Identification and characterization of a novel anti-inflammatory lipid isolated from Mycobacterium vaccae, a soil-derived bacterium with immunoregulatory and stress resilience properties.

RATIONALE: Mycobacterium vaccae (NCTC 11659) is an environmental saprophytic bacterium with anti-inflammatory, immunoregulatory, and stress resilience properties. Previous studies have shown that whole, heat-killed preparations of M. vaccae prevent allergic airway inflammation in a murine model of allergic asthma. Recent studies also demonstrate that immunization with M. vaccae prevents stress-induced exaggeration of proinflammatory cytokine secretion from mesenteric lymph node cells stimulated ex vivo, prevents stress-induced exaggeration of chemically induced colitis in a model of inflammatory bowel disease, and prevents stress-induced anxiety-like defensive behavioral responses. Furthermore, immunization with M. vaccae induces anti-inflammatory responses in the brain and prevents stress-induced exaggeration of microglial priming. However, the molecular mechanisms underlying anti-inflammatory effects of M. vaccae are not known. OBJECTIVES: Our objective was to identify and characterize novel anti-inflammatory molecules from M. vaccae NCTC 11659. METHODS: We have purified and identified a unique anti-inflammatory triglyceride, 1,2,3-tri [Z-10-hexadecenoyl] glycerol, from M. vaccae and evaluated its effects in freshly isolated murine peritoneal macrophages. RESULTS: The free fatty acid form of 1,2,3-tri [Z-10-hexadecenoyl] glycerol, 10(Z)-hexadecenoic acid, decreased lipopolysaccharide-stimulated secretion of the proinflammatory cytokine IL-6 ex vivo. Meanwhile, next-generation RNA sequencing revealed that pretreatment with 10(Z)-hexadecenoic acid upregulated genes associated with peroxisome proliferator-activated receptor alpha (PPARα) signaling in lipopolysaccharide-stimulated macrophages, in association with a broad transcriptional repression of inflammatory markers. We confirmed using luciferase-based transfection assays that 10(Z)-hexadecenoic acid activated PPARα signaling, but not PPARγ, PPARδ, or retinoic acid receptor (RAR) α signaling. The effects of 10(Z)-hexadecenoic acid on lipopolysaccharide-stimulated secretion of IL-6 were prevented by PPARα antagonists and absent in PPARα-deficient mice. CONCLUSION: Future studies should evaluate the effects of 10(Z)-hexadecenoic acid on stress-induced exaggeration of peripheral inflammatory signaling, central neuroinflammatory signaling, and anxiety- and fear-related defensive behavioral responses.

A Novel Glycolipid Antigen for NKT Cells That Preferentially Induces IFN-γ Production.

In this article, we characterize a novel Ag for invariant NKT (iNKT) cells capable of producing an especially robust Th1 response. This glycosphingolipid, DB06-1, is similar in chemical structure to the well-studied α-galactosylceramide (αGalCer), with the only change being a single atom: the substitution of a carbonyl oxygen with a sulfur atom. Although DB06-1 is not a more effective Ag in vitro, the small chemical change has a marked impact on the ability of this lipid Ag to stimulate iNKT cells in vivo, with increased IFN-γ production at 24 h compared with αGalCer, increased IL-12, and increased activation of NK cells to produce IFN-γ. These changes are correlated with an enhanced ability of DB06-1 to load in the CD1d molecules expressed by dendritic cells in vivo. Moreover, structural studies suggest a tighter fit into the CD1d binding groove by DB06-1 compared with αGalCer. Surprisingly, when iNKT cells previously exposed to DB06-1 are restimulated weeks later, they have greatly increased IL-10 production. Therefore, our data are consistent with a model whereby augmented and or prolonged presentation of a glycolipid Ag leads to increased activation of NK cells and a Th1-skewed immune response, which may result, in part, from enhanced loading into CD1d. Furthermore, our data suggest that strong antigenic stimulation in vivo may lead to the expansion of IL-10-producing iNKT cells, which could counteract the benefits of increased early IFN-γ production.

Peroxisome proliferator-activated receptor γ-regulated cathepsin D is required for lipid antigen presentation by dendritic cells.

