Cell Surface Binding Proteins Research Articles

Immunoinformatic and molecular docking approaches: siRNA prediction to silence cell surface binding protein of monkeypox virus

BackgroundMonkeypox virus (MPV), an endemic pathogen in Africa, shares clinical similarities with smallpox. Recent reports indicate a concerning increase in the number of MPV cases detected outside its endemic region, highlighting the emergence of a multi-country outbreak. Given the importance of the cell surface-binding protein E8L in facilitating viral attachment to host cells, this study aimed to identify potential small interfering RNAs (siRNAs) capable of silencing E8L and thereby serving as a basis for therapeutic development.ResultssiRNAs have emerged as promising candidates for genetic therapies and antiviral and antibacterial treatments. In this investigation, we employed computational assays, including GC content analysis, binding free energy assessment, folding properties evaluation, melting temperature determination, and siRNA efficacy prediction. Our comprehensive analysis identified five siRNAs with high potential for effectively silencing the cell surface-binding protein of the monkeypox virus. Among these siRNAs, molecular docking revealed that “S8” (Guide-UUAUGGAUCCAAUCACUUGAU, Passenger-CAAGUGAUUGGAUCCAUAAUC) demonstrated the strongest affinity with the human argonaute-2 protein.ConclusionsThe siRNA “S8” represents a promising therapeutic target for developing treatments against monkeypox virus infection by specifically silencing the cell surface-binding protein E8L. Our research lays the foundation for future endeavors in genome-level therapies. It can potentially create chemically produced RNA molecules as effective antiviral drugs targeting Monkeypox virus infection. These findings contribute to advancing therapeutic strategies and offer new avenues for combating the spread of MPV, particularly in regions affected by the multi-country outbreak.

Exploring the chemical space for potential inhibitors against cell surface binding protein of Mpox virus using molecular fingerprint based screening approach

Mpox virus is the latest member of the Poxviridae family of which small pox virus is a member. Monekypox virus has led to thousands of infections across the globe. Poxvirus gains entry into the cell making use of glycosaminoglycans like chondroitin sulphate and heparan sulphate. The interaction of the Mpox virus protein E8L also called cell surface binding protein is crucial for host cell attachment, membrane fusion and viral entry into the host cell leading to establishment of infection thus making this protein a very attractive therapeutic target. In this study we have tried to utilize the chondroitin sulphate binding groove present in the protein and identify molecules which are structurally similar to chondroitin sulphate. These molecules can thus occupy the same pocket but with a better binding affinity than chondroitin sulphate in order to outcompete the latter molecule from binding to the E8L protein and thus prevent it from performing its function. This study may pave the way for development of highly efficient therapeutics against the Mpox virus and further curb its infective potential. Communicated by Ramaswamy H. Sarma

In silico design and immunoinformatics analysis of a universal multi-epitope vaccine against monkeypox virus.

Monkeypox virus (MPXV) outbreaks have been reported in various countries worldwide; however, there is no specific vaccine against MPXV. In this study, therefore, we employed computational approaches to design a multi-epitope vaccine against MPXV. Initially, cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), linear B lymphocytes (LBL) epitopes were predicted from the cell surface-binding protein and envelope protein A28 homolog, both of which play essential roles in MPXV pathogenesis. All of the predicted epitopes were evaluated using key parameters. A total of 7 CTL, 4 HTL, and 5 LBL epitopes were chosen and combined with appropriate linkers and adjuvant to construct a multi-epitope vaccine. The CTL and HTL epitopes of the vaccine construct cover 95.57% of the worldwide population. The designed vaccine construct was found to be highly antigenic, non-allergenic, soluble, and to have acceptable physicochemical properties. The 3D structure of the vaccine and its potential interaction with Toll-Like receptor-4 (TLR4) were predicted. Molecular dynamics (MD) simulation confirmed the vaccine's high stability in complex with TLR4. Finally, codon adaptation and in silico cloning confirmed the high expression rate of the vaccine constructs in strain K12 of Escherichia coli (E. coli). These findings are very encouraging; however, in vitro and animal studies are needed to ensure the potency and safety of this vaccine candidate.

