SP Domain Research Articles

Impaired ligand-dependent MET activation caused by an extracellular SEMA domain missense mutation in lung cancer.

Aberrant activation of the MET/hepatocyte growth factor (HGF) receptor participates in the malignant behavior of cancer cells, such as invasion‐metastasis and resistance to molecular targeted drugs. Many mutations in the MET extracellular region have been reported, but their significance is largely unknown. Here, we report the dysregulation of mutant MET originally found in a lung cancer patient with Val370 to Asp370 (V370D) replacement located in the extracellular SEMA domain. MET‐knockout cells were prepared and reconstituted with WT‐MET or V370D‐MET. HGF stimulation induced MET dimerization and biological responses in cells reconstituted with WT‐MET, but HGF did not induce MET dimerization and failed to induce biological responses in V370D‐MET cells. The V370D mutation abrogated HGF‐dependent drug resistance of lung cancer cells to epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKI). Compared with WT‐MET cells, V370D‐MET cells showed different activation patterns in receptor tyrosine kinases upon exposure to survival/growth‐stressed conditions. Surface plasmon resonance analysis indicated that affinity between the extracellular region of V370D‐MET and HGF was reduced compared with that for WT‐MET. Further analysis of the association between V370D‐MET and the separate domains of HGF indicated that the SP domain of HGF was unchanged, but its association with the NK4 domain of HGF was mostly lost in V370D‐MET. These results indicate that the V370D mutation in the MET receptor impairs the functional association with HGF and is therefore a loss‐of‐function mutation. This mutation may change the dependence of cancer cell growth/survival on signaling molecules, which may promote cancer cell characteristics under certain conditions.

Multimethod Characterization of Volcanic Ashes from The Sunda Island Arc

Compared to other countries in the world, Indonesia has the highest number of volcanoes; many of these volcanoes are located in the Sunda Island Arc. Considering their importance’s as geological markers, identification of the sources of volcanic ashes is challenging and intriguing task. Standing on that reason, this study was aimed to characterize the magnetic and geochemical properties of several volcanic ashes from Sunda Island Arc to test whether these properties could serve as identifiers for the sources. This study used the ashes from Mount Sirung (in Pantar) and from Mounts Merapi and Slamet (in Java). The data would then be combined and compared with previous studies that used ashes from other volcanoes both inside as well as outside the Sunda Island Arc. The results showed that the magnetic susceptibility of Mounts Merapi and Slamet differed not too much, while that of Mount Sirung differed significantly due to low magnetic content. The plot of hysteresis parameters classified Mount Merapi into PSD domain and Mount Slamet into SP domain, while that of Mount Sirung could not be performed due to the weak signal obtained. Geochemical data also showed that ashes from Mount Sirung contained much lower concentration of Na2O + K2O compared to that in Mounts Merapi and Slamet. Each volcano had its distinct magnetic properties when these properties were compared to that of other volcanoes previously studied. The geochemical data also could reflect its magma properties regarding to its geological setting. It could be concluded that these multimethod approaches were able to differentiate volcanic ashes based on their sources.

Binding of PmClipSP2 to microbial cell wall components and activation of the proPO-activating system in the black tiger shrimp Penaeus monodon

Clip domain serine proteinases (ClipSPs) play an important role in the prophenoloxidase-activating (proPO) system. In the shrimp Penaeus monodon, the ClipSP PmClipSP2 has been previously shown to bind to microbial polysaccharides (LPS and β-1,3-glucan) and likely activates the proPO system. To reveal the binding site of the PmClipSP2 protein, the N-terminal clip domain (Clip-PmClipSP) and C-terminal SP domain (SP-PmClipSP2) were separately cloned. The recombinant proteins were then assayed for their binding properties and involvement in proPO activation. According to the ELISA-based binding assay, rSP-PmClipSP2, but not rClip-PmClipSP, can bind immobilized LPS and β-1,3-glucan as well as significantly activate PO activity. The binding site at the SP domain is proposed to have a pattern sequence (X-[PFY]-X-[AFILV]-[AFY]-[AITV]-X-[ILV]-X(5)-W-[IL]-X) that is located at the C-terminal region of the SP domain of PmClipSP2. Deletion of the pattern sequence abolished binding to LPS and β-1,3-glucan. Conversely, a recombinant protein containing the pattern sequence (rPT-PmClipSP2-TRX) had the ability to bind to cell wall components, confirming that the pattern sequence at the C-terminus of PmClipSP2 is responsible for binding to microbes, subsequently leading to activation of the proPO cascade.