It is well established that dendritic cells (DCs) take up, process, and present lipid Ags in complex with CD1d molecules to invariant NKT cells. The lipid-activated transcription factor, peroxisome proliferator-activated receptor γ (PPARγ), has previously been shown to regulate CD1d expression in human monocyte-derived DCs, providing a link between lipid metabolism and lipid Ag presentation. We report that PPARγ regulates the expression of a lysosomal protease, cathepsin D (CatD), in human monocyte-derived DCs. Inhibition of CatD specifically reduced the expansion of invariant NKT cells and furthermore resulted in decreased maturation of saposins, a group of lipid transfer proteins required for lysosomal lipid Ag processing and loading. These results reveal a novel mechanism of lipid Ag presentation and identify CatD as a key component of this machinery and firmly place PPARγ as the transcriptional regulator linking lipid metabolism and lipid Ag processing.

A molecular basis for the exquisite CD1d-restricted antigen specificity and functional responses of natural killer T cells.

Natural killer T (NKT) cells respond to a variety of CD1d-restricted antigens (Ags), although the basis for Ag discrimination by the NKT cell receptor (TCR) is unclear. Here we have described NKT TCR fine specificity against several closely related Ags, termed altered glycolipid ligands (AGLs), which differentially stimulate NKT cells. The structures of five ternary complexes all revealed similar docking. Acyl chain modifications did not affect the interaction, but reduced NKT cell proliferation, indicating an affect on Ag processing or presentation. Conversely, truncation of the phytosphingosine chain caused an induced fit mode of TCR binding that affected TCR affinity. Modifications in the glycosyl head group had a direct impact on the TCR interaction and associated cellular response, with ligand potency reflecting the t(1/2) life of the interaction. Accordingly, we have provided a molecular basis for understanding how modifications in AGLs can result in striking alterations in the cellular response of NKT cells.

A rapid fluorescence-based assay for classification of iNKT cell activating glycolipids.

Structural variants of α-galactosylceramide (αGC) that activate invariant natural killer T cells (iNKT cells) are being developed as potential immunomodulatory agents for a variety of applications. Identification of specific forms of these glycolipids that bias responses to favor production of proinflammatory vs anti-inflammatory cytokines is central to current efforts, but this goal has been hampered by the lack of in vitro screening assays that reliably predict the in vivo biological activity of these compounds. Here we describe a fluorescence-based assay to identify functionally distinct αGC analogues. Our assay is based on recent findings showing that presentation of glycolipid antigens by CD1d molecules localized to plasma membrane detergent-resistant microdomains (lipid rafts) is correlated with induction of interferon-γ secretion and Th1-biased cytokine responses. Using an assay that measures lipid raft residency of CD1d molecules loaded with αGC, we screened a library of ∼200 synthetic αGC analogues and identified 19 agonists with potential Th1-biasing activity. Analysis of a subset of these novel candidate Th1 type agonists in vivo in mice confirmed their ability to induce systemic cytokine responses consistent with a Th1 type bias. These results demonstrate the predictive value of this novel in vitro assay for assessing the in vivo functionality of glycolipid agonists and provide the basis for a relatively simple high-throughput assay for identification and functional classification of iNKT cell activating glycolipids.

Α-galactosylceramide analogs with weak agonist activity for human iNKT cells define new candidate anti-inflammatory agents.

CD1d-restricted natural killer T cells with invariant T cell receptor α chains (iNKT cells) are a unique lymphocyte subset that responds to recognition of specific lipid and glycolipid antigens. They are conserved between mice and humans and exert various immunoregulatory functions through their rapid secretion of a variety of cytokines and secondary activation of dendritic cells, B cells and NK cells. In the current study, we analyzed the range of functional activation states of human iNKT cells using a library of novel analogs of α-galactosylceramide (αGalCer), the prototypical iNKT cell antigen. Measurement of cytokines secreted by human iNKT cell clones over a wide range of glycolipid concentrations revealed that iNKT cell ligands could be classified into functional groups, correlating with weak versus strong agonistic activity. The findings established a hierarchy for induction of different cytokines, with thresholds for secretion being consistently lowest for IL-13, higher for interferon-γ (IFNγ), and even higher for IL-4. These findings suggested that human iNKT cells can be intrinsically polarized to selective production of IL-13 by maintaining a low level of activation using weak agonists, whereas selective polarization to IL-4 production cannot be achieved through modulating the strength of the activating ligand. In addition, using a newly designed in vitro system to assess the ability of human iNKT cells to transactivate NK cells, we found that robust secondary induction of interferon-γ secretion by NK cells was associated with strong but not weak agonist ligands of iNKT cells. These results indicate that polarization of human iNKT cell responses to Th2-like or anti-inflammatory effects may best be achieved through selective induction of IL-13 and suggest potential discrepancies with findings from mouse models that may be important in designing iNKT cell-based therapies in humans.