γ-Protocadherins control synapse formation and peripheral branching of touch sensory neurons

Light touch sensation begins with activation of low-threshold mechanoreceptor (LTMR) endings in the skin and propagation of their signals to the spinal cord and brainstem. We found that the clustered protocadherin gamma (Pcdhg) gene locus, which encodes 22 cell-surface homophilic binding proteins, is required in somatosensory neurons for normal behavioral reactivity to a range of tactile stimuli. Developmentally, distinct Pcdhg isoforms mediate LTMR synapse formation through neuron-neuron interactions and peripheral axonal branching through neuron-glia interactions. The Pcdhgc3 isoform mediates homophilic interactions between sensory axons and spinal cord neurons to promote synapse formation invivo and is sufficient to induce postsynaptic specializations invitro. Moreover, loss of Pcdhgs and somatosensory synaptic inputs to the dorsal horn leads to fewer corticospinal synapses on dorsal horn neurons. These findings reveal essential roles for Pcdhg isoform diversity in somatosensory neuron synapse formation, peripheral axonal branching, and stepwise assembly of central mechanosensory circuitry.

Identification of B and T Cell Epitopes to Design an Epitope-Based Peptide Vaccine against the Cell Surface Binding Protein of Monkeypox Virus: An Immunoinformatics Study.

Although the monkeypox virus-associated illness was previously confined to Africa, recently, it has started to spread across the globe and become a significant threat to human lives. Hence, this study was designed to identify the B and T cell epitopes and develop an epitope-based peptide vaccine against this virus's cell surface binding protein through an in silico approach to combat monkeypox-associated diseases. The analysis revealed that the cell surface binding protein of the monkeypox virus contains 30 B cell and 19 T cell epitopes within the given parameter. Among the T cell epitopes, epitope "ILFLMSQRY" was found to be one of the most potential peptide vaccine candidates. The docking analysis revealed an excellent binding affinity of this epitope with the human receptor HLA-B∗15:01 with a very low binding energy (-7.5 kcal/mol). The outcome of this research will aid the development of a T cell epitope-based peptide vaccine, and the discovered B and T cell epitopes will facilitate the creation of other epitope and multi-epitope-based vaccines in the future. This research will also serve as a basis for further in vitro and in vivo analysis to develop a vaccine that is effective against the monkeypox virus.

Immuno-informatics profiling of monkeypox virus cell surface binding protein for designing a next generation multi-valent peptide-based vaccine.

Monkeypox is a viral etiological agent with hallmarks analogous to those observed in smallpox cases in the past. The ongoing outbreak of Monkeypox viral infection is becoming a global health problem. Multi-valent peptide based next generation vaccines provides us a promising solution to combat these emerging infectious diseases by eliciting cell-mediated and humoral immune response. Considering the success rate of subtractive proteomics pipeline and reverse vaccinology approach, in this study, we have developed a novel, next-generation, multi-valent, in silico peptide based vaccine construct by employing cell surface binding protein. After analyzing physiochemical and biological properties of the selected target, the protein was subjected to B cell derived T cell epitope mapping. Iterative scrutinization lead to the identification of two highly antigenic, virulent, non-allergic, non-toxic, water soluble, and Interferon-gamma inducer epitopes i.e. HYITENYRN and TTSPVRENY. We estimated that the shortlisted epitopes for vaccine construction, roughly correspond to 99.74% of the world's population. UK, Finland and Sweden had the highest overall population coverage at 100% which is followed by Austria (99.99%), Germany (99.99%), France (99.98%), Poland (99.96), Croatia (99.93), Czech Republic (99.87%), Belgium (99.87), Italy (99.86%), China (97.83%), India (97.35%) and Pakistan (97.13%). The designed vaccine construct comprises of 150 amino acids with a molecular weight of 16.97242 kDa. Molecular docking studies of the modelled MEMPV (Multi-epitope Monkeypox Vaccine) with MHC I (PDB ID: 1I1Y), MHC II (PDB ID: 1KG0), and other immune mediators i.e. toll like receptors TLR3 (PDB ID: 2A0Z), and TLR4 (PDB ID: 4G8A) revealed strong binding affinity with immune receptors. Host immune simulation results predicted that the designed vaccine has strong potency to induce immune responses against target pathogen in the form of cellular and antibody-dependent immunity. Our findings suggest that the hypothesized vaccine candidate can be utilized as a potential therapeutic against Monkeypox however experimental study is required to validate the results and safe immunogenicity.

