Structure and properties of the Ca(2+)-binding CUB domain, a widespread ligand-recognition unit involved in major biological functions

CUB domains are 110-residue protein motifs exhibiting a β-sandwich fold and mediating protein-protein interactions in various extracellular proteins. Recent X-ray structural and mutagenesis studies have led to the identification of a particular CUB domain subset, cbCUB (Ca(2+)-binding CUB domain). Unlike other CUB domains, these harbour a homologous Ca(2+)-binding site that underlies a conserved binding site mediating ionic interaction between two of the three conserved acidic Ca(2+) ligands and a basic (lysine or arginine) residue of a protein ligand, similar to the interactions mediated by the low-density lipoprotein receptor family. cbCUB-mediated protein-ligand interactions usually involve multipoint attachment through several cbCUBs, resulting in high-affinity binding through avidity, despite the low affinity of individual interactions. The aim of the present review is to summarize our current knowledge about the structure and functions of cbCUBs, which represent the majority of the known CUB repertoire and are involved in a variety of major biological functions, including immunity and development, as well as in various cancer types. Examples discussed in the present review include a wide range of soluble and membrane-associated human proteins, as well as some archaeal and invertebrate proteins. The fact that these otherwise unrelated proteins share a common Ca(2+)-dependent ligand-binding ability suggests a mechanism inherited from very primitive ancestors. The information provided in the present review should stimulate further investigations on the crucial interactions mediated by cbCUB-containing proteins.     

IL1RN genetic variations and risk of IPF: a meta-analysis and mRNA expression study

Idiopathic pulmonary fibrosis (IPF) is a rare and devastating lung disease of unknown aetiology. Genetic variations in the IL1RN gene, encoding the interleukin-1 receptor antagonist (IL-1Ra), have been associated with IPF susceptibility. Several studies investigated the variable number tandem repeat (VNTR) or single nucleotide polymorphisms rs408392, rs419598 and rs2637988, with variable results. The aim of this study was to elucidate the influence of polymorphisms in IL1RN on IPF susceptibility and mRNA expression. We performed a meta-analysis of the five case–control studies that investigated an IL1RN polymorphism in IPF in a Caucasian population. In addition, we investigated whether IL1RN mRNA expression was influenced by IL1RN polymorphisms. The VNTR, rs408392 and rs419598 were in tight linkage disequilibrium, with D′ > 0.99. Furthermore, rs2637988 was in linkage disequilibrium with the VNTR (D′ = 0.90). A haploblock of VNTR*2 and the minor alleles of rs408392and rs419598 was constructed. Meta-analysis revealed that this VNTR*2 haploblock is associated with IPF susceptibility both with an allelic model (odds ratio = 1.42, p = 0.002) and a carriership model (odds ratio = 1.60, p = 0.002). IL1RN mRNA expression was significantly influenced by rs2637988, with lower levels found in carriers of the (minor) GG genotype (p < 0.001). From this meta-analysis, we conclude that the VNTR*2 haploblock is associated with susceptibility to IPF. In addition, polymorphisms in IL1RN influence IL-1Ra mRNA expression, suggesting that lower levels of IL-1Ra predispose to developing IPF. Together these findings demonstrate that the cytokine IL-1Ra plays a role in IPF pathogenesis.     

Molecular phylogeny of the Oriental butterfly genus Arhopala (Lycaenidae,Theclinae) inferred from mitochondrial and nuclear genes