Understanding the Dynamics of Full-Length Tissue Factor (TF1-263) in the Context of Allosteric Modulation of Factor VIIa : A Molecular Dynamics Simulation Approach

Tissue factor (TF) mediated blood clot is the key event in cellular physiology. During vascular injury or endothelial cell rupture, factor VII (FVII) which is present normally in flowing blood as zymogenic form, upon binding with its cofactor TF (present beneath endothelial cell), get converted into its activated form, FVIIa; makes TF-FVIIa protease complex for the conversion of downstream protease members into activated form. Upon successful transformation of FX to FXa, prothrombin to thrombin, soluble fibrinogen get converted into its insoluble form fibrin, which accumulates at the site of vascular injury in the presence of platelets, resulting into the blood clot.Recent studies have shown that TF is solely responsible for the structural modulation of FVIIa. However, due to lack of well-resolved crystal structure of the zymogenic form of FVII and full-length TF-FVIIa binary complex, the actual allosteric mechanism induced by TF and structural changes observed in FVIIa is not fully understood at atomic resolution. Molecular dynamic simulation is the only way to visual the potential interaction between protein-protein and protein-lipid at atomistic resolution. Here we did the simulation of complex and free FVIIa to understand the causative effect of TF in terms of allosteric modulation occurred in FVIIa. The present study was carried out to investigate the domain-wise structural modulation of FVIIa in the presence of full-length TF. We have done the comparative study among full-length (FL) TF1-263-FVIIa, sTF1-219-FVIIa and free FVIIa. Measurements of intrinsic dynamic changes clearly suggest that TF not only made changes in the allosteric modulation of FVIIa but inter as well intradomain communication made between FLTF and FVIIa is also responsible for the structural modulation in the protease domain of FVIIa. Among interdomain communication, two interactions between EGF2 domain and FLTF (Glu94-Ser47 and Asp104-Thr51) guide the potential effect of TF in the structural modulation of FVIIa. Examining the catalytic triad localization reveals that single as well as double interaction regulates the dynamism of protease part in substrate recognition and substrate catalysis, however in the case of no interaction the movement of CT is much higher and in a wide range. In a case of sTF-FVIIa complex, few inter-domain communications ware missing, which causes reduced affinity of sTF towards FVIIa and thus sTF-FVIIa shows less activity compare to FLTF. We proposed the hypothesis that binding of FLTF not only changes the allosteric alteration of SP domain but also induces the structural alteration in each domain of FVIIa, and these alterations in combination contribute to the enhancement of FLTF-FVIIa activity in which two interactions may be the controller for such dynamic changes in FVIIa. In summary, our study provides valuable information to understand the blood coagulation more intrinsically. DisclosuresNo relevant conflicts of interest to declare.

Thermal conductivity of giant mono- to few-layered CVD graphene supported on an organic substrate.

The thermal conductivity (k) of supported graphene is a critical property that reflects the graphene-substrate interaction, graphene structure quality, and is needed for thermal design of a graphene device. Yet the related k measurement has never been a trivial work and very few studies are reported to date, only at the μm level. In this work, for the first time, the k of giant chemical vapor decomposition (CVD) graphene supported on poly(methyl methacrylate) (PMMA) is characterized using our transient electro-thermal technique based on a differential concept. Our graphene size is ∼mm, far above the samples studied in the past. This giant graphene measurement eliminates the thermal contact resistance problems and edge phonon scattering encountered in μm-scale graphene k measurement. Such mm-scale measurement is critical for device/system-level thermal design since it reflects the effect of abundant grains in graphene. The k of 1.33-layered, 1.53-layered, 2.74-layered and 5.2-layered supported graphene is measured as 365 W m(-1) K(-1), 359 W m(-1) K(-1), 273 W m(-1) K(-1) and 33.5 W m(-1) K(-1), respectively. These values are significantly lower than the k of supported graphene on SiO2, and are about one order of magnitude lower than the k of suspended graphene. We speculate that the abundant C atoms in the PMMA promote more ready energy and momentum exchange with the supported graphene, and give rise to more phonon scattering than the SiO2 substrate. This leads to a lower k of CVD graphene on PMMA than that on SiO2. We attribute the existence of disorder in the sp(2) domain, graphene oxide (GO) and stratification in the 5.2-layered graphene to its more k reduction. The Raman linewidth (G peak) of the 5.2-layered graphene is also twice larger than that of the other three kinds of graphene, indicating the much more phonon scattering and shorter phonon lifetime in it. Also the electrical conductivity of the 5.2-layered graphene is about one-fifth of that for the other three. This further confirms the poor graphene quality of sample 4S, explaining its much lower k.