Evaluation of the antimicrobial function of Ginkgo biloba exocarp extract against clinical bacteria and its effect on Staphylococcus haemolyticus by disrupting biofilms

Ethnopharmacological relevanceThe fruit of Ginkgo biloba L. (Ginkgo nuts) has been used for a long time as a critical Chinese medicine material to treat cough and asthma, as well as a disinfectant. Similar records were written in the Compendium of Materia Medica (Ben Cao Gang Mu, pinyin in Chinese) and Sheng Nong's herbal classic (Shen Nong Ben Cao Jing, pinyin in Chinese). Recent research has shown that Ginkgo biloba exocarp extract (GBEE) has the functions of unblocking blood vessels and improving brain function, as well as antitumour activity and antibacterial activity. GBEE was shown to inhibit methicillin-resistant Staphylococcus aureus (MRSA) biofilm formation as a traditional Chinese herb in our previous report in this journal. Aim of the studyThe antibiotic resistance of clinical bacteria has recently become increasingly serious. Thus, this study aimed to investigate the Ginkgo biloba exocarp extract (GBEE) antibacterial lineage, as well as its effect and mechanism on S. haemolyticus biofilms. This study will provide a new perspective on clinical multidrug resistant (MDR) treatment with ethnopharmacology herbs. MethodsThe microbroth dilution assay was carried out to measure the antibacterial effect of GBEE on 13 types of clinical bacteria. Bacterial growth curves with or without GBEE treatment were drawn at different time points. The potential targets of GBEE against S. haemolyticus were screened by transcriptome sequencing. The effects of GBEE on bacterial biofilm formation and mature biofilm disruption were determined by crystal violet staining and scanning electron microscopy. The metabolic activity of bacteria inside the biofilm was assessed by colony-forming unit (CFU) counting and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2HY-tetrazolium bromide (MTT) assay. Quantitative polymerase chain reaction (qPCR) was used to measure the gene expression profile of GBEE on S. haemolyticus biofilm-related factors. ResultsThe results showed that GBEE has bacteriostatic effects on 3 g-positive (G+) and 2 g-negative (G-) bacteria among 13 species of clinical bacteria. The antibacterial effect of GBEE supernatant liquid was stronger than the antibacterial effect of GBEE supernviaould-like liquid. GBEE supernatant liquid inhibited the growth of S. epidermidis, S. haemolyticus, and E. faecium at shallow concentrations with minimum inhibitory concentrations (MICs) of 2 μg/ml, 4 μg/ml and 8 μg/ml, respectively. Genes involved in quorum sensing, two-component systems, folate biosynthesis, and ATP-binding cassette (ABC) transporters were differentially expressed in GBEE-treated groups compared with controls. Crystal violet, scanning electron microscopy (SEM) and MTT assays showed that GBEE suppressed S. haemolyticus biofilm formation in a dose-dependent manner. Moreover, GBEE supernatant liquid downregulated cidA, cidB and atl, which are involved in cell lysis and extracellular DNA (eDNA) release, as well as downregulated the cbp, ebp and fbp participation in encoding cell-surface binding proteins. ConclusionsGBEE has an excellent antibacterial effect on gram-positive bacteria and also inhibits the growth of gram-negative bacteria, such as A. baumannii (carbapenem-resistant Acinetobacter baumannii) CRABA and S. maltophilia. GBEE inhibits the biofilm formation of S. haemolyticus by altering the regulation and biofilm material-related genes, including the release of eDNA and cell-surface binding proteins.

