CD9 co-operation with syndecan-1 is required for a major staphylococcal adhesion pathway.

Epithelial colonization is a critical first step in bacterial pathogenesis. Staphylococcus aureus can utilize several host factors to associate with cells, including α5ß1 integrin and heparan sulfate proteoglycans, such as the syndecans. Here, we demonstrate that a partner protein of both integrins and syndecans, the host membrane adapter protein tetraspanin CD9, is essential for syndecan-mediated staphylococcal adhesion. Fibronectin is also essential in this process, while integrins are only critical for post-adhesion entry into human epithelial cells. Treatment of epithelial cells with CD9-derived peptide or heparin caused significant reductions in staphylococcal adherence, dependent on both CD9 and syndecan-1. Exogenous fibronectin caused a CD9-dependent increase in staphylococcal adhesion, whereas blockade of ß1 integrins did not affect adhesion but did reduce the subsequent internalization of adhered bacteria. CD9 disruption or deletion increased ß1 integrin-mediated internalization, suggesting that CD9 coordinates sequential staphylococcal adhesion and internalization. CD9 controls staphylococcal adhesion through syndecan-1, using a mechanism that likely requires CD9-mediated syndecan organization to correctly display fibronectin at the host cell surface. We propose that CD9-derived peptides or heparin analogs could be developed as anti-adhesion treatments to inhibit the initial stages of staphylococcal pathogenesis. IMPORTANCE Staphylococcus aureus infection is a significant cause of disease and morbidity. Staphylococci utilize multiple adhesion pathways to associate with epithelial cells, including interactions with proteoglycans or ß1 integrins through a fibronectin bridge. Interference with another host protein, tetraspanin CD9, halves staphylococcal adherence to epithelial cells, although CD9 does not interact directly with bacteria. Here, we define the role of CD9 in staphylococcal adherence and uptake, observing that CD9 coordinates syndecan-1, fibronectin, and ß1 integrins to allow efficient staphylococcal infection. Two treatments that disrupt this action are effective and may provide an alternative to antibiotics. We provide insights into the mechanisms that underlie staphylococcal infection of host cells, linking two known adhesion pathways together through CD9 for the first time.

Tetraspanin Cd9b plays a role in fertility in zebrafish.

In mice, CD9 expression on the egg is required for efficient sperm-egg fusion and no effects on ovulation or male fertility are observed in CD9 null animals. Here we show that cd9b knockout zebrafish also appear to have fertility defects. In contrast to mice, fewer eggs were laid by cd9b knockout zebrafish pairs and, of the eggs laid, a lower percentage were fertilised. These effects could not be linked to primordial germ cell numbers or migration as these were not altered in the cd9b mutants. The decrease in egg numbers could be rescued by exchanging either cd9b knockout partner, male or female, for a wildtype partner. However, the fertilisation defect was only rescued by crossing a cd9b knockout female with a wildtype male. To exclude effects of mating behaviour we analysed clutch size and fertilisation using in vitro fertilisation techniques. Number of eggs and fertilisation rates were significantly reduced in the cd9b mutants suggesting the fertility defects are not solely due to courtship behaviours. Our results indicate that CD9 plays a more complex role in fish fertility than in mammals, with effects in both males and females.

Tetraspanin Cd9b and Cxcl12a/Cxcr4b have a synergistic effect on the control of collective cell migration.

Collective cell migration is essential for embryonic development and homeostatic processes. During zebrafish development, the posterior lateral line primordium (pLLP) navigates along the embryo flank by collective cell migration. The chemokine receptors, Cxcr4b and Cxcr7b, as well as their cognate ligand, Cxcl12a, are essential for this process. We corroborate that knockdown of the zebrafish cd9 tetraspanin orthologue, cd9b, results in mild pLL abnormalities. Through generation of CRISPR and TALEN mutants, we show that cd9a and cd9b function partially redundantly in pLLP migration, which is delayed in the cd9b single and cd9a; cd9b double mutants. This delay led to a transient reduction in neuromast numbers. Loss of both Cd9a and Cd9b sensitized embryos to reduced Cxcr4b and Cxcl12a levels. Together these results provide evidence that Cd9 modulates collective cell migration of the pLLP during zebrafish development. One interpretation of these observations is that Cd9 contributes to more effective chemokine signalling.