Kinetics and cellular site of glycolipid loading control the outcome of natural killer T cell activation.

CD1d-restricted natural killer T cells (NKT cells) possess a wide range of effector and regulatory activities that are related to their ability to secrete both T helper 1 (Th1) cell- and Th2 cell-type cytokines. We analyzed presentation of NKT cell activating alpha galactosylceramide (alphaGalCer) analogs that give predominantly Th2 cell-type cytokine responses to determine how ligand structure controls the outcome of NKT cell activation. Using a monoclonal antibody specific for alphaGalCer-CD1d complexes to visualize and quantitate glycolipid presentation, we found that Th2 cell-type cytokine-biasing ligands were characterized by rapid and direct loading of cell-surface CD1d proteins. Complexes formed by association of these Th2 cell-type cytokine-biasing alphaGalCer analogs with CD1d showed a distinctive exclusion from ganglioside-enriched, detergent-resistant plasma membrane microdomains of antigen-presenting cells. These findings help to explain how subtle alterations in glycolipid ligand structure can control the balance of proinflammatory and anti-inflammatory activities of NKT cells.

Invariant natural killer T cell-natural killer cell interactions dictate transplantation outcome after alpha-galactosylceramide administration.

Invariant natural killer T cells (iNKT cells) have pivotal roles in graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects. iNKT cells are activated through their T-cell receptors by glycolipid moieties (typically the alpha-galactosylceramide [alpha-GalCer] derivative KRN7000) presented within CD1d. We investigated the ability of modified alpha-GalCer molecules to differentially modulate alloreactivity and GVL. KRN7000 and the N-acyl variant, C20:2, were administered in multiple well-established murine models of allogeneic stem cell transplantation. The highly potent and specific activation of all type I NKT cells with C20:2 failed to exacerbate and in most settings inhibited GVHD late after transplantation, whereas effects on GVL were variable. In contrast, the administration of KRN7000 induced hyperacute GVHD and early mortality in all models tested. Administration of KRN7000, but not C20:2, was found to result in downstream interleukin (IL)-12 and dendritic cell (DC)-dependent natural killer (NK)- and conventional T-cell activation. Specific depletion of host DCs, IL-12, or donor NK cells prevented this pathogenic response and the induction of hyperacute GVHD. These data demonstrate the ability of profound iNKT activation to modulate both the innate and adaptive immune response via the DC-NK-cell interaction and raise concern for the use of alpha-GalCer therapeutically to modulate GVHD and GVL effects.

Identification of distinct human invariant natural killer T-cell response phenotypes to alpha-galactosylceramide.