In situ and nonvolatile photoluminescence tuning and nanodomain writing demonstrated by all-solid-state devices based on graphene oxide.

In situ and nonvolatile tuning of photoluminescence (PL) has been achieved based on graphene oxide (GO), the PL of which is receiving much attention because of various potential applications of the oxide (e.g., display, lighting, and nano-biosensor). The technique is based on in situ and nonvolatile tuning of the sp(2) domain fraction to the sp(3) domain fraction (sp(2)/sp(3) fraction) in GO through an electrochemical redox reaction achieved by solid electrolyte thin films. The all-solid-state variable PL device was fabricated by GO and proton-conducting mesoporous SiO2 thin films, which showed an extremely low PL background. The device successfully tuned the PL peak wavelength in a very wide range from 393 to 712 nm, covering that for chemically tuned GO, by adjusting the applied DC voltage within several hundred seconds. We also demonstrate the sp(2)/sp(3) fraction tuning using a conductive atomic force microscope. The device achieved not only writing, but also erasing of the sp(2)/sp(3)-fraction-tuned nanodomain (both directions operation). The combination of these techniques is applicable to a wide range of nano-optoelectronic devices including nonvolatile PL memory devices and on-demand rewritable biosensors that can be integrated into nano- and microtips which are transparent, ultrathin, flexible, and inexpensive.

The role of the lys628 (192) residue of the complement protease, c1s, in interacting with Peptide and protein substrates.

The C1s protease of the classical complement pathway propagates the initial activation of this pathway of the system by cleaving and thereby activating the C4 and C2 complement components. This facilitates the formation of the classical pathway C3 convertase (C4bC2a). C1s has a Lys residue located at position 628 (192 in chymotrypsin numbering) of the SP domain that has the potential to partially occlude the S2–S2′ positions of the active site. The 192 residue of serine proteases generally plays an important role in interactions with substrates. We therefore investigated the role of Lys628 (192) in interactions with C4 by altering the Lys residue to either a Gln (found in many other serine proteases) or an Ala residue. The mutant enzymes had altered specificity profiles for a combinatorial peptide substrate library, suggesting that this residue does influence the active site specificity of the protease. Generally, the K628Q mutant had greater activity than wild type enzyme against peptide substrates, while the K628A residue had lowered activity, although this was not always the case. Against peptide substrates containing physiological substrate sequences, the K628Q mutant once again had generally higher activity, but the activity of the wild type and mutant enzymes against a C4 P4–P4′ substrate were similar. Interestingly, alteration of the K628 residue in C1s had a marked effect on the cleavage of C4, reducing cleavage efficiency for both mutants about fivefold. This indicates that this residue plays a different role in cleaving protein versus peptide substrates and that the Lys residue found in the wild type enzyme plays an important role in interacting with the C4 substrate. Understanding the basis of the interaction between C1s and its physiological substrates is likely to lead to insights that can be used to design efficient inhibitors of the enzyme for use in treating diseases caused by inflammation as result of over-activity of the classical complement pathway.

Surface plasmon enhancement of broadband photoluminescence emission from graphene oxide.

