SPE-51, a sperm secreted protein with an Immunoglobulin-like domain, is required for sperm-egg fusion inC. elegans

During the fertilization process, complex events are involved in the fusion between the reacted spermatozoa and the mature oocyte. Fusion implies that many proteins are present on the cell membrane of the gametes. Recently, a new protein, Izumo, has been shown to play a role in the sperm–egg fusion. Izumo was identified through the generation of a monoclonal antibody that inhibits this fusion. This protein belongs to the immunoglobulin superfamily type I transmembrane protein. Izumo can be detected only after acrosome reaction at the spermatozoal surface. The cashmere goat Izumo gene was identified and cloned by 3′ and 5′ rapid amplification of cDNA ends-PCR. The expression of cashmere goat Izumo was examined by RT-PCR and Northern blotting. The full-length cDNA of cashmere goat Izumo contains 1536 bp and an open reading frame of 1035 bp, encoding a polypeptide of 344 amino acids with a calculated molecular mass of 38.76 kDa and a theoretical isoelectric point of 8.18. This predicted amino acid sequence showed 89.82% amino acid identity with the bovine Izumo. The deduced amino acid sequence contained 3 conserved domains of the signal peptide, immunoglobulin-like domain, and transmembrane region. Reverse transcription-PCR and Northern blotting analysis showed that cashmere goat Izumo transcripts were highly expressed in the testis, caput, corpus, cauda epididymis. Cashmere goat Izumo may play a role in the biological process of fertilization. This work was supported by the National Natural Science Foundation (No. 30560103 and No.30740043), China, and the China Postdoctoral Science Foundation.

The L1 family neural cell adhesion molecules play key roles in specifying the formation and remodeling of the neural network, but their homophilic interaction that mediates adhesion is not well understood. We report two crystal structures of a dimeric form of the headpiece of neurofascin, an L1 family member. The four N-terminal Ig-like domains of neurofascin form a horseshoe shape, akin to several other immunoglobulin superfamily cell adhesion molecules such as hemolin, axonin, and Dscam. The neurofascin dimer, captured in two crystal forms with independent packing patterns, reveals a pair of horseshoes in trans-synaptic adhesion mode. The adhesion interaction is mediated mostly by the second Ig-like domain, which features an intermolecular β-sheet formed by the joining of two individual GFC β-sheets and a large but loosely packed hydrophobic cluster. Mutagenesis combined with gel filtration assays suggested that the side chain hydrogen bonds at the intermolecular β-sheet are essential for the homophilic interaction and that the residues at the hydrophobic cluster play supplementary roles. Our structures reveal a conserved homophilic adhesion mode for the L1 family and also shed light on how the pathological mutations of L1 affect its structure and function.

Aggregates of chondroitin-keratan sulfate proteoglycan (aggrecan) and hyaluronic acid (hyaluronan) are the major space-filling components of cartilage. A glycoprotein, link protein (LP; 40-48 kDa) stabilizes the aggregate by binding to both hyaluronic acid and aggrecan. In the absence of LP, aggregates are smaller (as estimated by rotary shadowing of electron micrographs) and less stable (they dissociate at pH 5) than they are in the presence of LP. The proteoglycan aggregate, including LP, is dissociated in the presence of chaotropes such as 4 M guanidine hydrochloride. On removal of the chaotrope, the complex will reassociate. This forms the basis of the isolation of LP from cartilage and has been described in detail elsewhere. Tryptic digestion of the proteoglycan aggregates results in a high molecular weight product that consists of hyaluronic acid to which is bound LP and the N-terminal globular domain of aggrecan (hyaluronic acid binding region; HABR) in a 1:1 stoichiometry. The amino acid sequences of LP and HABR are surprisingly similar. The amino acid sequence can be divided into three domains; an N-terminal domain that falls into the immunoglobulin super-family and two C-terminal domains that are similar to each other. The DNA structure echoes this similarity, in that the major domains are reflected in three separate exons in both LP and HABR. The two C-terminal domains are largely responsible for the association with HA and are related to two recently described hyaluronate-binding proteins, CD44 and TSG-6. A variety of approaches, including analysis of the forms of LP found in vivo, rotary shadowing and analysis of the sequence in the immunoglobulin-like domain, have shed considerable light on the structure-function relationships of LP. This review describes the structure and function of LP in detail, focusing on what can be inferred from the similarity of LP, HABR and related molecules such as immunoglobulins and lymphocyte HA-receptors.