ACE2-Independent Interaction of SARS-CoV-2 Spike Protein with Human Epithelial Cells Is Inhibited by Unfractionated Heparin.

Coronaviruses such as SARS-CoV-2, which is responsible for COVID-19, depend on virus spike protein binding to host cell receptors to cause infection. The SARS-CoV-2 spike protein binds primarily to ACE2 on target cells and is then processed by membrane proteases, including TMPRSS2, leading to viral internalisation or fusion with the plasma membrane. It has been suggested, however, that receptors other than ACE2 may be involved in virus binding. We have investigated the interactions of recombinant versions of the spike protein with human epithelial cell lines that express low/very low levels of ACE2 and TMPRSS2 in a proxy assay for interaction with host cells. A tagged form of the spike protein containing the S1 and S2 regions bound in a temperature-dependent manner to all cell lines, whereas the S1 region alone and the receptor-binding domain (RBD) interacted only weakly. Spike protein associated with cells independently of ACE2 and TMPRSS2, while RBD required the presence of high levels of ACE2 for interaction. As the spike protein has previously been shown to bind heparin, a soluble glycosaminoglycan, we tested the effects of various heparins on ACE2-independent spike protein interaction with cells. Unfractionated heparin inhibited spike protein interaction with an IC50 value of <0.05 U/mL, whereas two low-molecular-weight heparins were less effective. A mutant form of the spike protein, lacking the arginine-rich putative furin cleavage site, interacted only weakly with cells and had a lower affinity for unfractionated and low-molecular-weight heparin than the wild-type spike protein. This suggests that the furin cleavage site might also be a heparin-binding site and potentially important for interactions with host cells. The glycosaminoglycans heparan sulphate and dermatan sulphate, but not chondroitin sulphate, also inhibited the binding of spike protein, indicating that it might bind to one or both of these glycosaminoglycans on the surface of target cells.

Matriptase activation of Gq drives epithelial disruption and inflammation via RSK and DUOX.

Epithelial tissues are primed to respond to insults by activating epithelial cell motility and rapid inflammation. Such responses are also elicited upon overexpression of the membrane-bound protease, Matriptase, or mutation of its inhibitor, Hai1. Unrestricted Matriptase activity also predisposes to carcinoma. How Matriptase leads to these cellular outcomes is unknown. We demonstrate that zebrafish hai1a mutants show increased H2O2, NfκB signalling, and IP3R -mediated calcium flashes, and that these promote inflammation, but do not generate epithelial cell motility. In contrast, inhibition of the Gq subunit in hai1a mutants rescues both the inflammation and epithelial phenotypes, with the latter recapitulated by the DAG analogue, PMA. We demonstrate that hai1a has elevated MAPK pathway activity, inhibition of which rescues the epidermal defects. Finally, we identify RSK kinases as MAPK targets disrupting adherens junctions in hai1a mutants. Our work maps novel signalling cascades mediating the potent effects of Matriptase on epithelia, with implications for tissue damage response and carcinoma progression.

Cancer occurs when normal processes in the cell become corrupted or unregulated. Many proteins can contribute, including one enzyme called Matriptase that cuts other proteins at specific sites. Matriptase activity is tightly controlled by a protein called Hai1. In mice and zebrafish, when Hai1 cannot adequately control Matriptase activity, invasive cancers with severe inflammation develop. However, it is unclear how unregulated Matriptase leads to both inflammation and cancer invasion. One outcome of Matriptase activity is removal of proteins called Cadherins from the cell surface. These proteins have a role in cell adhesion: they act like glue to stick cells together. Without them, cells can dissociate from a tissue and move away, a critical step in cancer cells invading other organs. However, it is unknown exactly how Matriptase triggers the removal of Cadherins from the cell surface to promote invasion. Previous work has shown that Matriptase switches on a receptor called Proteinase-activated receptor 2, or Par2 for short, which is known to activate many enzymes, including one called phospholipase C. When activated, this enzyme releases two signals into the cell: a sugar called inositol triphosphate, IP3; and a lipid or fat called diacylglycerol, DAG. It is possible that these two signals have a role to play in how Matriptase removes Cadherins from the cell surface. To find out, Ma et al. mapped the effects of Matriptase in zebrafish lacking the Hai1 protein. This revealed that Matriptase increases IP3 and DAG levels, which initiate both inflammation and invasion. IP3 promotes inflammation by switching on pro-inflammatory signals inside the cell such as the chemical hydrogen peroxide. At the same time, DAG promotes cell invasion by activating a well-known cancer signalling pathway called MAPK. This pathway activates a protein called RSK. Ma et al. show that this protein is required to remove Cadherins from the surface of cells, thus connecting Matriptase's activation of phospholipase C with its role in disrupting cell adhesion. An increase in the ratio of Matriptase to HAI-1 (the human equivalent of Hai1) is present in many cancers. For this reason, the signal cascades described by Ma et al. may be of interest in developing treatments for these cancers. Understanding how these signals work together could lead to more direct targeted anti-cancer approaches in the future.