Abstract. We present a phylogeny for a selection of species of the butterfly genus Arhopala Boisduval, 1832 based on molecular characters. We sequenced 1778 bases of the mitochondrial genes Cytochrome Oxidase 1 and 2 including tRNA^Leu, and a 393‐bp fragment of the nuclear wingless gene for a total of 42 specimens of 33 species, representing all major species groups. Analyses of mtDNA and wingless genes show congruent phylogenetic signal. The phylogeny presented here confirms the monophyly of the centaurus, eumolphus, camdeo and epimuta groups and the amphimuta subgroup. It confirms close relationships between species within the agelastus group, that together with the amphimuta subgroup, centaurus and camdeo groups form a monophyletic group. However, incongruencies with previous taxonomic studies also occur; the amphimuta and silhetensis groups are not monophyletic, as is the genus Arhopala itself. One enigmatic species, A. kinabala, was evaluated further for topology and the support for basal placement of this species is due mainly to the wingless gene. However, in the Parsimony analysis, and subsequent Maximum Likelihood evaluations, certain nodes could not be resolved due to insufficient support. The mtDNA shows extreme AT bias with compositional heterogeneity at 3rd codon positions, which may result in saturation. By contrast, the wingless gene does not show compositional bias, suggesting that poor support is not due solely to saturation. The evaluation of morphological characters used in previous studies on Arhopala systematics on the molecular tree indicates that the macular pattern and the absence of tails at the hind wings show extensive homoplasy. A significant phylogenetic signal (as indicated by T‐PTP tests) is present in several of these morphological characters, which are nevertheless of limited use in phylogenetic studies due to their labile nature.     

Crystallization and preliminary X-ray study of porcine trypsin, free and complexed with Ecballium elaterium trypsin inhibitor, a member of the squash inhibitors family

Porcine trypsin has been crystallized either free or complexed with synthetic Ecballium elaterium trypsin inhibitor II, a 28-residue peptide with three disulfide bridges. The crystals diffract beyond 2.0 A. Crystals are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 77.32 A, b = 53.81 A, c = 46.91 A, for the free trypsin, and a = 62.25 A, b = 62.27 A, c = 84.66 A for the complex with E. elaterium trypsin inhibitor II.     

Crystal structure of the CUB1-EGF-CUB2 domain of human MASP-1/3 and identification of its interaction sites with mannan-binding lectin and ficolins

MASP-1 and MASP-3 are homologous proteases arising from alternative splicing of the MASP1/3 gene. They include an identical CUB(1)-EGF-CUB(2)-CCP(1)-CCP(2) module array prolonged by different serine protease domains at the C-terminal end. The x-ray structure of the CUB(1)-EGF-CUB(2) domain of human MASP-1/3, responsible for interaction of MASP-1 and -3 with their partner proteins mannan-binding lectin (MBL) and ficolins, was solved to a resolution of 2.3A(.) The structure shows a head-to-tail homodimer mainly stabilized by hydrophobic interactions between the CUB(1) module of one monomer and the epidermal growth factor (EGF) module of its counterpart. A Ca(2+) ion bound primarily to both EGF modules stabilizes the intra- and inter-monomer CUB(1)-EGF interfaces. Additional Ca(2+) ions are bound to each CUB(1) and CUB(2) module through six ligands contributed by Glu(49), Asp(57), Asp(102), and Ser(104) (CUB(1)) and their counterparts Glu(216), Asp(226), Asp(263), and Ser(265) (CUB(2)), plus one and two water molecules, respectively. To identify the residues involved in interaction of MASP-1 and -3 with MBL and L- and H-ficolins, 27 point mutants of human MASP-3 were generated, and their binding properties were analyzed using surface plasmon resonance spectroscopy. These mutations map two homologous binding sites contributed by modules CUB(1) and CUB(2), located in close vicinity of their Ca(2+)-binding sites and stabilized by the Ca(2+) ion. This information allows us to propose a model of the MBL-MASP-1/3 interaction, involving a major electrostatic interaction between two acidic Ca(2+) ligands of MASP-1/3 and a conserved lysine of MBL. Based on these and other data, a schematic model of a MBL.MASP complex is proposed.     

The two major oligomeric forms of human mannan-binding lectin: chemical characterization, carbohydrate-binding properties, and interaction with MBL-associated serine proteases