The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor that binds to diverse ligands and initiates a downstream proinflammatory signaling cascade. RAGE activation has been linked to diabetic complications, Alzheimer disease, infections, and cancers. RAGE is known to mediate cell signaling and downstream proinflammatory gene transcription activation, although the precise mechanism surrounding receptor-ligand interactions is still being elucidated. Recent fluorescence resonance energy transfer evidence indicates that RAGE may form oligomers on the cell surface and that this could be related to signal transduction. To investigate whether RAGE forms oligomers, protein-protein interaction assays were carried out. Here, we demonstrate the interaction between RAGE molecules via their N-terminal V domain, which is an important region involved in ligand recognition. By protein cross-linking using water-soluble and membrane-impermeable cross-linker bis(sulfosuccinimidyl) suberate and nondenaturing gels, we show that RAGE forms homodimers at the plasma membrane, a process potentiated by S100B and advanced glycation end products. Soluble RAGE, the RAGE inhibitor, is also capable of binding to RAGE, similar to V peptide, as shown by surface plasmon resonance. Incubation of cells with soluble RAGE or RAGE V domain peptide inhibits RAGE dimerization, subsequent phosphorylation of intracellular MAPK proteins, and activation of NF-kappaB pathways. Thus, the data indicate that dimerization of RAGE represents an important component of RAGE-mediated cell signaling.

Here, we report the analysis of the structure-function relationship of the extracellular region of human interleukin 6 receptor (IL-6R). Upon binding of IL-6, IL-6R becomes associated extracellularly with a non-IL-6-binding but signal transducing molecule, gp130, and the IL-6 signal is generated. In this region, the cytokine receptor family domain, but not the immunoglobulin-like domain, was responsible both for IL-6 binding and for signal transduction through gp130. Because a soluble, extracellular portion of IL-6R (sIL-6R) could bind IL-6 and mediate IL-6 functions through gp130, amino acid substitutions were introduced into sIL-6R by site-directed mutagenesis. The results, together with the previously proposed tertiary structure model, suggested that the amino acid residues critical for IL-6 binding have a tendency to be distributed to the hinge region between the two 'barrel'-like fibronectin type III modules and to the same side of these two 'barrels'. Amino acid residues, of which substitutions barely affected the IL-6-binding but did abolish the IL-6 signalling capability of sIL-6R, were identified and found to be located mainly in the membrane proximal half of the second barrel. sIL-6R mutants carrying such substitutions lacked the capacity to associate with gp130 in the presence of IL-6.

BIT/SHPS-1/SIRPα/P84 is a unique molecule with a high degree of homology with immune antigen recognition molecules (immunoglobulin, T-cell receptor and MHC), and is highly expressed in the brain. The extracellular region contains three immunoglobulin-like domains (V-type, C1-type and C1-type), and the intracellular region contains two signalling motifs that interact with SHP-2 protein tyrosine phosphatase. BIT-coated plates support cell-substrate adhesion and neurite extension of neurons, and BIT participates in neuronal signal transduction. Diversity of the V-type domain sequences of human BIT has been reported. In the present study we analysed the structure of the mouse BIT gene (Bit). The protein coding region consists of eight exons corresponding to a signal peptide, a V-type domain, a C1-type domain, a C1-type domain, a transmembrane region and three parts of one cytoplasmic region. The two signalling motifs are encoded in one exon. Four splicing forms of mouse BIT were revealed. We also found the sequence diversity in three mouse strains, namely BALB/c, 129/Sv and C57BL/6. The substitution patterns of amino acids and nucleotides indicate positive pressure to alter the amino acids in the V-type domain in evolution. Immunoblot analyses showed that mouse BIT and human BITα are predominantly expressed in the brain. On the bases of these findings we discuss the possibility that BIT contributes to the genetic individuality and diversity of the brain.