Tetraspanins are involved in Burkholderia pseudomallei-induced cell-to-cell fusion of phagocytic and non-phagocytic cells.

Tetraspanins are four-span transmembrane proteins of host cells that facilitate infections by many pathogens. Burkholderia pseudomallei is an intracellular bacterium and the causative agent of melioidosis, a severe disease in tropical regions. This study investigated the role of tetraspanins in B. pseudomallei infection. We used flow cytometry to determine tetraspanins CD9, CD63, and CD81 expression on A549 and J774A.1 cells. Their roles in B. pseudomallei infection were investigated in vitro using monoclonal antibodies (MAbs) and recombinant large extracellular loop (EC2) proteins to pretreat cells before infection. Knockout of CD9 and CD81 in cells was performed using CRISPR Cas9 to confirm the role of tetraspanins. Pretreatment of A549 cells with MAb against CD9 and CD9-EC2 significantly enhanced B. pseudomallei internalization, but MAb against CD81 and CD81-EC2 inhibited MNGC formation. Reduction of MNGC formation was consistently observed in J774.A1 cells pretreated with MAbs specific to CD9 and CD81 and with CD9-EC2 and CD81-EC2. Data from knockout experiments confirmed that CD9 enhanced bacterial internalization and that CD81 inhibited MNGC formation. Our data indicate that tetraspanins are host cellular factors that mediated internalization and membrane fusion during B. pseudomallei infection. Tetraspanins may be the potential therapeutic targets for melioidosis.

A role for tetraspanin proteins in regulating fusion induced by Burkholderia thailandensis.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high morbidity that is endemic in South East Asia and northern Australia. An unusual feature of the bacterium is its ability to induce multinucleated giant cell formation (MNGC), which appears to be related to bacterial pathogenicity. The mechanism of MNGC formation is not fully understood, but host cell factors as well as known bacterial virulence determinants are likely to contribute. Since members of the tetraspanin family of membrane proteins are involved in various types of cell:cell fusion, their role in MNGC formation induced by Burkholderia thailandensis, a mildly pathogenic species closely related to B. pseudomallei, was investigated. The effect of antibodies to tetraspanins CD9, CD81, and CD63 in MNGC formation induced by B. thailandensis in infected mouse J774.2 and RAW macrophage cell lines was assessed along with that of recombinant proteins corresponding to the large extracellular domain (EC2) of the tetraspanins. B. thailandensis-induced fusion was also examined in macrophages derived from CD9 null and corresponding WT mice, and in J774.2 macrophages over-expressing CD9. Antibodies to CD9 and CD81 promoted MNGC formation induced by B. thailandensis, whereas EC2 proteins of CD9, CD81, and CD63 inhibited MNGC formation. Enhanced MNGC formation was observed in CD9 null macrophages, whereas a decrease in MNGC formation was associated with overexpression of CD9. Overall our findings show that tetraspanins are involved in MNGC formation induced by B. thailandensis and by implication, B. pseudomallei, with CD9 and CD81 acting as negative regulators of this process.

The Use of Plant-Derived Ribosome Inactivating Proteins in Immunotoxin Development: Past, Present and Future Generations.

Ribosome inactivating proteins (RIPs) form a class of toxins that was identified over a century ago. They continue to fascinate scientists and the public due to their very high activity and long-term stability which might find useful applications in the therapeutic killing of unwanted cells but can also be used in acts of terror. We will focus our review on the canonical plant-derived RIPs which display ribosomal RNA N-glycosidase activity and irreversibly inhibit protein synthesis by cleaving the 28S ribosomal RNA of the large 60S subunit of eukaryotic ribosomes. We will place particular emphasis on therapeutic applications and the generation of immunotoxins by coupling antibodies to RIPs in an attempt to target specific cells. Several generations of immunotoxins have been developed and we will review their optimisation as well as their use and limitations in pre-clinical and clinical trials. Finally, we endeavour to provide a perspective on potential future developments for the therapeutic use of immunotoxins.