Mannan-binding lectin (MBL) is an oligomeric C-type lectin assembled from homotrimeric structural units that binds to neutral carbohydrates on microbial surfaces. It forms individual complexes with MBL-associated serine proteases (MASP)-1, -2, -3 and a truncated form of MASP-2 (MAp19) and triggers the lectin pathway of complement through MASP-2 activation. To characterize the oligomerization state of the two major MBL forms present in human serum, both proteins were analyzed by mass spectrometry. Mass values of 228,098 +/- 170 Da (MBL-I) and 304,899 +/- 229 Da (MBL-II) were determined for the native proteins, whereas reduction of both species yielded a single chain with an average mass of 25,340 +/- 18 Da. This demonstrates that MBL-I and -II contain 9 and 12 disulfide-linked chains, respectively, and therefore are trimers and tetramers of the structural unit. As shown by surface plasmon resonance spectroscopy, trimeric and tetrameric MBL bound to immobilized mannose-BSA and N-acetylglucosamine-BSA with comparable K(D) values (2.2 and 0.55 nM and 1.2 and 0.96 nM, respectively). However, tetrameric MBL exhibited significantly higher maximal binding capacity and lower dissociation rate constants for both carbohydrates. In contrast, no significant difference was detected for binding of the recombinant MASPs or MAp19 to immobilized trimeric or tetrameric MBL. As shown by gel filtration, both MBL species formed 1:2 complexes with MASP-3 or MAp19. These results provide the first precise analysis of the major human MBL oligomers. The oligomerization state of MBL has a direct effect on its carbohydrate-binding properties, but no influence on the interaction with the MASPs.     

The stress response protein Hsp12p increases the flexibility of the yeast Saccharomyces cerevisiae cell wall

The yeast S. cerevisiae cell wall comprising a 10 nm thick layer of polysaccharides, predominantly beta(1,3)-glucan and proteins, is the interface between the cell and the neighbouring environment. As such it is not a static entity but rather one that is dynamically remodelled in response to changes in the environmental conditions. We have recently proposed from studies using yeast cells lacking the gene encoding Hsp12p (Deltahsp12 yeast) and from incorporation of Hsp12p into agarose, used as a model system for the beta-glucan layer of the cell wall, that the hydrophilic stress response cell wall protein Hsp12p acts as a cell wall plasticizer. In this report we have used force spectroscopy to confirm that Deltahsp12 yeast are indeed less flexible than the wild type strain. The spring constant of the cell wall of Deltahsp12 yeast, kcw was determined to be 72+/-3 mN m-1 as compared to 17+/-5 mN m-1 obtained for the wild type strain. A similar result was found on the basis of a quantitative analysis of the electrophoretic mobilities measured for the two yeast strains. Those indicated that the hydrodynamic permeability quantified through the softness parameter of the external layer of Deltahsp12 cells was smaller than the one of wild type cells. We proposed from surface infrared spectroscopy measurements that yeast compensate for the lack of Hsp12p by reducing the carbohydrate/proteins ratio of the cell wall or increasing the cell wall chitin content.     

Plasticity results in delayed breeding in a long‐distant migrant seabird

A major question for conservationists and evolutionary biologists is whether natural populations can adapt to rapid environmental change through micro‐evolution or phenotypic plasticity. Making use of 17 years of data from a colony of a long‐distant migratory seabird, the common tern (Sterna hirundo), we examined phenotypic plasticity and the evolutionary potential of breeding phenology, a key reproductive trait. We found that laying date was strongly heritable (0.27 ± 0.09) and under significant fecundity selection for earlier laying. Paradoxically, and in contrast to patterns observed in most songbird populations, laying date became delayed over the study period, by about 5 days. The discrepancy between the observed changes and those predicted from selection on laying date was explained by substantial phenotypic plasticity. The plastic response in laying date did not vary significantly among individuals. Exploration of climatic factors showed individual responses to the mean sea surface temperature in Senegal in December prior to breeding: Common terns laid later following warmer winters in Senegal. For each 1°C of warming of the sea surface in Senegal, common terns delayed their laying date in northern Germany by 6.7 days. This suggests that warmer waters provide poorer wintering resources. We therefore found that substantial plastic response to wintering conditions can oppose natural selection, perhaps constraining adaptation.     

Hydrophobic cluster analysis: an efficient new way to compare and analyse amino acid sequences

A new method for comparing and aligning protein sequences is described. This method, hydrophobic cluster analysis (HCA), relies upon a two-dimensional (2D) representation of the sequences. Hydrophobic clusters are determined in this 2D pattern and then used for the sequence comparisons. The method does not require powerful computer resources and can deal with distantly related proteins, even if no 3D data are available. This is illustrated in the present report by a comparison of human haemoglobin with leghaemoglobin, a comparison of the two domains of liver rhodanese (thiosulphate sulphurtransferase) and a comparison of plastocyanin and azurin.