ANGT_HUMAN[448-462], an Anorexigenic Peptide Identified Using Plasma Peptidomics.

The discovery of bioactive peptides is an important research target that enables the elucidation of the pathophysiology of human diseases and provides seeds for drug discovery. Using a large number of native peptides previously identified using plasma peptidomics technology, we sequentially synthesized selected sequences and subjected them to functional screening using human cultured cells. A 15-amino-acid residue proangiotensinogen-derived peptide, designated ANGT_HUMAN[448–462], elicited cellular responses and bound to cultured human cells. Synthetic fluorescent-labeled and biotinylated ANGT_HUMAN[448–462] peptides were rendered to bind to cell- and tissue-derived proteins and peptide-cell protein complexes were retrieved and analyzed using liquid chromatography–tandem mass spectrometry, revealing the β-subunit of ATP synthase as its cell-surface binding protein. Because ATP synthase mediates the effects of anorexigenic peptides, the ability of ANGT_HUMAN[448–462] to modulate eating behavior in mice was investigated. Both intraperitoneal and intracerebroventricular injections of low doses of ANGT_HUMAN[448–462] suppressed spontaneous food and water intake throughout the dark phase of the diurnal cycle without affecting locomotor activity. Immunoreactive ANGT_HUMAN[448–462], distributed throughout human tissues and in human-derived cells, is mostly co-localized with angiotensin II and is occasionally present separately from angiotensin II. In this study, an anorexigenic peptide, ANGT_HUMAN[448–462], was identified by exploring cell surface target proteins of the human native peptides identified using plasma peptidomics.

Structural Features Mediating Zinc Binding and Transfer in the AztABCD Zinc Transporter System.

Many bacteria require ATP binding cassette (ABC) transporters for the import of the essential metal zinc from limited environments. These systems rely on a periplasmic or cell-surface solute binding protein (SBP) to bind zinc with high affinity and specificity. AztABCD is one such zinc transport system recently identified in a large group of diverse bacterial species. In addition to a classical SBP (AztC), the operon also includes a periplasmic metallochaperone (AztD) shown to transfer zinc directly to AztC. Crystal structures of both proteins from Paracoccus denitrificans have been solved and suggest several structural features on each that may be important for zinc binding and transfer. Here we determine zinc binding affinity, dissociation kinetics, and transfer kinetics for several deletion mutants as well as a crystal structure for one of them. The results indicate specific roles for loop structures on AztC and an N-terminal motif on AztD in zinc binding and transfer. These data are consistent with a structural transfer model proposed previously and provide further mechanistic insight into the processes of zinc binding and transfer.

Characterization of Macrophage Galactose-type Lectin (MGL) ligands in colorectal cancer cell lines

BackgroundThe Ca2+-dependent C-type lectin receptor Macrophage Galactose-type Lectin (MGL) is highly expressed by tolerogenic dendritic cells (DC) and macrophages. MGL exhibits a high binding specificity for terminal alpha- and beta-linked GalNAc residues found in Tn, sTn and LacdiNAc antigens. These glycan epitopes are often overexpressed in colorectal cancer (CRC), and, as such, MGL can be used to discriminate tumor from the corresponding healthy tissues. Moreover, the high expression of MGL ligands is associated with poor disease-free survival in stage III of CRC tumors. Nonetheless, the glycoproteins expressed by tumor cells that are recognized by MGL have hitherto remained elusive. MethodsUsing a panel of three CRC cell lines (HCT116, HT29 and LS174T), recapitulating CRC diversity, we performed FACS staining and pull-down assays using a recombinant soluble form of MGL (and a mutant MGL as control) combined with mass spectrometry-based (glyco)proteomics. ResultsHCT116 and HT29, but not LS174T, are high MGL-binding CRC cell lines. On these cells, the major cell surface binding proteins are receptors (e.g. MET, PTK7, SORL1, PTPRF) and integrins (ITGB1, ITGA3). From these proteins, several N- and/or O-glycopeptides were identified, of which some carried either a LacdiNAc or Tn epitope. ConclusionsWe have identified cell surface MGL-ligands on CRC cell lines. General significanceAdvances in (glyco)proteomics have led to identification of candidate key mediators of immune-evasion and tumor growth in CRC.