The photoluminescence (PL) emission studies of both graphene oxide (GO) and partially reduced graphene oxide (rGO) have been investigated. It has been observed that GO has broadband emission from the green to near infrared range and upon reduction rGO shows blue PL emission. The broadband PL emission is due to the recombination of the electron-hole pair in the sp(2) domain embedded within the sp(3) matrix. The broadband PL emission also suggests the existence of various sizes of the sp(2) domain within the same matrix. Furthermore, PL emission from GO in the presence of an Au metal thin film has been investigated. It has been observed that the entire broadband emission from GO in the green to near infrared wavelength region is enhanced significantly at room temperature. The Au-GO interface exhibits surface plasmon resonance in the visible wavelength region and is responsible for over 10 fold enhancement in the photoluminescence at ∼2.36 eV. The electrical property measurements on the GO and rGO thin films suggested that the rGO exhibits significantly higher electrical conductivity compared to that of the GO thin film. Furthermore, the GO thin film exhibits semiconducting behaviour. These properties make the material quite suitable for fabrication of new generation photonic devices.

In Situ and Non‐Volatile Bandgap Tuning of Multilayer Graphene Oxide in an All‐Solid‐State Electric Double‐Layer Transistor

The sp(2) /sp(3) domain fraction of multilayer graphene oxide (GO) is tuned in situ and in a non-volatile manner by using a proton-induced redox reaction at the interface of GO and the yttria-stabilized zirconia proton-conductor. This method opens the door for transparent, ultrathin, flexible, and low cost carbon-nanoionics devices that can control not only electronic transport, but also other properties such as photoluminescence and optics.

Direct Observation of Spatially Heterogeneous Single-Layer Graphene Oxide Reduction Kinetics

Graphene oxide (GO) is an important precursor in the production of chemically derived graphene. During reduction, GO's electrical conductivity and band gap change gradually. Doping and chemical functionalization are also possible, illustrating GO's immense potential in creating functional devices through control of its local hybridization. Here we show that laser-induced photolysis controllably reduces individual single-layer GO sheets. The reaction can be followed in real time through sizable decreases in GO's photoluminescence efficiency along with spectral blueshifts. As-produced reduced graphene oxide (rGO) sheets undergo additional photolysis, characterized by dramatic emission enhancements and spectral redshifts. Both GO's reduction and subsequent conversion to photobrightened rGO are captured through movies of their photoluminescence kinetics. Rate maps illustrate sizable spatial and temporal heterogeneities in sp(2) domain growth and reveal how reduction "flows" across GO and rGO sheets. The observed heterogeneous reduction kinetics provides mechanistic insight into GO's conversion to chemically derived graphene and highlights opportunities for overcoming its dynamic, chemical disorder.

Phase I/II Trial Of Immucin a Pan HLA, Anti-MUC1 Signal Peptide Vaccine, In Multiple Myeloma Patients With Residual Disease Or Progression Following Autologous Stem Cell Transplantation

