Analysis of serum proteomics data identifies a quantitative association between beta-defensin 2 at baseline and clinical response to IL-17 blockade in psoriatic arthritis.

OBJECTIVES: Despite several effective targeted therapies, biomarkers that predict whether a patient with psoriatic arthritis (PsA) will respond to a particular treatment are currently lacking. METHODS: We analysed proteomics data from serum samples of nearly 2000 patients with PsA in placebo-controlled phase-III clinical trials of the interleukin-17 inhibitor secukinumab. To discover predictive biomarkers of clinical response, we used statistical learning with controlled feature selection. The top candidate was validated using an ELISA and was separately assessed in a trial of almost 800 patients with PsA treated with secukinumab or the tumour necrosis factor inhibitor adalimumab. RESULTS: Serum levels of beta-defensin 2 (BD-2) at baseline were found to be robustly associated with subsequent clinical response (eg, American College of Rheumatology definition of 20%, 50% and 70% improvement) to secukinumab, but not to placebo. This finding was validated in two independent clinical studies not used for discovery. Although BD-2 is known to be associated with psoriasis severity, the predictivity of BD-2 was independent of baseline Psoriasis Area and Severity Index. The association between BD-2 and response to secukinumab was observed as early as 4 weeks and maintained up to 52 weeks. BD-2 was also found to predict response to treatment with adalimumab. Unlike in PsA, BD-2 was not predictive of response to secukinumab in rheumatoid arthritis. CONCLUSIONS: In PsA, BD-2 at baseline is quantitatively associated with clinical response to secukinumab. Patients with high levels of BD-2 at baseline reach and sustain higher rates of clinical response after treatment with secukinumab.

The Aspergillus fumigatus dihydroxyacid dehydratase Ilv3A/IlvC is required for full virulence.

Dihydroxyacid dehydratase (DHAD) is a key enzyme in the branched-chain amino acid biosynthetic pathway that exists in a variety of organisms, including fungi, plants and bacteria, but not humans. In this study we identified four putative DHAD genes from the filamentous fungus Aspergillus fumigatus by homology to Saccharomyces cerevisiae ILV3. Two of these genes, AFUA_2G14210 and AFUA_1G03550, initially designated AfIlv3A and AfIlv3B for this study, clustered in the same group as S. cerevisiae ILV3 following phylogenetic analysis. To investigate the functions of these genes, AfIlv3A and AfIlv3B were knocked out in A. fumigatus. Deletion of AfIlv3B gave no apparent phenotype whereas the Δilv3A strain required supplementation with isoleucine and valine for growth. Thus, AfIlv3A is required for branched-chain amino acid synthesis in A. fumigatus. A recombinant AfIlv3A protein derived from AFUA_2G14210 was shown to have DHAD activity in an in vitro assay, confirming that AfIlv3A is a DHAD. In addition we show that mutants lacking AfIlv3A and ilv3B exhibit reduced levels of virulence in murine infection models, emphasising the importance of branched-chain amino acid biosynthesis in fungal infections, and hence the potential of targeting this pathway with antifungal agents. Here we propose that AfIlv3A/AFUA_2G2410 be named ilvC.

A public resource for metabolic pathway mapping of Aspergillus fumigatus Af293.

Our understanding of the human pathogenic fungus Aspergillus fumigatus has recently benefitted from much work at the genomics level, including genome sequencing and comparative genome analyses. The next stage in this process is to develop a higher-level appreciation of the organism's biology and to this end the Pathway Tools software has been used to map the metabolic pathways of A. fumigatus Af293. The resulting web-based resource shows 242 pathways which can be viewed at a variety of resolutions. Some pathways have been manually curated (e.g., ergosterol biosynthesis, 4-hydroxymandelate degradation, fatty acid ß-oxidation, fatty acid ω-oxidation, the glyoxylate cycle, palmitate biosynthesis, pyridoxal 5'-phosphate salvage, sphingolipid metabolism, ubiquinone biosynthesis and very long chain fatty acid biosynthesis) while others can be used as a starting point for more detailed research. The pathways can be viewed via the Scientific Information:Genomes section of the Aspergillus website ( www.aspergillus.org.uk ).