BACKGROUND: Human CD1d-restricted, invariant natural killer T cells (iNKT) are a unique class of T lymphocytes that recognise glycolipid antigens such as alpha-galactosylceramide (alphaGalCer) and upon T cell receptor (TCR) activation produce both Th1 and Th2 cytokines. iNKT cells expand when cultured in-vitro with alphaGalCer and interleukin 2 (IL-2) in a CD1d-restricted manner. However, the expansion ratio of human iNKT cells varies between individuals and this has implications for attempts to manipulate this pathway therapeutically. We have studied a panel of twenty five healthy human donors to assess the variability in their in-vitro iNKT cell expansion responses to stimulation with CD1d ligands and investigated some of the factors that may influence this phenomenon. RESULTS: Although all donors had comparable numbers of circulating iNKT cells their growth rates in-vitro over 14 days in response to a range of CD1d ligands and IL-2 were highly donor-dependent. Two reproducible donor response patterns of iNKT expansion were seen which we have called 'strong' or 'poor' iNKT responders. Donor response phenotype did not correlate with age, gender, frequency of circulating iNKT, or with the CD1d ligand utilised. Addition of exogenous recombinant human interleukin 4 (IL-4) to 'poor' responder donor cultures significantly increased their iNKT proliferative capacity, but not to levels equivalent to that of 'strong' responder donors. However in 'strong' responder donors, addition of IL-4 to their cultures did not significantly alter the frequency of iNKT cells in the expanded CD3+ population. CONCLUSION: (i) in-vitro expansion of human iNKT cells in response to CD1d ligand activation is highly donor variable, (ii) two reproducible patterns of donor iNKT expansion were observed, which could be classified into 'strong' and 'poor' responder phenotypes, (iii) donor iNKT response phenotypes did not correlate with age, gender, frequency of circulating iNKT cells, or with the CD1d ligand utilised, (iv) addition of IL-4 to 'poor' but not 'strong' responder donor cultures significantly increased their in-vitro iNKT cell expansion to alphaGalCer.

Serum lipids regulate dendritic cell CD1 expression and function.

Dendritic cells (DCs) are highly potent antigen-presenting cells (APCs) and play a vital role in stimulating naïve T cells. Treatment of human blood monocytes with the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 stimulates them to develop into immature dendritic cells (iDCs) in vitro. DCs generated by this pathway have a high capacity to prime and activate resting T cells and prominently express CD1 antigen-presenting molecules on the cell surface. The presence of human serum during the differentiation of iDCs from monocytes inhibits the expression of CD1a, CD1b and CD1c, but not CD1d. Correspondingly, T cells that are restricted by CD1c showed poor responses to DCs that were generated in the presence of human serum, while the responses of CD1d-restricted T cells were enhanced. We chemically fractionated human serum to isolate the bioactive factors that modulate surface expression of CD1 proteins during monocyte to DC differentiation. The human serum components that affected CD1 expression partitioned with polar organic soluble fractions. Lysophosphatidic acid and cardiolipin were identified as lipids present in normal human serum that potently modulate CD1 expression. Control of CD1 expression was mediated at the level of gene transcription and correlated with activation of the peroxisome proliferator-activated receptor (PPAR) nuclear hormone receptors. These findings indicate that the ability of human DCs to present lipid antigens to T cells through expression of CD1 molecules is sensitively regulated by lysophosphatidic acid and cardiolipin in serum, which are ligands that can activate PPAR transcription factors.

Alteration of the relative levels of iNKT cell subsets is associated with chronic mycobacterial infections.

CD1d-restricted invariant natural killer T cells (iNKT cells) have been identified as an important type of effector and regulatory T cell, but their roles in the chronic infectious diseases caused by Mycobacterium tuberculosis and Mycobacterium leprae remain poorly defined. Here, we studied circulating human iNKT cells in blood samples from tuberculosis (TB) and leprosy patients. We found that the percentages of iNKT cells among total circulating T cells in TB and leprosy patients were not significantly different from those in normal controls. However, both TB and leprosy patients showed a selective reduction of the proinflammatory CD4(-)CD8beta(-) (DN) iNKT cells with a proportionate increase in the CD4(+) iNKT cells. Similar phenotypic alterations in circulating iNKT cells were observed in a mouse model of M. tuberculosis infection. Taken together, these findings indicate that the selective reduction of circulating DN iNKT cells is associated with chronic infections caused by M. tuberculosis and M. leprae.

Phage display-derived recombinant antibodies with TCR-like specificity against alpha-galactosylceramide and its analogues in complex with human CD1d molecules.

