Saposins utilize two strategies for lipid transfer and CD1 antigen presentation

Funding: We are grateful to A.N. Odyniec, M. Brigl, G.F.M. Watts, and T.Y. Cheng for suggestions and excellent technical assistance. This work was supported by National Institutes of Health (NIH) Grants AI028973 and AI063428 (to M.B.B.), DK36729 and NS36681 (to G.A.G.), and AR048632 and AI049313 (to D.B.M. and A.K.); a Howard Hughes Medical Institute Gilliam Fellowship (to L.L.); the Burroughs Wellcome Fund (D.B.M. and A.K.); a Personal Research Chair from Mr. James Bardrick (to V.B., N.V., and G.S.B.); a Royal Society Wolfson Research Merit Award (to V.B., N.V., and G.S.B.); the Medical Research Council (V.B., N.V., and G.S.B.); Wellcome Trust Grant 084923/B/08/Z (to V.B., N.V., and G.S.B.); and a Netherlands Organization for Scientific Research Grant (to A.J.M.Transferring lipid antigens from membranes into CD1 antigen-presenting proteins represents a major molecular hurdle necessary for T-cell recognition. Saposins facilitate this process, but the mechanisms used are not well understood. We found that saposin B forms soluble saposin protein-lipid complexes detected by native gel electrophoresis that can directly load CD1 proteins. Because saposin B must bind lipids directly to function, we found it could not accommodate long acyl chain containing lipids. In contrast, saposin C facilitates CD1 lipid loading in a different way. It uses a stable, membrane-associated topology and was capable of loading lipid antigens without forming soluble saposin-lipid antigen complexes. These findings reveal how saposins use different strategies to facilitate transfer of structurally diverse lipid antigens.publishersversionpublishe

Comparative Genomics of Cell Envelope Components in Mycobacteria

Mycobacterial cell envelope components have been a major focus of research due to their unique features that confer intrinsic resistance to antibiotics and chemicals apart from serving as a low-permeability barrier. The complex lipids secreted by Mycobacteria are known to evoke/repress host-immune response and thus contribute to its pathogenicity. This study focuses on the comparative genomics of the biosynthetic machinery of cell wall components across 21-mycobacterial genomes available in GenBank release 179.0. An insight into survival in varied environments could be attributed to its variation in the biosynthetic machinery. Gene-specific motifs like ‘DLLAQPTPAW’ of ufaA1 gene, novel functional linkages such as involvement of Rv0227c in mycolate biosynthesis; Rv2613c in LAM biosynthesis and Rv1209 in arabinogalactan peptidoglycan biosynthesis were detected in this study. These predictions correlate well with the available mutant and coexpression data from TBDB. It also helped to arrive at a minimal functional gene set for these biosynthetic pathways that complements findings using TraSH

Symmetrical and unsymmetrical analogues of isoxyl; active agents against mycobacterium tuberculosis

Symmetrical and unsymmetrical analogues of the antimycobacterial agent isoxyl-have been synthesized and tested against Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG, some showing an increased bactericidal effect. In particular, compounds 1-(p-n-butylphenyl)-3-(4-propoxy-phenyl) thiourea (10) and 1-(p-n-butylphenyl)-3-(4-n-butoxy-phenyl) thiourea (11) showed an approximate 10-fold increase in in vitro potency compared to isoxyl, paralleled by increased inhibition of mycolic acid biosynthesis in M. bovis BCG. Interestingly, these isoxyl analogues showed relatively poor inhibition of oleate production, suggesting that the modifications have changed the spectrum of biological activity

Crystal structure of bovine CD1b3 with endogenously bound ligands

The CD1 family of Ag-presenting molecules is able to display lipids to T cells by binding them within a hydrophobic groove connected to the protein surface. In particular, the CD1b isotype is capable of binding ligands with greatly varying alkyl chain lengths through a complex network of interconnected hydrophobic pockets. Interestingly, mycobacterial lipids such as glucose monomycolate exclusively bind to CD1b.We determined the crystal structure of one of the three expressed bovine CD1b proteins, CD1b3, in complex with endogenous ligands, identified by mass spectrometry as a mixture of phosphatidylcholine and phosphatidylethanolamine, and analyzed the ability of the protein to bind glycolipids in vitro. The structure reveals a complex binding groove architecture, similar to the human ortholog but with consequential differences. Intriguingly, in bovine CD1b3 only the A’, C’ and F’ pockets are present, whereas the T’ pocket previously described in human CD1b is closed. This different pocket conformation could affect the ability of boCD1b3 to recognize lipids with long acyl chains such as glucose monomycolate. However, even in the absence of a T’ tunnel, bovine CD1b3 is able to bind mycolates from Rhodococcus ruber in vitro