WAIF1 Is a Cell-Surface CTHRC1 Binding Protein Coupling Bone Resorption and Formation.

The osteoclast-derived collagen triple helix repeat containing 1 (CTHRC1) protein stimulates osteoblast differentiation, but the underlying mechanism remains unclear. Here, we identified Wnt-activated inhibitory factor 1 (WAIF1)/5T4 as a cell-surface protein binding CTHRC1. The WAIF1-encoding Trophoblast glycoprotein (Tpbg) gene, which is abundantly expressed in the brain and bone but not in other tissues, showed the same expression pattern as Cthrc1. Tpbg downregulation in marrow stromal cells reduced CTHRC1 binding and CTHRC1-stimulated alkaline phosphatase activity through PKCδ activation of MEK/ERK, suggesting a novel WAIF1/PKCδ/ERK pathway triggered by CTHRC1. Unexpectedly, osteoblast lineage-specific deletion of Tpbg downregulated Rankl expression in mouse bones and reduced both bone formation and resorption; importantly, it impaired bone mass recovery following RANKL-induced resorption, reproducing the phenotype of osteoclast-specific Cthrc1 deficiency. Thus, the binding of osteoclast-derived CTHRC1 to WAIF1 in stromal cells activates PKCδ-ERK osteoblastogenic signaling and serves as a key molecular link between bone resorption and formation during bone remodeling. © 2018 American Society for Bone and Mineral Research.

Novel electrospun nanofibers of modified gelatin-tyrosine in cartilage tissue engineering

In natural cartilage tissues, chondrocytes are linked to extracellular matrix (ECM) through cell-surface binding proteins. Surface modification of gelatin can provide a new generation of biopolymers and fibrous scaffolds with chemical, mechanical, and biological properties. In this study tyrosine protein and 1,2,3-triazole ring were utilized to functionalize gelatin without Cu catalyst. Their molecular structure was characterized by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (1HNMR). Chemical cross-linkers such as glutaraldehyde (GA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysulfosuccinimide (NHS) were used to electrospin the modified gelatin. The modification of gelatin and cross-linking effects were confirmed by scanning electron microscopy (SEM), contact angle measurement, and mechanical tests. MTT assay using chondrocyte cells showed cell viability of electrospun modified gelatin scaffolds. In vitro cell culture studies showed that electrospun engineered protein scaffolds would support the attachment and growth of cells. The results also showed that cross-linked nanofibers with EDC/NHS could be considered excellent matrices in cell adhesion and proliferation before electrospinning process and their potential substrate in tissue engineering applications, especially in the field of cartilage engineering.

Probing cell-surface carbohydrate binding proteins with dual-modal glycan-conjugated nanoparticles.

Dual-modal fluorescent magnetic glyconanoparticles have been prepared and shown to be powerful in probing lectins displayed on pathogenic and mammalian cell surfaces. Blood group H1- and Le(b)-conjugated nanoparticles were found to bind to BabA displaying Helicobacter pylori, and Le(a)- and Le(b)-modified nanoparticles are both recognized by and internalized into DC-SIGN and SIGN-R1 expressing mammalian cells via lectin-mediated endocytosis. In addition, glyconanoparticles block adhesion of H. pylori to mammalian cells, suggesting that they can serve as inhibitors of infection of host cells by this pathogen. It has been also shown that owing to their magnetic properties, glyconanoparticles are useful tools to enrich lectin expressing cells. The combined results indicate that dual-modal glyconanoparticles are biocompatible and that they can be employed in lectin-associated biological studies and biomedical applications.