The glycolipid alpha-galactosylceramide (alpha-GalCer) is a potent activator of invariant natural killer T (iNKT) cells and has been shown to be an effective agent against cancer, infections and autoimmune diseases. The effectiveness of alpha-GalCer and its alkyl chain analogues depends on efficient loading and presentation by the antigen-presenting molecule CD1d. To monitor the ability of CD1d to present the glycolipids, we have used a phage display strategy to generate recombinant antibodies with T cell receptor-like (TCRL) specificity against the human CD1d (hCD1d)-alpha-GalCer complex. These Fab fragments were able to detect specifically hCD1d-alpha-GalCer complexes in cell-free systems such as surface plasmon resonance and ELISA, as well as on the surface of hCD1d(+) antigen-presenting cells (APC) by flow cytometry and immunofluorescence microscopy, the latter of which could also detect intracellular complexes. We show that our TCRL antibodies can stain dendritic cells from CD11c-hCD1d-transgenic mice administered in vivo with alpha-GalCer and its analogues. Furthermore, the antibody was also able to detect the presentation by hCD1d molecules of analogues of alpha-GalCer with the same polar head structure. Using this reagent, we were able to confirm directly that the alpha-GalCer analogue C20:2 preferentially loads onto cell surface CD1d rapidly without the need for internalization, while the loading of alpha-GalCer is improved with longer incubation times on professional APC. This reagent will be essential for assessing the loading and presenting capabilities of hCD1d of alpha-GalCer and its analogues.

Modulation of invariant natural killer T cell cytokine responses by indoleamine 2,3-dioxygenase.

The intracellular enzyme indoleamine 2,3-dioxygenase (IDO), which degrades the rare and essential amino acid tryptophan and converts it into a series of biologically active catabolites, has been linked to the regulation of immune tolerance by specific dendritic cell subsets, and to the downmodulation of exacerbated immune responses. Although the immunoregulatory effects of IDO may be in part due to generalized suppression of cell proliferation caused by tryptophan starvation, there is also evidence that tryptophan catabolites could be directly responsible for some of the observed effects. In this report, we investigated the consequences of IDO activity, particularly with regard to the effects of tryptophan-derived catabolites, on the cytokine responses of activated invariant natural killer T (iNKT) cells, a specialized T cell subset known to have immunoregulatory properties. Our results showed that pharmacologic inhibition of IDO skewed cytokine responses of iNKT cells towards a Th1 profile. In contrast, the presence at low micromolar concentrations of the tryptophan catabolites l-kynurenine, 3-hydroxy-kynurenine, or 3-hydroxy-anthranilic acid shifted the cytokine balance towards a Th2 pattern. These findings have implications for our current understanding of immunoregulation, and the mechanisms by which iNKT cells participate in the modulation of immune responses.

Modulation of human natural killer T cell ligands on TLR-mediated antigen-presenting cell activation.

Invariant natural killer T (iNKT) cells are a subset of nonconventional T cells recognizing endogenous and/or exogenous glycolipid antigens in the context of CD1d molecules. It remains unclear whether innate stimuli can modify the profile of endogenous lipids recognized by iNKT cells on the surface of antigen-presenting cells (APCs). We report that activation of human APCs by Toll-like receptor ligands (TLR-L) modulates the lipid biosynthetic pathway, resulting in enhanced recognition of CD1d-associated lipids by iNKT cells, as defined by IFN-gamma secretion. APC-derived soluble factors further increase CD1d-restricted iNKT cell activation. Finally, using soluble tetrameric iNKT T cell receptors (TCR) as a staining reagent, we demonstrate specific up-regulation of CD1d-bound ligand(s) on TLR-mediated APC maturation. The ability of innate stimuli to modulate the lipid profile of APCs resulting in iNKT cell activation and APC maturation underscores the role of iNKT cells in assisting priming of antigen-specific immune responses.

Rapid identification of immunostimulatory alpha-galactosylceramides using synthetic combinatorial libraries.

Two 60+-membered libraries of alpha-galactosylceramides have been prepared by reactions between activated ester resins and two core, fully deprotected galactosylated sphingoid bases. The libraries were evaluated for their ability to stimulate CD1d-restricted NKT cells, using in vitro stimulation of a murine NKT cell hybridoma line and for their ability to induce the expansion of NKT cells from peripheral blood mononuclear cells (PBMC) of a normal human subject. Our results showed that many compounds constructed on a C18-phytosphingosine base had significant stimulatory activity in both assays. Because no product purification was required, this approach is particularly attractive as a method for rapid synthesis of large libraries of potential immunomodulatory glycosylceramides.

Distinct endosomal trafficking requirements for presentation of autoantigens and exogenous lipids by human CD1d molecules.