The receptor for the cytokine leukemia inhibitory factor (LIF) associates the low affinity binding component gp190 and the high affinity converter gp130. Both are members of the hematopoietic receptors family characterized by the cytokine receptor homology (CRH) domain, which consists of two barrel-like modules of around 100 amino acids each. The gp190 is among the very few members of this large family to contain two CRH domains. The membrane-distal one (herein called D1) is followed by an immunoglobulin-like domain, a membrane-proximal CRH domain called D2, and three type III fibronectin-like repeats. A minimal D1IgD2 fragment is required for binding LIF. By using transmembrane forms of deletion mutants in gp190 ectodomain, we demonstrated that removal of D1 led to spontaneous activation of the receptor and that this property was devoted to a peptidic sequence localized within the last 42 amino acids of the carboxyl-terminal module of D2. By using soluble forms of deletion mutants made by progressive truncations from the end of the D1IgD2 fragment, we demonstrated that the carboxyl-terminal module of D2 was dispensable for LIF binding and that the correct conformation of the D1Ig fragment required a full amino-terminal module of D2. Therefore, the two constitutive modules of the membrane-proximal CRH domain D2 of gp190 fulfill two distinct roles in gp190 function, i.e. in stabilizing the conformation of gp190 allowing LIF binding and in activating the receptor.

Interleukin-1 alpha and -1 beta (IL-1 alpha and IL-1 beta) are cytokines that participate in the regulation of immune responses, inflammatory reactions, and hematopoiesis. A direct expression strategy was used to clone the receptor for IL-1 from mouse T cells. The product of the cloned complementary DNA binds both IL-1 alpha and IL-1 beta in a manner indistinguishable from that of the native T cell IL-1 receptor. The extracellular, IL-1 binding portion of the receptor is 319 amino acids in length and is composed of three immunoglobulin-like domains. The cytoplasmic portion of the receptor is 217 amino acids long.

Cell surface molecules with restricted spatial and temporal distributions are good candidates for mediators of the cell-cell interactions that are necessary for the development of the nervous system. A monoclonal antibody (MAb 23A7) was produced that selectively and transiently labeled a limited subset of axons in the chick embryo spinal cord. Determination of the N-terminal amino acid sequence and immunoprecipitation experiments demonstrated that the 23A7 antigen is identical to Bravo/Nr-CAM, a previously described cell adhesion molecule with immunoglobulin-like domains (E.J. de la Rosa, J.F. Kayyem, J.M. Roman, Y.-D. Stierhof, W.J. Dreyer, and U. Schwartz [1989] J. Cell Biol. 111:3087-3096; M. Grumet, V. Mauro, M.P. Goon, G.M. Edelman, and B.A. Cunningham [1991] J. Cell Biol. 113:1399-1412). The temporal distribution of the 23A7 antigen is unusual in that, immunohistochemically, MAb 23A7 binding greatly decreases after 7 days of development, whereas Western blot analysis indicates increasing levels of the antigen until 17 days of development. In contrast, an antiserum against purified Nr-CAM, which also binds only to the 23A7 antigen, labels nearly all the axons in the tissue throughout all the later stages of development. These anomalous observations are apparently not the result of differential sensitivity of the 23A7 epitope to fixation, the use of suboptimal concentrations of the MAb, or selective MAb binding to a subset of Bravo/Nr-CAM molecules produced by alternative splicing of the transcript or by posttranslational modification. These findings could indicate the existence of multiple states of Bravo/Nr-CAM, which during development, vary in the accessibility of their extracellular domains to the MAb. This suggests the existence of multiple conformation or aggregation states of this cell adhesion molecule, each of which might be performing a different function.

Vascular endothelial growth factor (VEGF) is involved in the promotion of endothelial cell proliferation, migration, and capillary formation. These activities are mainly mediated by the VEGFR2 receptor tyrosine kinase that upon stimulation, promotes the activation of numerous proteins including phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase (PI3K), Akt, Src, and ERK1/2. However, the VEGFR2-proximal signaling events leading to the activation of these targets remain ill defined. We have identified the Gab1 adapter as a novel tyrosine-phosphorylated protein in VEGF-stimulated cells. In bovine aortic endothelial cells, Gab1 associates with VEGFR2, Grb2, PI3K, SHP2, Shc, and PLCgamma, and its overexpression enhances VEGF-dependent cell migration. Importantly, silencing of Gab1 using small interfering RNAs leads to the impaired activation of PLCgamma, ERK1/2, Src, and Akt; blocks VEGF-induced endothelial cell migration; and perturbs actin reorganization and capillary formation. In addition, co-expression of VEGFR2 with Gab1 mutants unable to bind SHP2 or PI3K in human embryonic kidney 293 cells and bovine aortic endothelial cells mimics the defects observed in Gab1-depleted cells. Our work thus identifies Gab1 as a novel critical regulatory component of endothelial cell migration and capillary formation and reveals its key role in the activation of VEGF-evoked signaling pathways required for angiogenesis.

