediate lipidexchange/editing processes with CD1b and point to a pathway whereby the repertoire of lipid antigens presented by human dendritic cells might be expanded.3D structure | glycolipid antigen presentation | human CD1b | lipid antigen editing | lipid transfer protein F our transmembrane CD1 molecules (CD1a, are expressed in different cell-specific combinations by human immune cells and, among these, in dendritic cells (DCs), the professional antigen-presenting cells (APCs). These proteins present self or microbial lipid antigens to T cells, thus participating in innate and adaptive immunity (1, 2). Myeloid DCs also express a fifth isoform, CD1e, which indirectly participates in glycolipid antigen presentation. This protein has been found to facilitate the processing of complex mycobacterial hexamannosylated phosphatidylinositol (PIM 6 ) by lysosomal α-mannosidase into dimannosylated forms (PIM 2 ) that activate CD1b-restricted T-cell clones (3).In several respects, CD1e behaves differently from the other human CD1 family members. After biosynthesis, all membraneanchored CD1 molecules reach the Golgi compartments. Apart from CD1d, which is also delivered directly to endosomes (4), CD1a-d molecules are then transported to the plasma membrane, where they have been shown to bind some antigens. Subsequently, CD1 molecules are constitutively internalized into the endocytic network, where they capture antigenic ligands (4). Finally, the CD1-antigen complexes cycle back to the plasma membrane to activate specific T lymphocytes. In contrast, CD1e remains exclusively intracellular. After reaching the Golgi compartments, CD1e is addressed to sorting endosomes and from there to CD1...
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