CD1d molecules present both self Ags and microbial lipids to NKT cells. Previous studies have established that CD1d lysosomal trafficking is required for presentation of autoantigens to murine invariant NKT cells. We show in this study that this is not necessary for autoantigen presentation by human CD1d, but significantly affects the presentation of exogenous Ags. Wild-type and tail-deleted CD1d molecules stimulated similar autoreactive responses by human NKT clones, whereas presentation of exogenous lipids by tail-deleted CD1d was highly inefficient. Chloroquine treatment markedly inhibited exogenous Ag presentation by wild-type CD1d transfectants, but did not affect NKT autoreactive responses. Conversely, APC expression of HLA-DRalphabeta and the invariant chain (Ii) was associated with faster internalization of CD1d into the endocytic system and enhanced CD1d-mediated presentation of exogenous Ags, but did not appear to augment NKT autoreactivity. Knockdown of the Ii by small interfering RNA resulted in reduced CD1d surface expression and slower internalization in HLA-DR+ APCs, but not HLA-DR- APCs, demonstrating a direct effect of MHC/Ii complexes on CD1d trafficking. CD1d-mediated presentation of exogenous Ags was much more efficient in immature dendritic cells, which actively recycle MHC class II molecules through the endocytic system, than in mature dendritic cells that have stabilized MHC class II expression at the cell surface, suggesting a physiological role for MHC/Ii complexes in modulating CD1d function. These results indicate that autoantigens and exogenous lipids are acquired by human CD1d at distinct cellular locations, and that Ii trafficking selectively regulates CD1d-mediated presentation of extracellular Ags.

The length of lipids bound to human CD1d molecules modulates the affinity of NKT cell TCR and the threshold of NKT cell activation.

CD1d-restricted lymphocytes recognize a broad lipid range. However, how CD1d-restricted lymphocytes translate T cell receptor (TCR) recognition of lipids with similar group heads into distinct biological responses remains unclear. Using a soluble invariant NKT (iNKT) TCR and a newly engineered antibody specific for alpha-galactosylceramide (alpha-GalCer)-human CD1d (hCD1d) complexes, we measured the affinity of binding of iNKT TCR to hCD1d molecules loaded with a panel of alpha-GalCer analogues and assessed the rate of dissociation of alpha-GalCer and alpha-GalCer analogues from hCD1d molecules. We extended this analysis by studying iNKT cell synapse formation and iNKT cell activation by the same panel of alpha-GalCer analogues. Our results indicate the unique role of the lipid chain occupying the hCD1d F' channel in modulating TCR binding affinity to hCD1d-lipid complexes, the formation of stable immunological synapse, and cell activation. These data are consistent with previously described conformational changes between empty and loaded hCD1d molecules (Koch, M., V.S. Stronge, D. Shepherd, S.D. Gadola, B. Mathew, G. Ritter, A.R. Fersht, G.S. Besra, R.R. Schmidt, E.Y. Jones, and V. Cerundolo. 2005. Nat. Immunol 6:819-826), suggesting that incomplete occupation of the hCD1d F' channel results in conformational differences at the TCR recognition surface. This indirect effect provides a general mechanism by which lipid-specific lymphocytes are capable of recognizing both the group head and the length of lipid antigens, ensuring greater specificity of antigen recognition.

Production and characterization of monoclonal antibodies against complexes of the NKT cell ligand alpha-galactosylceramide bound to mouse CD1d.

The alpha-galactosylceramide (alpha-GalCer) known as KRN7000 remains the best studied ligand of the lipid-binding MHC class I-like protein CD1d. The KRN7000:CD1d complex is highly recognized by invariant natural killer T (iNKT) cells, an evolutionarily conserved subset of T lymphocytes that express an unusual semi-invariant T cell antigen receptor, and mediate a variety of proinflammatory and immunoregulatory functions. To facilitate the study of glycolipid antigen presentation to iNKT cells by CD1d, we undertook the production of mouse monoclonal antibodies (mAbs) specific for complexes of KRN7000 bound to mouse CD1d (mCD1d) proteins. Three such monoclonal antibodies were isolated that bound only to mCD1d proteins that were loaded with KRN7000 or closely-related forms of alpha-GalCer. These mAbs showed no reactivity with mCD1d proteins that were not loaded with alpha-GalCer, nor did they bind to complexes formed by loading mCD1d with the self-glycolipid and putative iNKT cell ligand isoglobotrihexosylceramide. These complex-specific monoclonal antibodies allow the direct detection and monitoring of complexes formed by the binding of KRN7000 and other alpha-GalCer analogues to mCD1d. The availability of these mAbs should facilitate a wide range of studies on the biology and potential clinical applications of CD1d-restricted iNKT cells.