Deletion of the SPARC acidic domain or EGF-like module reduces SPARC-induced migration and signaling through p38 MAPK/HSP27 in glioma

We previously demonstrated that secreted protein acidic and rich in cysteine (SPARC) increases heat shock protein 27 (HSP27) expression and phosphorylation and promotes glioma cell migration through the p38 mitogen-activated protein kinase (MAPK)/HSP27 signaling pathway. As different regions of the SPARC protein mediate different SPARC functions, elucidating which SPARC domains regulate HSP27 expression, signaling and migration might provide potential therapeutic strategies to target these functions. To investigate the roles of specific domains, we used an SPARC-green fluorescent protein (GFP) fusion protein and constructs of SPARC-GFP with deletions of either the acidic domain (ΔAcidic) or the epidermal growth factor (EGF)-like module (ΔEGF). GFP, SPARC-GFP and the two deletion mutants were expressed in U87MG glioma cells. Characterization of the derived stable clones by confocal imaging and western blotting suggests proper folding, processing and secretion of the deletion constructs. Uptake of the constructs by naive cells suggests enhanced internalization of ΔAcidic and reduced internalization of ΔEGF. Wound and transwell migration assays and western blot analysis confirm our previous results and indicate that ΔAcidic reduces SPARC-induced migration and p38 MAPK/HSP27 signaling and ΔEGF decreases SPARC-induced migration and dramatically decreases the expression and phosphorylation of HSP27 but is poorly internalized. Loss of the EGF-like module suppresses the enhanced HSP27 protein stability conferred by SPARC. In conclusion, deletions of the acidic domain and EGF-like module have differential effects on cell surface binding and HSP27 protein stability; however, both regions regulate SPARC-induced migration and signaling through HSP27. Our data link the domains of SPARC with different functions and suggest one or both of the constructs as potential therapeutic agents to inhibit SPARC-induced migration.

Staphylococcus aureus immunodominant surface antigen B is a cell-surface associated nucleic acid binding protein

BackgroundStaphylococcus aureus immunodominant surface antigen B (IsaB) elicits an immune response during septicemia and is generally classified as a virulence factor, but its biological function remains completely undefined. In an attempt to identify staphylococcal RNA-binding proteins, we designed an RNA Affinity Chromatography assay and subsequently isolated IsaB.ResultsWestern analysis indicated that IsaB was both secreted and cell-surface associated. Gel Shift analysis confirmed the RNA binding activity but revealed that IsaB bound to any nucleic acid without sequence specificity. IsaB exhibited the highest affinity for double-stranded DNA followed by single-stranded DNA and RNA. Because extracellular DNA has been shown to play a role in biofilm formation, we investigated the biofilm-forming capacity of an isogenic isaB deletion mutant but we found that IsaB did not contribute to biofilm formation under any conditions tested.ConclusionIsaB is an extracellular nucleic acid binding protein, with little to no sequence specificity, but its role in virulence remains unclear.