BackgroundAnti-multiple myeloma (MM) vaccinations have recently emerged as a strategy to eradicate MM cells in patients with residual tumor mass. However, most vaccination studies reported transient immune responses, involving CD8+ T-cells or antibodies responses in patients with predefined MHC repertoire.Signal peptide (SP) domains, a 13-50 amino acid long peptides, have been demonstrated to present an exceptionally high number of antigen specific MHC class I, class II and B-cell epitopes per sequence length. Therefore, they are suitable to induce a robust immune response combining CD8+, CD4+ T-cells and antibodies across HLA barrier. Following this rationale we developed ImMucin, A 21-mer therapeutic vaccine encoding the entire SP domain of the MUC1 tumor-associated antigen (TAA), which is over expressed by most carcinomas and hematological tumors including MM. MethodsA phase I/II study to assess the safety (primary endpoint) and efficacy (secondary endpoint) of ImMucin was conducted in MM subjects with stable or progressive residual asymptomatic MUC1+ disease who have already had upfront auto-SCT. Patients recruited into the study initially received 6 bi-weekly vaccination of intra-dermal 100ug ImMucin plus 250ug hGM-CSF. Based on immune and clinical response eligible patient could receive additional 6 bi-weekly vaccination of 100ug or 250ug ImMucin plus 250ug hGM-CSF.Immunomonitoring analysis included; ImMucin-specific IFN-g production in CD4+ and CD8+ T-cells, HLA-A2.1 tetramer binding in CD8+ T-cells, proliferation and antibodies production. Positive response in these assays vs. pre vaccination levels were; x2 increase in IFN-g and >0.5%+ T-cells, any increase in tetramer binding levels, x2 increase and stimulation index (SI)>2 and any increase in anti-ImMucin titer respectively. Results15 patients, median age 58 years participated in the study. The median number of prior therapies was 2. Median time from diagnosis and from auto-SCT to vaccination were 25 months (12-143) and 15 months (3-134) respectively. 9 patients had post auto-SCT residual MM and 6 had an evidence for biochemical progression. 11 patients completed the vaccination program while 4 discontinued the program due to PD. ImMucin was shown to be safe with no vaccine related grade ≥3 adverse events (AEs). Patients had temporary, grade 1,2 AEs, including injection related rash (n=8), fatigue (n=6), weakness, self-resolving fever and muscle pain (n=5).IFN-g production response to ImMucin was highly positive in all patients with 4-80 folds increase for CD4+ and 18-80 folds increase for CD8+. Mean baseline and peak post vaccination levels for ImMucin-specific CD4+ T-cells were 0.21% and 4.07%, respectively (P<0.001, t-test) and of CD8+ T-cells 0.21% and 11.76%, respectively (P<0.0001, t-test).All 4 HLA-A2.1 positive patients demonstrated < x2 increase in tetramer-positive cells after vaccination, with mean levels of 0.33% and 2.11% at baseline and peak post vaccination, respectively. Proliferative response to ImMucin increased signifcantly in all patients with mean baseline and peak of 3.24 and 15.92, respectively (P<0.024, t-test). An increased ImMucin humoral response of up to 40 folds from baseline levels was detected in 10 of 15 patients (66.6%). The vaccination efficacy was further confirmed by CD8+ T-cells and antibody mediated cytotoxicity against autologous BM cells. In these assays MUC1 control epitope didn't show positive activation.Since ImMucin target MUC1's SP domain which is not part of soluble MUC1 (sMUC1), a reduction in sMUC1 following vaccination can reflect a specific tumor destruction. A reduction of up to 17 folds, 5129.44 pg/ml as a mean levels at baseline vs. 792.33 pg/ml at maximal response (P<0.002, t-test) was detected in 9 of 10 patients, initially presenting abnormally elevated sMUC1 levels.Response assessment revealed disease stabilization in the majority of the patients, lasting for up to 29 months post study completion (ongoing follow up), including in patients entering study with PD. ConclusionsThe ImMucin therapeutic vaccine is directed to the less studied SP domain of MUC1 TAA. The current phase I/II results confirmed its high safety profile. ImMucin induced a robust CD8+ and CD4+ specific T-cell response in all patients and a marked anti-tumor humoral response. Further studies exploring ImMucin's efficacy in patients with residual myeloma are under development. Disclosures:Carmon:Vaxil BioTherapeutics Ltd. : Employment, Equity Ownership, Patents & Royalties. Avivi:Vaxil BioTherapeutics Ltd. : Consultancy. Riva:Vaxil BioTherapeutics Ltd. : Employment. Shapira:Vaxil BioTherapeutics Ltd. : Consultancy.

Supercoiled pseudocircular domains in single‐twisted DNAs under tension: Elastic constants and unwinding dynamics in complexes with topo I

Extension versus twist data of Koster et al. (Nature 2005, 434, 671-674) are analyzed to obtain C for the main-chain segments and the twist energy parameter (ET ) for the supercoiled pseudocircular (sp) domain(s) from which C is estimated via simulations. The torsional rigidity in the tension-free sp domain(s) (C = 163 fJ fm) is typical of the unstrained DNA and is less than half the value in the main-chain segments under tension (C = 350-410 fJ fm). Tension is suggested to induce a structural transition to a torsionally stiffer state. Data of Koster et al. for the rate of extension owing to unwinding of a covalent complex of DNA with human Topoisomerase Ib (H Topo I) are analyzed to determine the torque and rate of rotation from which an effective friction coefficient is obtained. A Langevin equation for the unwinding motion in a supercoiled DNA:H Topo I complex is solved to obtain the temporal trajectory of the average winding angle and the time-dependent distribution of winding angles. The mean rate constant for the religation reaction is estimated from the measured probability of reaction per turn. We predict that unwinding proceeds rather far during a single-cleavage and religation cycle, and is effectively completely equilibrated during the 3.2 cleavage and religation cycles that occur during each noncovalent binding and dissociation event. H Topo I is predicted to be completely processive as in accord with observations on calf-thymus Topo I (Brewood et al., Biochemistry 2010, 49, 3367-3380).