Saposin B is the dominant saposin that facilitates lipid binding to human CD1d molecules.

CD1d molecules bind lipid antigens in the endocytic pathway, and access to the pathway is important for the development of CD1d-restricted natural killer T (NKT) cells. Saposins, derived from a common precursor, prosaposin, are small, heat-stable lysosomal glycoproteins required for lysosomal degradation of sphingolipids. Expression of prosaposin is required for efficient lipid binding and recognition of human CD1d molecules by NKT cells. Despite high sequence homology among the four saposins, they have different specificities for lipid substrates and different mechanisms of action. To determine the saposins involved in promoting lipid binding to CD1d, we expressed prosaposin deletion mutants lacking individual saposins in prosaposin-negative, CD1d-positive cells. No individual saposin proved to be absolutely essential, but the absence of saposin B resulted in the lowest recognition of alpha-galactosylceramide by NKT cells. When recombinant exogenous saposins were added to the prosaposin-negative cells, saposin B was the most efficient in restoring CD1d recognition. Saposin B was also the most efficient in mediating alpha-galactosylceramide binding to recombinant plate-bound CD1d and facilitating NKT cell activation. Saposin B could also mediate lipid binding to soluble CD1d molecules in a T cell-independent assay. The optimal pH for saposin B-mediated lipid binding to CD1d, pH 6, is higher than that of lysosomes, suggesting that saposin B may facilitate lipid binding to CD1d molecules throughout the endocytic pathway.

Inactivation of Corynebacterium glutamicum NCgl0452 and the role of MgtA in the biosynthesis of a novel mannosylated glycolipid involved in lipomannan biosynthesis.

Mycobacterium tuberculosis PimB has been demonstrated to catalyze the addition of a mannose residue from GDP-mannose to a monoacylated phosphatidyl-myo-inositol mannoside (Ac(1)PIM(1)) to generate Ac(1)PIM(2). Herein, we describe the disruption of its probable orthologue Cg-pimB and the chemical analysis of glycolipids and lipoglycans isolated from wild type Corynebacterium glutamicum and the C. glutamicum::pimB mutant. Following a careful analysis, two related glycolipids, Gl-A and Gl-X, were found in the parent strain, but Gl-X was absent from the mutant. The biosynthesis of Gl-X was restored in the mutant by complementation with either Cg-pimB or Mt-pimB. Subsequent chemical analyses established Gl-X as 1,2-di-O-C(16)/C(18:1)-(alpha-d-mannopyranosyl)-(1-->4)-(alpha-d-glucopyranosyluronic acid)-(1-->3)-glycerol (ManGlcAGroAc(2)) and Gl-A as the precursor, GlcAGroAc(2). In addition, C. glutamicum::pimB was still able to produce Ac(1)PIM(2), suggesting that Cg-PimB catalyzes the synthesis of ManGlcAGroAc(2) from GlcAGroAc(2). Isolation of lipoglycans from C. glutamicum led to the identification of two related lipoglycans. The larger lipoglycan possessed a lipoarabinomannan-like structure, whereas the smaller lipoglycan was similar to lipomannan (LM). The absence of ManGlcA-GroAc(2) in C. glutamicum::pimB led to a severe reduction in LM. These results suggested that ManGlcAGroAc(2) was further extended to an LM-like molecule. Complementation of C. glutamicum::pimB with Cg-pimB and Mt-pimB led to the restoration of LM biosynthesis. As a result, Cg-PimB, which we have assigned as MgtA, is now clearly defined as a GDP-mannose-dependent alpha-mannosyltransferase from our in vitro analyses and is involved in the biosynthesis of ManGlcAGroAc(2).