Defect of Hepatocyte Growth Factor Activator Inhibitor Type 1/Serine Protease Inhibitor, Kunitz Type 1 (Hai-1/Spint1) Leads to Ichthyosis-Like Condition and Abnormal Hair Development in Mice

Hepatocyte growth factor activator inhibitor type 1 (HAI-1)/serine protease inhibitor, Kunitz type 1 (SPINT1) is a membrane-bound, serine proteinase inhibitor initially identified as an inhibitor of hepatocyte growth factor activator. It also inhibits matriptase and prostasin, both of which are membrane-bound serine proteinases that have critical roles in epidermal differentiation and function. In this study, skin and hair phenotypes of mice lacking the Hai-1/Spint1 gene were characterized. Previously, we reported that the homozygous deletion of Hai-1/Spint1 in mice resulted in embryonic lethality attributable to impaired placental development. To test the role of Hai-1/Spint1 in mice, the placental function of Hai-1/Spint1-mutant mice was rescued. Injection of Hai-1/Spint1(+/+) blastocysts with Hai-1/Spint1(-/-) embryonic stem cells successfully generated high-chimeric Hai-1/Spint1(-/-) embryos (B6Hai-1(-/-High)) with normal placentas. These embryos were delivered without apparent developmental abnormalities, confirming that embryonic lethality of Hai-1/Spint1(-/-) mice was caused by placental dysfunction. However, newborn B6Hai-1(-/-High) mice showed growth retardation and died by 16 days. These mice developed scaly skin because of hyperkeratinization, reminiscent of ichthyosis, and abnormal hair shafts that showed loss of regular cuticular septation. The interfollicular epidermis showed acanthosis with enhanced Akt phosphorylation. Immunoblot analysis revealed altered proteolytic processing of profilaggrin in Hai-1/Spint1-deleted skin with impaired generation of filaggrin monomers. These findings indicate that Hai-1/Spint1 has critical roles in the regulated keratinization of the epidermis and hair development.

Crystal Structure of the Resuscitation-Promoting Factor ΔDUFRpfB from M. tuberculosis

Mycobacterium tuberculosis is able to establish a non-replicating state and survive in an intracellular habitat for years. Resuscitation of dormant M. tuberculosis bacteria is promoted by resuscitation-promoting factors (Rpfs), which are secreted from slowly replicating bacteria close to dormant bacteria. Here we report the crystal structure of a truncated form of RpfB (residues 194–362), the sole indispensable Rpf of the five Rpfs encoded in this bacterium genome. The structure, denoted as ΔDUFRpfB, exhibits a comma-like shape formed by a lysozyme-like globular catalytic domain and an elongated G5 domain, which is widespread among cell surface binding proteins. The G5 domain, whose structure was previously uncharacterised, presents some peculiar features. The basic structural motif of this domain, which represents the tail of the comma-like structure, is a novel super-secondary-structure element, made of two β-sheets interconnected by a pseudo-triple helix. This intricate organisation leads to the exposure of several backbone hydrogen-bond donors/acceptors. Mutagenesis analyses and solution studies indicate that this protein construct as well as the full-length form are elongated monomeric proteins. Although ΔDUFRpfB does not self-associate, the exposure of structural elements (backbone H-bond donors/acceptors and hydrophobic side chains) that are usually buried in globular proteins is typically associated with adhesive properties. This suggests that the RpfB G5 domain has a cell-wall adhesive function, which allows the catalytic domain to be properly oriented for the cleavage reaction. Interestingly, sequence comparisons indicate that these structural features are also shared by G5 domains involved in biofilm formation.

The Regulated Cell Surface Zymogen Activation of the Proprotein Convertase PC5A Directs the Processing of Its Secretory Substrates

The proprotein convertases are synthesized as zymogens that acquire activity upon autocatalytic removal of their NH(2)-terminal prosegment. Based on the convertase furin, to fold properly and gain activity, the convertases PC5A, PACE4, and PC7 are presumed to undergo two sequential prosegment cleavages in the endoplasmic reticulum and then in the trans-Golgi network. However, biochemical and immunocytochemical experiments revealed that mouse PC5A is complexed to its prosegment at the plasma membrane. This labeling is lost upon treatment with heparin and is increased by overexpressing members of the syndecan family and CD44, suggesting attachment of secreted PC5A-prosegment complex to heparan sulfate proteoglycans. Following stimulation of Y1 cells with adrenocorticotropic hormone or 8-bromo-cyclic AMP, the cell surface labeling of the prosegment of PC5A is greatly diminished, whereas the signal for mature PC5A is increased. Moreover, after stimulation, the protease activity of PC5A is enhanced, as evidenced by the cleavage of the PC5A substrates Lefty, ADAMTS-4, endothelial lipase, and PCSK9. Our data suggest a novel mechanism for PC5A activation and substrate cleavage at the cell surface, through a regulated removal of its prosegment. A similar mechanism may also apply to the convertase PACE4, thereby extending our knowledge of the molecular details of the zymogen activation and functions of these heparan sulfate proteoglycan-bound convertases.