Pentosan polysulfate increases affinity between ADAMTS-5 and TIMP-3 through formation of an electrostatically driven trimolecular complex

The semi-synthetic sulfated polysaccharide PPS (pentosan polysulfate) increases affinity between the aggrecan-degrading ADAMTSs (adamalysins with thrombospondin motifs) and their endogenous inhibitor, TIMP (tissue inhibitor of metalloproteinases)-3. In the present study we demonstrate that PPS mediates the formation of a high-affinity trimolecular complex with ADAMTS-5 and TIMP-3. A TIMP-3 mutant that lacks extracellular-matrix-binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Sp (spacer) domain had reduced PPS-binding ability and sensitivity to the affinity increase. PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of eight saccharide units, indicating the involvement of extended or multiple protein-interaction sites. The formation of a high-affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl. These results suggest that PPS enhances the affinity between ADAMTS-5 and TIMP-3 by forming electrostatically driven trimolecular complexes under physiological conditions.

The Unconserved Groucho Central Region Is Essential for Viability and Modulates Target Gene Specificity

Groucho (Gro) is a Drosophila corepressor required by numerous DNA-binding repressors, many of which are distributed in gradients and provide positional information during development. Gro contains well-conserved domains at its N- and C-termini, and a poorly conserved central region that includes the GP, CcN, and SP domains. All lethal point mutations in gro map to the conserved regions, leading to speculation that the unconserved central domains are dispensable. However, our sequence analysis suggests that the central domains are disordered leading us to suspect that the lack of lethal mutations in this region reflects a lack of order rather than an absence of essential functions. In support of this conclusion, genomic rescue experiments with Gro deletion variants demonstrate that the GP and CcN domains are required for viability. Misexpression assays using these same deletion variants show that the SP domain prevents unrestrained and promiscuous repression by Gro, while the GP and CcN domains are indispensable for repression. Deletion of the GP domain leads to loss of nuclear import, while deletion of the CcN domain leads to complete loss of repression. Changes in Gro activity levels reset the threshold concentrations at which graded repressors silence target gene expression. We conclude that co-regulators such as Gro are not simply permissive components of the repression machinery, but cooperate with graded DNA-binding factors in setting borders of gene expression. We suspect that disorder in the Gro central domains may provide the flexibility that allows this region to mediate multiple interactions required for repression.

Identification and characterization of two novel types of non-clip domain serine proteases (PtSP and PtSPH1) from cDNA haemocytes library of swimming crab Portunus trituberculatus

In our previous studies, five serine proteases containing clip domain were characterized from the swimming crab Portunus trituberculatus. To further investigate the characterization and function of serine proteases, one serine protease (PtSP) and one serine protease homolog (PtSPH1) without clip domain were identified from haemocytes cDNA library in this paper. They both possessed an SP or SP-like domain at the C-terminal. In contrast to PtSP, absence of Ser catalytic residue resulted in the loss of serine protease activity of PtSPH1. Phylogenetic analysis suggested either SPs or SPHs might not have a single origin in gene evolution. Six introns presented in PtSP genomic DNA with one uncommon splice site (GG) was discovered at exon 1/intron 1 boundary region. Four introns with common splice sites were found in PtSPH1 genomic DNA. RT-PCR results showed that PtSP mRNA was mainly distributed in haemocytes, gill and eyestalk, whereas PtSPH1 transcript was mainly expressed in stomach. PtSP showed slight increase during the first 48 h compared to control groups except 8 h point after Micrococcus luteus challenge. However, significant up-regulation was observed in the expression level of PtSPH1 challenged by Gram-negative bacteria Vibrio alginolyticus, Gram-positive bacteria M. luteus and fungi Pichia pastoris during the first 48 h. It indicates that PtSPH1 might be more sensitive to microorganism challenges compared with PtSP.