The transposon impala is activated by low temperatures: use of a controlled transposition system to identify genes critical for viability of Aspergillus fumigatus.

Genes that are essential for viability represent potential targets for the development of anti-infective agents. However, relatively few have been determined in the filamentous fungal pathogen Aspergillus fumigatus. A novel solution employing parasexual genetics coupled with transposon mutagenesis using the Fusarium oxysporum transposon impala had previously enabled the identification of 20 essential genes from A. fumigatus; however, further use of this system required a better understanding of the mode of action of the transposon itself. Examination of a range of conditions indicated that impala is activated by prolonged exposure to low temperatures. This newly identified property was then harnessed to identify 96 loci that are critical for viability in A. fumigatus, including genes required for RNA metabolism, organelle organization, protein transport, ribosome biogenesis, and transcription, as well as a number of noncoding RNAs. A number of these genes represent potential targets for much-needed novel antifungal drugs.

Two families of extracellular phospholipase C genes are present in aspergilli.

Fungi secrete extracellular enzymes to enable them to harvest nutrients from the environment. In the case of pathogenic fungi these enzymes can also be pathogenesis factors. Here we report the identification in fungi of a complex family of extracellular phospholipase C (PLC) enzymes, homologous to the Pseudomonas aeruginosa PLCH_PSEAE. Database searches and phylogenetic analysis showed that the PLCs clustered into two groups with different evolutionary histories. One group, subdivided into PLC-A, -B, -C and -D, was found only in aspergilli and Neosartorya fischeri. Each species only ever showed three of the four PLCs except N. fischeri which had all four PLCs plus duplicate PLC-A, -B and -C genes. Modelling studies indicated that these PLCs had mechanistic similarities to phosphoesterases and aryl sulphatases, but that they probably did not differ in substrate specificity. The second group, PLC-E, was seen in a wider range of fungi including some species of aspergilli and was always found in a head-to-head arrangement with a copper oxidase, similar to the laccases. The PLC genes appear to have arisen from separate gene transfer events from bacteria or lower eukaryotes. Thus, aspergilli have acquired PLCs twice in the course of evolution.

ADAMs are present in fungi: identification of two novel ADAM genes in Aspergillus fumigatus.

The ADAMs are a family of integral membrane proteases involved in shedding and fusion events in animal tissues. Here, we report the identification of two ADAMs, ADM-A and ADM-B, in the pathogenic fungus Aspergillus fumigatus. The domain structure of metazoan ADAMs was seen in ADM-A and -B, although with some differences. ADAMs were identified in other filamentous fungi and phylogenetic analysis indicated that the fungal ADAMs were monophyletic and most closely related to metazoan ADAM 10 and 17. Recombinant ADM-B protease specifically cleaved casein and albumin while recombinant propeptide+protease was inactive. A sheddase function is therefore proposed for fungal ADAMs.

The rapid assignment of ruminal fungi to presumptive genera using ITS1 and ITS2 RNA secondary structures to produce group-specific fingerprints.

Identification of microbial community members in complex environmental samples is time consuming and repetitive. Here, ribosomal sequences and hidden Markov models are used in a novel approach to rapidly assign fungi to their presumptive genera. The ITS1 and ITS2 fragments from a range of axenic, anaerobic gut fungal cultures, including several type strains, were isolated and the RNA secondary structures predicted for these sequences were used to generate a fingerprinting program. The methodology was then tested and the algorithms improved using a collection of environmentally derived sequences, providing a rapid indicator of the fungal diversity and numbers of novel sequence groups within the environmental sample from which they were derived. While the methodology was developed to assist in investigations involving the rumen ecosystem, it has potential generic application in studying diversity and population dynamics in other microbial ecosystems.

Identification of a novel class of annexin genes.