A role for the tetraspanin proteins in Salmonella infection of human macrophages.

OBJECTIVE: Infected macrophages play a role in the dissemination of Salmonella and may serve as a reservoir of infection in asymptomatic carriers. However, relatively little is known about the early molecular interactions of the bacteria with these cells. We have recently shown that members of the tetraspanin family of membrane proteins are involved in the initial adhesion of a range of bacteria to host cells. This study investigated the role of tetraspanins in Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) infection of human monocyte-derived macrophages (MDM). METHODS: The role of tetraspanins was studied by the use of tetraspanins recombinant proteins as well as monoclonal antibodies targeted against different tetraspanins. Knockdown of the tetraspanin CD63 was carried out by siRNA to further study the role of CD63 in Salmonella uptake. RESULTS: Recombinant proteins representing the large extracellular domains of tetraspanins inhibited binding of S. Typhimurium to human MDM by ∼50%, whereas tetraspanin-specific antibodies showed varying effects, with some enhancing (anti-CD37) and some inhibiting (anti-CD81, anti-CD63) binding. Inhibition of the S. Typhimurium-MDM interaction by anti-CD63 mAb appeared to be mediated by antibody induced internalization, suggesting that surface expression of CD63 is required for S. Typhimurium binding. Knockdown of CD63 in human MDM using siRNA greatly reduced S. Typhimurium binding, confirming the importance of CD63. However, ectopic expression of CD63 in the non-phagocytic cell line HEK293 was insufficient to mediate bacterial binding. CONCLUSION: Bacterial adhesion is the first step in infection by pathogens that invade and replicate within host cells. Taken together, the results here describe a role for tetraspanins in binding of S. Typhimurium to human macrophages and highlight the particular importance of CD63 in this process.

Tspan2: a tetraspanin protein involved in oligodendrogenesis and cancer metastasis.

Tetraspanin 2 (Tspan2) is one of the less well-characterised members of the tetraspanin superfamily, and its precise function in different human tissue types remains to be explored. Initial studies have highlighted its possible association in neuroinflammation and carcinogenesis. In the central nervous system, Tspan2 may contribute to the early stages of the oligodendrocyte differentiation into myelin-forming glia. Furthermore, in human lung cancer, Tspan2 could be involved in the progression of the tumour metastasis by modulating cancer cell motility and invasion functions. In this review, we discuss the available evidence for the potential role of Tspan2 and introduce possible strategies for disease targeting.

Peptides from Tetraspanin CD9 Are Potent Inhibitors of Staphylococcus Aureus Adherence to Keratinocytes.

Staphylococcus aureus is one of the primary causative agents of skin and wound infections. As bacterial adherence is essential for infection, blocking this step can reduce invasion of host tissues by pathogens. An anti-adhesion therapy, based on a host membrane protein family, the tetraspanins, has been developed that can inhibit the adhesion of S. aureus to human cells. Synthetic peptides derived from a keratinocyte-expressed tetraspanin, CD9, were tested for anti-adhesive properties and at low nanomolar concentrations were shown to inhibit bacterial adhesion to cultured keratinocytes and to be effective in a tissue engineered model of human skin infection. These potential therapeutics had no effect on keratinocyte viability, migration or proliferation, indicating that they could be a valuable addition to current treatments for skin infection.

Distinct regions of the large extracellular domain of tetraspanin CD9 are involved in the control of human multinucleated giant cell formation.

