Siderocalin (Lcn 2) Also Binds Carboxymycobactins, Potentially Defending against Mycobacterial Infections through Iron Sequestration

SummarySiderocalin, a member of the lipocalin family of binding proteins, is found in neutrophil granules, uterine secretions, and at markedly elevated levels in serum and synovium during bacterial infection; it is also secreted from epithelial cells in response to inflammation or tumorigenesis. Identification of high-affinity ligands, bacterial catecholate-type siderophores (such as enterochelin), suggested a possible function for siderocalin: an antibacterial agent, complementing the general antimicrobial innate immune system iron-depletion strategy, sequestering iron as ferric siderophore complexes. Supporting this hypothesis, siderocalin is a potent bacteriostatic agent in vitro under iron-limiting conditions and, when knocked out, renders mice remarkably susceptible to bacterial infection. Here we show that siderocalin also binds soluble siderophores of mycobacteria, including M. tubercu losis: carboxymycobactins. Siderocalin employs a degenerate recognition mechanism to cross react with these dissimilar types of siderophores, broadening the potential utility of this innate immune defense

Crystal Structure of the MHC Class I Homolog MIC-A, a γδ T Cell Ligand

AbstractThe major histocompatibility complex (MHC) class I homolog MIC-A functions as a stress-inducible antigen that is recognized by a subset of γδ T cells independent of β2-microglobulin and bound peptides. Its crystal structure reveals a dramatically altered MHC class I fold, both in detail and overall domain organization. The only remnant of a peptide-binding groove is a small cavity formed as the result of disordering a large section of one of the groove-defining helices. Loss of β2-microglobulin binding is due to a restructuring of the interaction interfaces. Structural mapping of sequence variation suggests potential receptor binding sites on the underside of the platform on the side opposite of the surface recognized by αβ T cell receptors on MHC class I–peptide complexes

A conserved surface on Toll-like receptor 5 recognizes bacterial flagellin

The molecular basis for Toll-like receptor (TLR) recognition of microbial ligands is unknown. We demonstrate that mouse and human TLR5 discriminate between different flagellins, and we use this difference to map the flagellin recognition site on TLR5 to 228 amino acids of the extracellular domain. Through molecular modeling of the TLR5 ectodomain, we identify two conserved surface-exposed regions. Mutagenesis studies demonstrate that naturally occurring amino acid variation in TLR5 residue 268 is responsible for human and mouse discrimination between flagellin molecules. Mutations within one conserved surface identify residues D295 and D367 as important for flagellin recognition. These studies localize flagellin recognition to a conserved surface on the modeled TLR5 structure, providing detailed analysis of the interaction of a TLR with its ligand. These findings suggest that ligand binding at the β sheets results in TLR activation and provide a new framework for understanding TLR–agonist interactions

Autoreactivity and Exceptional CDR Plasticity (but Not Unusual Polyspecificity) Hinder Elicitation of the Anti-HIV Antibody 4E10

The broadly-neutralizing anti-HIV antibody 4E10 recognizes an epitope in the membrane-proximal external region of the HIV envelope protein gp41. Previous attempts to elicit 4E10 by vaccination with envelope-derived or reverse-engineered immunogens have failed. It was presumed that the ontogeny of 4E10-equivalent responses was blocked by inherent autoreactivity and exceptional polyreactivity. We generated 4E10 heavy-chain knock-in mice, which displayed significant B cell dysregulation, consistent with recognition of autoantigen/s by 4E10 and the presumption that tolerance mechanisms may hinder the elicitation of 4E10 or 4E10-equivalent responses. Previously proposed candidate 4E10 autoantigens include the mitochondrial lipid cardiolipin and a nuclear splicing factor, 3B3. However, using carefully-controlled assays, 4E10 bound only weakly to cardiolipin-containing liposomes, but also bound negatively-charged, non-cardiolipin-containing liposomes comparably poorly. 4E10/liposome binding was predominantly mediated by electrostatic interactions rather than presumed hydrophobic interactions. The crystal structure of 4E10 free of bound ligands showed a dramatic restructuring of the combining site, occluding the HIV epitope binding site and revealing profound flexibility, but creating an electropositive pocket consistent with non-specific binding of phospholipid headgroups. These results strongly suggested that antigens other than cardiolipin mediate 4E10 autoreactivity. Using a synthetic peptide library spanning the human proteome, we determined that 4E10 displays limited and focused, but unexceptional, polyspecificity. We also identified a novel autoepitope shared by three ER-resident inositol trisphosphate receptors, validated through binding studies and immunohistochemistry. Tissue staining with 4E10 demonstrated reactivity consistent with the type 1 inositol trisphosphate receptor as the most likely candidate autoantigen, but is inconsistent with splicing factor 3B3. These results demonstrate that 4E10 recognition of liposomes competes with MPER recognition and that HIV antigen and autoepitope recognition may be distinct enough to permit eliciting 4E10-like antibodies, evading autoimmunity through directed engineering. However, 4E10 combining site flexibility, exceptional for a highly-matured antibody, may preclude eliciting 4E10 by conventional immunization strategies