Identification of a potent microbial lipid antigen for diverse NKT cells

Invariant Natural Killer T (iNKT) cells are a well-characterized CD1d-restricted T cell subset. The availability of potent antigens and tetramers for iNKT cells has allowed this population to be extensively studied and has revealed their central roles in infection, autoimmunity, and tumor immunity. In contrast, diverse Natural Killer T (dNKT) cells are poorly understood because the lipid antigens they recognize are largely unknown. We sought to identify dNKT cell lipid antigen(s) by interrogating a panel of dNKT mouse cell hybridomas with lipid extracts from the pathogen Listeria monocytogenes. We identified Listeria phosphatidylglycerol (PG) as a microbial antigen that was significantly more potent than a previously characterized dNKT cell antigen, mammalian PG. Further, while mammalian PG loaded CD1d tetramers did not stain dNKT cells, the Listeria-derived PG loaded tetramers did. The structure of Listeria PG was distinct from mammalian PG since it contained shorter, fully-saturated anteiso fatty acid lipid tails. CD1d binding lipid displacement studies revealed that the microbial PG antigen binds significantly better to CD1d than counterparts with the same headgroup. These data reveal a highly-potent microbial lipid antigen for a subset of dNKT cells and provide an explanation for its increased antigen potency compared to the mammalian counterpart

Identification of a Potent Microbial Lipid Antigen for Diverse NKT Cells

Semi-invariant/type I NKT cells are a well-characterized CD1d-restricted T cell subset. The availability of potent Ags and tetramers for semi-invariant/type I NKT cells allowed this population to be extensively studied and revealed their central roles in infection, autoimmunity, and tumor immunity. In contrast, diverse/type II NKT (dNKT) cells are poorly understood because the lipid Ags that they recognize are largely unknown. We sought to identify dNKT cell lipid Ag(s) by interrogating a panel of dNKT mouse cell hybridomas with lipid extracts from the pathogen Listeria monocytogenes. We identified Listeria phosphatidylglycerol as a microbial Ag that was significantly more potent than a previously characterized dNKT cell Ag, mammalian phosphatidylglycerol. Further, although mammalian phosphatidylglycerol-loaded CD1d tetramers did not stain dNKT cells, the Listeria-derived phosphatidylglycerol-loaded tetramers did. The structure of Listeria phosphatidylglycerol was distinct from mammalian phosphatidylglycerol because it contained shorter, fully-saturated anteiso fatty acid lipid tails. CD1d-binding lipid-displacement studies revealed that the microbial phosphatidylglycerol Ag binds significantly better to CD1d than do counterparts with the same headgroup. These data reveal a highly potent microbial lipid Ag for a subset of dNKT cells and provide an explanation for its increased Ag potency compared with the mammalian counterpart

Determinants of Intima-Media Thickness in the Young: The ALSPAC Study

Objectives: This study characterized the determinants of carotid intima-media thickness (cIMT) in a large (n > 4,000) longitudinal cohort of healthy young people age 9 to 21 years. Background: Greater cIMT is commonly used in the young as a marker of subclinical atherosclerosis, but its evolution at this age is still poorly understood. Methods: Associations between cardiovascular risk factors and cIMT were investigated in both longitudinal (ages 9 to 17 years) and cross-sectional (ages 17 and 21 years) analyses, with the latter also related to other measures of carotid structure and stress. Additional use of ultra-high frequency ultrasound in the radial artery at age 21 years allowed investigation of the distinct layers (i.e., intima or media) that may underlie observed differences. Results: Fat-free mass (FFM) and systolic blood pressure were the only modifiable risk factors positively associated with cIMT (e.g., mean difference in cIMT per 1-SD increase in FFM at age 17: 0.007 mm: 95% confidence interval [CI]: 0.004 to 0.010; p < 0.001), whereas fat mass was negatively associated with cIMT (difference: −0.0032; 95% CI: 0.004 to −0.001; p = 0.001). Similar results were obtained when investigating cumulative exposure to these factors throughout adolescence. An increase in cIMT maintained circumferential wall stress in the face of increased mean arterial pressure when increases in body mass were attributable to increased FFM, but not fat mass. Risk factor−associated differences in the radial artery occurred in the media alone, and there was little evidence of a relationship between intimal thickness and any risk factor. Conclusions: Subtle changes in cIMT in the young may predominantly involve the media and represent physiological adaptations as opposed to subclinical atherosclerosis. Other vascular measures may be more appropriate for the identification of arterial disease before adulthood. © 2021 The Author