Multinucleated giant cells, formed by the fusion of monocytes/macrophages, are features of chronic granulomatous inflammation associated with infections or the persistent presence of foreign material. The tetraspanins CD9 and CD81 regulate multinucleated giant cell formation: soluble recombinant proteins corresponding to the large extracellular domain (EC2) of human but not mouse CD9 can inhibit multinucleated giant cell formation, whereas human CD81 EC2 can antagonise this effect. Tetraspanin EC2 are all likely to have a conserved three helix sub-domain and a much less well-conserved or hypervariable sub-domain formed by short helices and interconnecting loops stabilised by two or more disulfide bridges. Using CD9/CD81 EC2 chimeras and point mutants we have mapped the specific regions of the CD9 EC2 involved in multinucleated giant cell formation. These were primarily located in two helices, one in each sub-domain. The cysteine residues involved in the formation of the disulfide bridges in CD9 EC2 were all essential for inhibitory activity but a conserved glycine residue in the tetraspanin-defining 'CCG' motif was not. A tyrosine residue in one of the active regions that is not conserved between human and mouse CD9 EC2, predicted to be solvent-exposed, was found to be only peripherally involved in this activity. We have defined two spatially-distinct sites on the CD9 EC2 that are required for inhibitory activity. Agents that target these sites could have therapeutic applications in diseases in which multinucleated giant cells play a pathogenic role.

Regulation of zebrafish hatching by tetraspanin cd63.

Tetraspanins cause the clustering of membrane proteins into a level of organisation essential for cellular function. Given the importance and complicated nature of this mechanism, we attempted a novel approach to identify the function of a single component in a biologically relevant context. A morpholino knockdown strategy was used to investigate the role of cd63, a membrane protein associated with intracellular transport and a melanoma marker, in embryonic zebrafish. By using three separate morpholinos targeting cd63, we were able to identify a specific phenotype. Strikingly, morphant fish failed to hatch due to the lack of secreted proteolytic enzymes required for chorion-softening. The morphology of the hatching gland at both the cellular and intracellular levels was disorganised, suggesting a role for cd63 in the functioning of this organ. This work identifies a specific role for cd63 in the zebrafish embryo and provides evidence for the suitability of zebrafish as a model system for the investigation of tetraspanin enriched microdomains.

Cooperative role for tetraspanins in adhesin-mediated attachment of bacterial species to human epithelial cells.

The tetraspanins are a superfamily of transmembrane proteins with diverse functions and can form extended microdomains within the plasma membrane in conjunction with partner proteins, which probably includes receptors for bacterial adhesins. Neisseria meningitidis, the causative agent of meningococcal disease, attaches to host nasopharyngeal epithelial cells via type IV pili and opacity (Opa) proteins. We examined the role of tetraspanin function in Neisseria meningitidis adherence to epithelial cells. Tetraspanins CD9, CD63, and CD151 were expressed by HEC-1-B and DETROIT 562 cells. Coincubation of cells with antibodies against all three tetraspanin molecules used individually or in combination, with recombinant tetraspanin extracellular domains (EC2), or with small interfering RNAs (siRNAs) significantly reduced adherence of Neisseria meningitidis. In contrast, recombinant CD81, a different tetraspanin, had no effect on meningococcal adherence. Antitetraspanin antibodies reduced the adherence to epithelial cells of Neisseria meningitidis strain derivatives expressing Opa and pili significantly more than isogenic strains lacking these determinants. Adherence to epithelial cells of strains of Staphylococcus aureus, Neisseria lactamica, Escherichia coli, and Streptococcus pneumoniae was also reduced by pretreatment of cells with tetraspanin antibodies and recombinant proteins. These data suggest that tetraspanins are required for optimal function of epithelial adhesion platforms containing specific receptors for Neisseria meningitidis and potentially for multiple species of bacteria.

Strategies for targeting tetraspanin proteins: potential therapeutic applications in microbial infections.

The identification of novel targets and strategies for therapy of microbial infections is an area of intensive research due to the failure of conventional vaccines or antibiotics to combat both newly emerging diseases (e.g. viruses such as severe acute respiratory syndrome (SARS) and new influenza strains, and antibiotic-resistant bacteria) and entrenched, pandemic diseases exemplified by HIV. One clear approach to this problem is to target processes of the host organism rather than the microbe. Recent data have indicated that members of the tetraspanin superfamily, proteins with a widespread distribution in eukaryotic organisms and 33 members in humans, may provide such an approach. Tetraspanins traverse the membrane four times, but are distinguished from other four-pass membrane proteins by the presence of conserved charged residues in the transmembrane domains and a defining 'signature' motif in the larger of the two extracellular domains (the EC2). They characteristically form promiscuous associations with one another and with other membrane proteins and lipids to generate a specialized type of microdomain: the tetraspanin-enriched microdomain (TEM). TEMs are integral to the main role of tetraspanins as 'molecular organizers' involved in functions such as membrane trafficking, cell-cell fusion, motility, and signaling. Increasing evidence demonstrates that tetraspanins are used by intracellular pathogens as a means of entering and replicating within human cells. Although previous investigations focused mainly on viruses such as hepatitis C and HIV, it is now becoming clear that other microbes associate with tetraspanins, using TEMs as a 'gateway' to infection. In this article we review the properties and functions of tetraspanins/TEMs that are relevant to infective processes and discuss the accumulating evidence that shows how different pathogens exploit these properties in infection and in the pathogenesis of disease. We then investigate the novel and exciting possibilities of targeting tetraspanins for the treatment of infectious disease, using specific antibodies, recombinant EC2 domains, small-molecule mimetics, and small interfering RNA. Such therapies, directed at host-cell molecules, may provide alternative options for combating fast-mutating or newly emerging pathogens, where conventional approaches face difficulties.