The annexins are a family of calcium- and phospholipid-binding proteins that have been widely studied in animals. Investigation of annexins in the fungus Aspergillus fumigatus identified a novel annexin-like gene (ANXC4) as well as two conventional annexins (ANXC3.1 and ANXC3.2). The genes were initially identified by bioinformatics, and sequences were then determined experimentally. Reverse transcription polymerase chain reaction indicated that all three genes were expressed. ANXC4 lacked calcium-binding consensus sequences and had a 553 residue N-terminal tail. However, bioinformatics indicated that ANXC4 is an annexin and homologues were identified in other filamentous fungi. ANXC4 therefore represents a new grouping within the annexin family.

M142.2 (cut-6), a novel Caenorhabditis elegans matrix gene important for dauer body shape.

The cuticle of the nematode Caenorhabditis elegans is a collagenous extracellular matrix which forms the exoskeleton and defines the shape of the worm. We have characterized the C. elegans gene M142.2, and we show that this is a developmentally regulated gene important for cuticle structure. Transgenic worms expressing M142.2 promoter fused to green fluorescent protein showed that M142.2 is expressed in late embryos and L2d predauers, in the hypodermal cells which synthesize the cuticle. The same temporal pattern was seen by RT-PCR using RNA purified from specific developmental stages. A recombinant fragment of M142.2 was expressed in Escherichia coli and used to raise an antiserum. Immunohistochemistry using the antiserum localized M142.2 to the periphery of the alae of L1 and dauers, forming two longitudinal ribbons over the hypodermal cells. Loss-of-function of M142.2 by RNAi resulted in a novel phenotype: dumpy dauers which lacked alae. M142.2 therefore plays a major role in the assembly of the alae and the morphology of the dauer cuticle; because of its similarity to the other cut genes of the cuticle, we have named the gene cut-6.

The alpha2beta1 integrin inhibitor rhodocetin binds to the A-domain of the integrin alpha2 subunit proximal to the collagen-binding site.

Rhodocetin is a snake venom protein that binds to alpha2beta1 integrin, inhibiting its interaction with its endogenous ligand collagen. We have determined the mechanism by which rhodocetin inhibits the function of alpha2beta1. The interaction of alpha2beta1 with collagen and rhodocetin differed: Ca(2+) ions and slightly acidic pH values increased the binding of alpha2beta1 integrin to rhodocetin in contrast with their attenuating effect on collagen binding, suggesting that rhodocetin preferentially binds to a less active conformation of alpha2beta1 integrin. The alpha2A-domain [von Willebrand factor domain A homology domain (A-domain) of the integrin alpha2 subunit] is the major site for collagen binding to alpha2beta1. Recombinant alpha2A-domain bound rhodocetin, demonstrating that the A-domain is also the rhodocetin-binding domain. Although the interaction of alpha2beta1 with rhodocetin is affected by altering divalent cations, the interaction of the A-domain was divalent-cation-independent. The rhodocetin-binding site on the alpha2A-domain was mapped first by identifying an anti-alpha2 antibody that blocked rhodocetin binding and then mapping the epitope of the antibody using human-mouse alpha2A-domain chimaeras; and secondly, by binding studies with alpha2A-domain, which bear point mutations in the vicinity of the mapped epitope. In this way, the rhodocetin-binding site was identified as the alpha3-alpha4 loop plus adjacent alpha-helices. This region is known to form part of the collagen-binding site, thus attaining a mainly competitive mode of inhibition by rhodocetin.

A novel and highly conserved collagen (pro(alpha)1(XXVII)) with a unique expression pattern and unusual molecular characteristics establishes a new clade within the vertebrate fibrillar collagen family.