Relatively little is known about the genes and brain structures that enable virgin female Drosophila to make the decision to mate or not. Classical genetic approaches have identified several mutant females that have a reluctance-to-mate phenotype, but most of these have additional behavioral defects. However, the icebox (ibx) mutation was previously reported to lower the sexual receptivity of females, without apparently affecting any other aspect of female behavior. We have shown that the ibx mutation maps to the 7F region of the Drosophila X chromosome to form a complex complementation group with both lethal and viable alleles of neuroglian (nrg). The L1-type cell adhesion molecule encoded by nrg consists of six immunoglobulin-like domains, five fibronectin-like domains, one transmembrane domain and one alternatively spliced intracellular domain. The ibx strain has a missense mutation causing a glycine-to-arginine change at amino acid 92 in the first immunoglobulin domain of nrg. Defects in the central brain of ibx mutants are similar to those observed in another nrg mutant, central brain deranged(1) (ceb(1)). However, both ceb(1) homozygous and ceb(1)/ibx heterozygous females are receptive. The expression of a transgene containing the non-neural isoform of nrg rescues both the receptivity and the brain structure phenotypes of ibx females.

During development, neuronal growth cones navigate over long distances to reach their target and establish appropriate connections. This process is usually described as a step-by-step mechanism of growth recruiting several guidance cues with attractive and repulsive properties. Among these signals, the semaphorins define a large family of more than 20 members divided into eight classes according to their phylogenic relationship and the existence of differential structural domains or sequence motifs 1 Fig. 1). Classes I and II are found in invertebrates and classes III, IV and VII in vertebrates while class V contains members from both vertebrates and invertebrates. The class VIII corresponds to viral semaphorins. Semaphorins can be soluble proteins (classes II and III), transmembrane proteins (classes I, IV, V and VI) or membrane-bound through a glycosylphosphatidylinositol anchor (class VII). Together with plexins and scatter factor receptors, semaphorins belong to the semaphorin superfamily whose defining feature is the existence of a conserved domain: the semaphorin domain. This domain of more than 500 amino acids located at the mature protein N-terminus was first identified by Kolodkin and collaborators in 1993 2. All semaphorins also contain N-glycosylation sites and their C-terminal part is differing from one class to another. Indeed, class V contains seven copies of thrombospondin repeats, classes II–V and VII contain an immunoglobulin-like domain while class III has an N-terminus basic motif. Semaphorins are widely expressed in the developing nervous system. Initially described as guidance cues ensuring axon targeting, further studies showed that they are also key regulators of cell migration, cell death or synapse formation during nervous system development. Like other guidance cues, semaphorins are also clearly implicated in various aspects of organogenesis 3 (including lung and kidney formation or angiogenesis) and during tumor progression 4. Here, we review the major functions of semaphorins in the nervous system together with the signaling mechanisms involved both at the level of receptor complex formation and recruitment of selective intracellular pathways.

Molecular characterization and expression of a novel human leukocyte cell-surface marker homologous to mouse Ly-9

The shift from unicellular life to multicellular, integrated organisms has been accompanied by the acquisition of adhesion proteins/receptors. Recently we succeeded to clone some genes coding for such proteins from the lowest multicellular animals, the marine sponges (model: the siliceous sponge Geodia cydonium). G. cydonium contains e.g. several lectins; cDNAs for two of them were cloned. Both lectins have a framework sequence of 38 conserved amino acids which are characteristic for the carbohydrate binding site of vertebrate S-type lectins. Next, the cDNA coding for a receptor tyrosine kinase of class II was isolated and characterized. The deduced aa sequence shows two characteristic domains; (i) the tyrosine kinase domain and (ii) an immunoglobulin-like domain. The latter part displays high homology to the vertebrate type immunoglobulin domain. This result together with the lectin data demonstrates that binding domains of such adhesion proteins are not recent achievements of higher animals but exist already in animals (sponges) which have diverged from other organisms about 800 million years ago. Considering the fact that during embryogenesis of sponges a typical anteroposterior organization pattern is seen a 'home-otic' organ-like transformation has been postulated. The subsequent search for genes provided with the homeodomain-like sequence was successful. These data support the view that the kingdom Animalia is of monophyletic origin.