Moderate reduction of Norrin signaling activity associated with the causative missense mutations identified in patients with familial exudative vitreoretinopathy

Mutations in Norrin signaling genes (NDP, FZD4 and LRP5) have been found in patients with familial exudative vitreoretinopathy (FEVR) and the altered signaling is suspected to play a critical role in its pathogenesis. To better understand this relationship, we systematically performed functional analyses on previously identified single nucleotide variants of LRP5, FZD4 and NDP, utilizing the Norrin dependent Topflash reporter assay. Cell surface binding assays and protein electrophoresis analysis of Norrin were also performed. Seven causative mutations and five possibly causative but indecisive variants were examined. We found: (1) a nonsense mutation in FZD4 completely abolished its signaling activity, while single missense mutations in LRP5 and FZD4 caused a moderate level of reduction (ranging from 26 to 48, 36% on average) and a double missense mutation in both genes caused a severe reduction in activity (71%). These observations correlated roughly with clinical phenotypes. (2) A mutational effect is suggested in four of five indecisive variants by signaling reductions comparable to those of missense mutations. (3) Norrin mutants demonstrated variable effects on signal transduction, and no apparent correlation with clinical phenotypes was observed. (4) The Norrin mutants examined demonstrated impaired cell surface binding, and some may have partially lost their ability to form a complex with unknown high molecular weight material(s). Our results illustrate the nature of FEVR in relation to Norrin signaling and further suggest the complexity of its disease causing mechanism.

Identification and characterization of p63 (CKAP4/ERGIC-63/CLIMP-63), a surfactant protein A binding protein, on type II pneumocytes

Surfactant protein A (SP-A) binds to alveolar type II cells through a specific high-affinity cell membrane receptor, although the molecular nature of this receptor is unclear. In the present study, we have identified and characterized an SP-A cell surface binding protein by utilizing two chemical cross-linkers: profound sulfo-SBED protein-protein interaction reagent and dithiobis(succinimidylpropionate) (DSP). Sulfo-SBED-biotinylated SP-A was cross-linked to the plasma membranes isolated from rat type II cells, and the biotin label was transferred from SP-A to its receptor by reduction. The biotinylated SP-A-binding protein was identified on blots by using streptavidin-labeled horseradish peroxidase. By using DSP, we cross-linked SP-A to intact mouse type II cells and immunoprecipitated the SP-A-receptor complex using anti-SP-A antibody. Both of the cross-linking approaches showed a major band of 63 kDa under reduced conditions that was identified as the rat homolog of the human type II transmembrane protein p63 (CKAP4/ERGIC-63/CLIMP-63) by matrix-assisted laser desorption ionization and nanoelectrospray tandem mass spectrometry of tryptic fragments. Thereafter, we confirmed the presence of p63 protein in the cross-linked SP-A-receptor complex by immunoprobing with p63 antibody. Coimmunoprecipitation experiments and functional assays confirmed specific interaction between SP-A and p63. Antibody to p63 could block SP-A-mediated inhibition of ATP-stimulated phospholipid secretion. Both intracellular and membrane localized pools of p63 were detected on type II cells by immunofluorescence and immunobloting. p63 colocalized with SP-A in early endosomes. Thus p63 closely interacts with SP-A and may play a role in the trafficking or the biological function of the surfactant protein.