Distinct roles for tetraspanins CD9, CD63 and CD81 in the formation of multinucleated giant cells.

Members of the tetraspanin superfamily of proteins are implicated in a variety of complex cell processes including cell fusion. However, the contribution of individual tetraspanins to these processes has proved difficult to define. Here we report the use of recombinant extracellular regions of tetraspanins to investigate the role of specific members of this family in the fusion of monocytes to form multinucleated giant cells (MGC). In contrast to their positive requirement in sperm-egg fusion, previous studies using antibodies and knockout mice have indicated a negative regulatory role for tetraspanins CD9 and CD81 in this process. In an in vitro model of fusion using human monocytes, we have confirmed observations that antibodies to CD9 and CD81 enhance MGC formation; however, in contrast to previous investigations, we found that all members of a panel of antibodies to CD63 inhibited fusion. Moreover, recombinant proteins corresponding to the large extracellular domains (EC2s) of CD63 and CD9 inhibited MGC formation, whereas the EC2s of CD81 and CD151 had no effect. The potent inhibition of fusion and binding of labelled CD63 EC2 to monocytes under fusogenic conditions suggest a direct interaction with a membrane component required for fusion. Our findings indicate that the tetraspanins CD9, CD63 and CD81 are all involved in MGC formation, but play distinct roles.

Characterisation of receptor binding by the chemotaxis inhibitory protein of Staphylococcus aureus and the effects of the host immune response.

The chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is reported to bind to the receptors for C5a and formylated peptides and has been proposed as a promising lead for the development of new anti-inflammatory compounds. Here we have examined the receptor specificity and mode of action of recombinant CHIPS(28-149) and also the immune response to CHIPS(28-149) in patients with S. aureus infections and in uninfected controls. Recombinant CHIPS(28-149) bound with high affinity to the human C5a receptor (C5aR), but had low affinity for the second C5a receptor, C5L2, and the formyl peptide receptor, FPR. Although ligand binding to C5aR was potently inhibited, CHIPS(28-149) had much weaker effects on ligand binding to C5L2 and FPR. Similarly, CHIPS(28-149) potently inhibited the ligand-induced activation of C5aR but was less potent at inhibition via FPR. NMR studies showed that CHIPS(28-149) bound directly to the N-terminus of C5aR but not C5L2, and CHIPS(28-149) residues involved in the interaction were identified by chemical shift analysis. All human sera examined contained high titres of IgG and IgA reactivity against CHIPS(28-149), and no correlation was observed between infection status at the time of serum collection and antibody titre. Individual serum samples promoted or inhibited the binding of CHIPS(28-149) to C5aR, or had no effect. IgG depletion of serum samples abrogated the effects on CHIPS binding, demonstrating that these were antibody mediated. Sera from infected individuals were more likely to inhibit CHIPS(28-149) binding than sera from healthy controls. However, high antibody titres correlated well with both inhibition and enhancement of CHIPS(28-149) binding to C5aR; this suggests that the inhibitory effect relates to epitope specificity rather than greater antibody binding. We conclude that CHIPS is likely to be too immunogenic to be used as an anti-inflammatory treatment but that some antibodies against CHIPS may be useful in the treatment of S. aureus infections.

The role of the N-terminal domain of the complement fragment receptor C5L2 in ligand binding.