The type XXVII collagen gene codes for a novel vertebrate fibrillar collagen that is highly conserved in man, mouse, and fish (Fugu rubripes). The pro(alpha)1(XXVII) chain has a domain structure similar to that of the type B clade chains (alpha1(V), alpha3(V), alpha1(XI), and alpha2(XI)). However, compared with other vertebrate fibrillar collagens (types I, II, III, V, and XI), type XXVII collagen has unusual molecular features such as no minor helical domain, a major helical domain that is short and interrupted, and a short chain selection sequence within the NC1 domain. Pro(alpha)1(XXVII) mRNA is 9 kb and expressed by chondrocytes but also by a variety of epithelial cell layers in developing tissues including stomach, lung, gonad, skin, cochlear, and tooth. By Western blotting, type XXVII antisera recognized multiple bands of 240-110 kDa in tissue extracts and collagenous bands of 150-140 kDa in the conditioned medium of the differentiating chondrogenic ATDC5 cell line. Phylogenetic analyses revealed that type XXVII, together with the closely related type XXIV collagen gene, form a new, third clade (type C) within the vertebrate fibrillar collagen family. Furthermore, the exon structure of the type XXVII collagen gene is similar to, but distinct from, those of the genes coding for the type A or B clade pro(alpha) chains.

Fibrillar collagen: the key to vertebrate evolution? A tale of molecular incest.

Fibril-forming (fibrillar) collagens are extracellular matrix proteins conserved in all multicellular animals. Vertebrate members of the fibrillar collagen family are essential for the formation of bone and teeth, tissues that characterise vertebrates. The potential role played by fibrillar collagens in vertebrate evolution has not been considered previously largely because the family has been around since the sponge and it was unclear precisely how and when those particular members now found in vertebrates first arose. We present evidence that the classical vertebrate fibrillar collagens share a single common ancestor that arose at the very dawn of the vertebrate world and prior to the associated genome duplication events. Furthermore, we present a model, 'molecular incest', that not only accounts for the characteristics of the modern day vertebrate fibrillar collagen family but demonstrates the specific effects genome or gene duplications may have on the evolution of multimeric proteins in general.

Identification and analysis of collagen alpha 1(XXI), a novel member of the FACIT collagen family.

The FACIT collagens bind to the surface of collagen fibrils linking them with other matrix molecules. Bioinformatics analysis of cDNA clone DKFZp564B052 showed that it resembled the FACIT collagens and was therefore designated collagen alpha 1(XXI). Phylogenetic analyses of the N-terminal NC3 domains of alpha 1(XXI) and other FACIT collagens showed that (i) alpha 1(XXI) clustered with the FACIT collagens; (ii) collagen alpha 1(XXI) arose before the divergence of alpha 1(XII), alpha 1(XIV) and alpha 1(XX); (iii) collagen alpha 1(XIV) derived from the C-terminal region of the NC3 domain of a collagen alpha 1(XII)-like molecule; and (iv) collagen alpha 1(XX) derived from a collagen alpha 1(XIV)-like molecule. This study provides a framework for the evolution of the FACIT collagens which will be of value in linking NC3 domains with their functions.

A novel gain-of-function mutation of the integrin alpha2 VWFA domain.

Integrin alpha2beta1 is the major receptor for collagens in human tissues, being involved in cell adhesion and the control of collagen and collagenase gene expression. The collagen binding site of alpha2beta1 has been localized to the alpha2 von Willebrand Factor type A (VWFA) domain (A-domain or I-domain) and the residues responsible for the interaction with collagen have been mapped. We report a study of alpha2 VWFA domain in which residue E318, which lies outside the collagen binding site, is mutated to tryptophan, showing that this is a gain-of-function mutation. Recombinant alpha2-E318W VWFA domain showed elevated and specific binding to collagen I compared with the wild-type. Side chain hydrophobicity was important for the gain-of-function as elevated binding was seen with E318I and E318Y, but not with E318R. The E318W mutation had additional effects on VWFA domain properties as alpha2-E318W VWFA domain differed from the wild-type in its cation preferences for ligand binding and in binding to monoclonal antibody JA203, which bound at a site distal to E318. The gain-of-function effect was not restricted to binding to collagen I as alpha2-E318W also showed elevated binding to collagen IV, collagen I C-propeptide, laminin and E-cadherin. Binding to these ligands was inhibited by collagen peptide containing the GFOGER motif, indicating that these bound to the VWFA domain by a similar mechanism to collagen I. These data indicate that residue E318 plays a novel and important role in modulating alpha2 VWFA domain--ligand binding and may be involved in the conformational changes associated with its regulation.