Ontogeny of Recognition Specificity and Functionality for the Broadly Neutralizing Anti-HIV Antibody 4E10

The process of antibody ontogeny typically improves affinity, on-rate, and thermostability, narrows polyspecificity, and rigidifies the combining site to the conformer optimal for binding from the broader ensemble accessible to the precursor. However, many broadly-neutralizing anti-HIV antibodies incorporate unusual structural elements and recognition specificities or properties that often lead to autoreactivity. The ontogeny of 4E10, an autoreactive antibody with unexpected combining site flexibility, was delineated through structural and biophysical comparisons of the mature antibody with multiple potential precursors. 4E10 gained affinity primarily by off-rate enhancement through a small number of mutations to a highly conserved recognition surface. Controverting the conventional paradigm, the combining site gained flexibility and autoreactivity during ontogeny, while losing thermostability, though polyspecificity was unaffected. Details of the recognition mechanism, including inferred global effects due to 4E10 binding, suggest that neutralization by 4E10 may involve mechanisms beyond simply binding, also requiring the ability of the antibody to induce conformational changes distant from its binding site. 4E10 is, therefore, unlikely to be re-elicited by conventional vaccination strategies

Radioactive 26Al and massive stars in the Galaxy

Gamma-rays from radioactive 26Al (half life ~7.2 10^5 yr) provide a 'snapshot' view of ongoing nucleosynthesis in the Galaxy. The Galaxy is relatively transparent to such gamma-rays, and emission has been found concentrated along the plane of the Galaxy. This led to the conclusion1 that massive stars throughout the Galaxy dominate the production of 26Al. On the other hand, meteoritic data show locally-produced 26Al, perhaps from spallation reactions in the protosolar disk. Furthermore, prominent gamma-ray emission from the Cygnus region suggests that a substantial fraction of Galactic 26Al could originate in localized star-forming regions. Here we report high spectral resolution measurements of 26Al emission at 1808.65 keV, which demonstrate that the 26Al source regions corotate with the Galaxy, supporting its Galaxy-wide origin. We determine a present-day equilibrium mass of 2.8 (+/-0.8) M_sol of 26Al. We use this to estimate that the frequency of core collapse (i.e. type Ib/c and type II) supernovae to be 1.9(+/- 1.1) events per century.Comment: accepted for publication in Nature, 24 pages including Online Supplements, 11 figures, 1 tabl

Effects of HLA single chain trimer design on peptide presentation and stability

MHC class I “single-chain trimer” molecules, coupling MHC heavy chain, β2-microglobulin, and a specific peptide into a single polypeptide chain, are widely used in research. To more fully understand caveats associated with this design that may affect its use for basic and translational studies, we evaluated a set of engineered single-chain trimers with combinations of stabilizing mutations across eight different classical and non-classical human class I alleles with 44 different peptides, including a novel human/murine chimeric design. While, overall, single-chain trimers accurately recapitulate native molecules, care was needed in selecting designs for studying peptides longer or shorter than 9-mers, as single-chain trimer design could affect peptide conformation. In the process, we observed that predictions of peptide binding were often discordant with experiment and that yields and stabilities varied widely with construct design. We also developed novel reagents to improve the crystallizability of these proteins and confirmed novel modes of peptide presentation

Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors

A key challenge for the academic and biopharmaceutical communities is the rapid and scalable production of recombinant proteins for supporting downstream applications ranging from therapeutic trials to structural genomics efforts. Here, we describe a novel system for the production of recombinant mammalian proteins, including immune receptors, cytokines and antibodies, in a human cell line culture system, often requiring <3 weeks to achieve stable, high-level expression: Daedalus. The inclusion of minimized ubiquitous chromatin opening elements in the transduction vectors is key for preventing genomic silencing and maintaining the stability of decigram levels of expression. This system can bypass the tedious and time-consuming steps of conventional protein production methods by employing the secretion pathway of serum-free adapted human suspension cell lines, such as 293 Freestyle. Using optimized lentiviral vectors, yields of 20–100 mg/l of correctly folded and post-translationally modified, endotoxin-free protein of up to ~70 kDa in size, can be achieved in conventional, small-scale (100 ml) culture. At these yields, most proteins can be purified using a single size-exclusion chromatography step, immediately appropriate for use in structural, biophysical or therapeutic applications