C5L2 is a new cellular receptor found to interact with the human anaphylatoxins complement factor C5a and its C-terminal cleavage product C5a des Arg. The classical human C5a receptor (C5aR) preferentially binds C5a, with a 10-100-fold lower affinity for C5a des Arg. In contrast, C5L2 binds both ligands with nearly equal affinity. C5aR presents acidic and tyrosine residues in its N terminus that interact with the core of C5a while a hydrophobic pocket formed by the transmembrane helices interacts with residues in the C terminus of C5a. Here, we have investigated the molecular basis for the increased affinity of C5L2 for C5a des Arg. Rat and mouse C5L2 preferentially bound C5a des Arg, whereas rodent C5aR showed much higher affinity for intact C5a. Effective peptidic and non-peptidic ligands for the transmembrane hydrophobic pocket of C5aR were poor inhibitors of ligand binding to C5L2. An antibody raised against the N terminus of human C5L2 did not affect the binding of C5a to C5L2 but did inhibit C5a des Arg binding. A chimeric C5L2, containing the N terminus of C5aR, had little effect on the affinity for C5a des Arg. Mutation of acidic and tyrosine residues in the N terminus of human C5L2 revealed that 3 residues were critical for C5a des Arg binding but had little involvement in C5a binding. C5L2 thus appears to bind C5a and C5a des Arg by different mechanisms, and, unlike C5aR, C5L2 uses critical residues in its N-terminal domain for binding only to C5a des Arg.

Recombinant extracellular domains of tetraspanin proteins are potent inhibitors of the infection of macrophages by human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) infection of human macrophages can be inhibited by antibodies which bind to the tetraspanin protein CD63, but not by antibodies that bind to other members of the tetraspanin family. This inhibitory response was limited to CCR5 (R5)-tropic virus and was only observed using macrophages, but not T cells. Here, we show that recombinant soluble forms of the large extracellular domain (EC2) of human tetraspanins CD9, CD63, CD81, and CD151 produced as fusion proteins with glutathione S-transferase (GST) can all potently and completely inhibit R5 HIV-1 infection of macrophages with 50% inhibitory concentration values of 0.11 to 1.2 nM. Infection of peripheral blood mononuclear cells could also be partly inhibited, although higher concentrations of EC2 proteins were required. Inhibition was largely coreceptor independent, as macrophage infections by virions pseudotyped with CXCR4 (X4)-tropic HIV-1 or vesicular stomatitis virus (VSV)-G glycoproteins were also inhibited, but was time dependent, since addition prior to or during, but not after, virus inoculation resulted in potent inhibition. Incubation with tetraspanins did not decrease CD4 or HIV-1 coreceptor expression but did block virion uptake. Colocalization of fluorescently labeled tetraspanin EC2 proteins and HIV-1 virions within, and with CD4 and CXCR4 at the cell surfaces of, macrophages could be detected, and internalized tetraspanin EC2 proteins were directed to vesicular compartments that contained internalized dextran and transferrin. Collectively, the data suggest that the mechanism of inhibition of HIV-1 infection by tetraspanins is at the step of virus entry, perhaps via interference with binding and/or the formation of CD4-coreceptor complexes within microdomains that are required for membrane fusion events.

Crystallization of a carbamatase catalytic antibody Fab fragment and its complex with a transition-state analogue.

Catalytic antibodies showing carbamatase activity have significant potential in antibody-directed prodrug therapy against tumours. The Fab fragment of an IgG1 mouse monoclonal carbamatase catalytic antibody JC1 raised against a transition-state analogue, ethyl N-(3,5-dicarboxyphenyl)-P-[N-[5'-(2",5"-dioxo-1"-pyrrolidinyl)oxy-1',5'-dioxopentyl]-4-aminophenylmethyl]phosphonamidate, was obtained by digestion of the whole antibody with papain and was purified by two-step ion-exchange chromatography. Using hanging-drop vapour-diffusion crystallization techniques, three different crystal forms of the Fab fragment were obtained in the presence and absence of the transition-state analogue. All crystals diffract X-rays to between 3.5 and 3.2 A resolution. The two crystal forms grown in the presence of the transition-state analogue contain up to four or eight copies of the Fab in the asymmetric unit and diffract to 3.5 and 3.2 A, respectively. The crystal of the Fab alone is most likely to contain only two copies of the Fab in the asymmetric unit and diffracts to beyond 3.5 A. Determination of the structure will provide insights into the active-site arrangement of this antibody and will help to increase our understanding of the molecular mechanisms by which the immune system can evolve catalytic function.