The correct formation of disulfide bonds is a crucial step in the folding of many virulence factors in bacteria. In many Gram-negative bacteria, redox machinery in the periplasm ensures this occurs efficiently and correctly for secreted and membrane proteins through the processes of disulfide bond oxidation and isomerisation. This machinery has been well characterised in Escherichia coli (strain K-12) where it consists of four key disulfide bond forming (Dsb) proteins, DsbA, DsbB, DsbC and DsbD. E. coli DsbA is the prototypical bacterial oxidase, which catalyses the formation of disulfide bonds in its protein substrates. When the active site cysteines of DsbA become reduced, DsbA needs to be reoxidised by its redox partner, DsbB, to continue to function. DsbA often forms disulfide bonds incorrectly and DsbC corrects this by isomerising or shuffling the disulfide bonds in the substrate protein. As DsbC is only active when its catalytic cysteines are reduced, it also has a redox partner, DsbD. Over the past 27 years, Dsb-like proteins from various bacteria have been studied and it has become clear that there is great diversity in the number and type of proteins that contribute to the redox machinery in different bacteria. Some bacteria have multiple Dsb proteins and distinct Dsb-like systems that have specific roles. The suppressor of copper sensitivity (Scs) proteins form one such system. Originally identified in Salmonella enterica serovar Typhimurium, the Scs locus encodes four proteins (ScsA, ScsB, ScsC and ScsD). The S. Typhimurium locus was found to confer copper tolerance to a copper sensitive E. coli mutant, so it is thought that these proteins play a role in bacterial response to copper stress. Three Scs proteins (ScsB, ScsC and ScsD) have predicted thioredoxin folds and catalytic cysteines, similar to the Dsb proteins. This thesis focuses on the characterisation of the ScsC and ScsB proteins from the uropathogen Proteus mirabilis. Prior to this work, ScsC had only been studied in two organisms, S. Typhimurium and Caulobacter crescentus. Chapter 2 is a Nature Communications paper that describes the genetic, biochemical and structural characterisation of P. mirabilis ScsC (PmScsC). This work revealed that PmScsC is a highly dynamic, trimeric disulfide isomerase that increases the swarming motility of P. mirabilis under copper stress. An N-terminally truncated PmScsC variant, PmScsCΔN, is monomeric and has little in vitro disulfide isomerase activity but is highly active as a dithiol oxidase. This variant could not complement the loss of ScsC in P. mirabilis, suggesting that PmScsC functions as a trimeric disulfide isomerase in vivo. The three crystal structures of PmScsC revealed that the region responsible for the highly dynamic nature of the protein is an 11 amino acid flexible linker between the trimerisation domain and catalytic domain. Replacement of this flexible linker with a rigid linker revealed that it was necessary for the disulfide isomerase activity of PmScsC. Chapter 3 describes the structural characterisation of the PmScsC variant, PmScsCΔN and the biochemical and structural characterisation of another variant PmScsCΔLinker. The structure of PmScsCΔN is very similar to the catalytic domains of the native PmScsC structures, suggesting that it is the lack of the trimerisation stem and thus change in oligomerisation state that converts it from an isomerase to an oxidase. To assess the role of the flexible linker in PmScsC, the linker residues (39-49) were deleted. This deletion mutant (PmScsCΔLinker) crystallised as a trimer but exists in different oligomeric states in solution and does not function as an oxidase or an isomerase. The characterisation of these variants adds to our understanding of the key structural features of PmScsC and their influence on the function of this trimeric disulfide isomerase. This chapter has been submitted to Acta Crystallographica Section D for publication.Based on work performed in C. crescentus, we hypothesised that the N-terminal periplasmic domain of PmScsB (PmScsBα) is the redox partner of PmScsC. Chapter 4 is a Journal of Biological Chemistry publication that describes the experiments performed to confirm this hypothesis. This publication also reports the crystal structure of PmScsBα, (the first ScsBα structure to be solved) and a SANS derived model of the complex formed between PmScsC and PmScsBα. I found that PmScsBα consists of two immunoglobulin-like domains, both of which interact with one PmScsC protomer in the SANS model.To gain more information about PmScsB, I wanted to produce the full-length transmembrane protein for further characterisation. Chapter 5 describes the work performed towards the design of robust expression and purification protocols for the production of PmScsB. In summary, this thesis describes the characterisation of PmScsC, PmScsC mutants, PmScsBα and the interaction between PmScsC and PmScsBα. It also contains the steps taken toward the production of the full-length transmembrane protein PmScsB for future analysis. This work adds to our understanding about the Suppressor of copper sensitivity proteins and the diversity of redox machinery in bacteria.