Multiple domains of MASP-2, an initiating complement protease, are required for interaction with its substrate C4

The complement system is fundamental to both innate and adaptive immunity and can be initiated via the classical, lectin or alternative pathways. Cleavage of C4 by MASP-2, the initiating protease of the lectin pathway, is a crucial event in the activation of this pathway, preceding the eventual formation of the C3 convertase (C4bC2a) complex on the pathogen surface. Interactions required for the cleavage of C4 by MASP-2 are likely to be facilitated by the initial binding of C4 to an exosite on the protease. We have shown that both proteolytically active and catalytically inactive CCP1–CCP2–serine protease (CCP1–CCP2–SP) forms bind C4 with similar affinity. Interestingly, proteins containing the CCP1–CCP2 domains or the SP domain alone bound C4 with much lower affinity than the CCP1–CCP2–SP protein, suggesting that the CCP domains cooperate positively with the active site to mediate efficient binding and cleavage of C4. In addition, mutation of residue K342 to alanine in the CCP1 domain abolished binding to both C4 and C4b in its CCP1–CCP2 form, suggesting a key electrostatic role for this amino acid. The presented data indicates that all of the domains are required in order to mediate high affinity interaction with C4.

EphrinB1 interacts with the transcriptional co-repressor Groucho/xTLE4

Ephrin signaling is involved in various morphogenetic events, such as axon guidance, hindbrain segmentation, and angiogenesis. We conducted a yeast two-hybrid screen using the intracellular domain (ICD) of EphrinB1 to gain biochemical insightinto the function of the EphrinB1 ICD. We identified the transcriptional co-repressor xTLE1/Groucho as an EphrinB1 interacting protein. Whole-mount in situ hybridization of Xenopus embryos confirmed the co-localization of EphrinB1 and a Xenopus counterpart to TLE1, xTLE4, during various stages of development. The EphrinB1/xTLE4 interaction was confirmed by co-immunoprecipitation experiments. Further characterization of the interaction revealed that the carboxy-terminal PDZ binding motif of EphrinB1 and the SP domain of xTLE4 are required for binding. Additionally, phosphorylation of EphrinB1 by a constitutively activated fibroblast growth factor receptor resulted in loss of the interaction, suggesting that the interaction is modulated by tyrosine phosphorylation of the EphrinB1 ICD.

Clip domain serine protease and its homolog respond to Vibrio challenge in Chinese white shrimp, Fenneropenaeus chinensis

Clip domain serine proteases and their homologs are involved in invertebrate innate immunity, including hemolymph coagulation, antimicrobial peptide synthesis, cell adhesion, and melanization. Recognition of pathogens by pattern recognition receptors can trigger activation of a serine protease cascade. We report here the cDNA cloning of a serine protease ( FcSP) and a serine protease homolog ( FcSPH) from Chinese white shrimp, Fenneropenaeus chinensis. Both FcSP and FcSPH possess a clip domain at the N-terminal and an SP or SP-like domain at the C-terminal. In contrast to FcSP, FcSPH lacks a catalytic residue and is catalytically inactive. Tissue distribution and time course qRT-PCR analysis indicates that FcSP and FcSPH can respond to Vibrio anguillarum challenge in hemocytes, hepatopancreas and intestine. In situ hybridization analysis shows that FcSP is distributed in hemocytes and gills, and originated mainly from the hemocytes. FcSPH protein is expressed in gills and stomach of non-challenged shrimp. Its expression in gill mainly originates from the hemocytes in it. Two immunoreactive bands of FcSP can be detected in gills and stomach of non-challenged shrimp. FcSP protein is partially cleaved in non-challenged shrimp, while FcSPH protein is unprocessed in unchallenged shrimp and is partially cleaved after V. anguillarum challenge. Our results suggest that this Clip domain serine protease and its homolog may be involved in the serine protease cascade and play an important role in innate immunity of the shrimp.

Gene silencing of a prophenoloxidase activating enzyme in the shrimp, Penaeus monodon, increases susceptibility to Vibrio harveyi infection

The prophenoloxidase (proPO) activating system is an important innate immune response against microbial infections in invertebrates. The major enzyme, phenoloxidase (PO), is synthesized as an inactive precursor and its activation to an active enzyme is mediated by a cascade of clip domain serine proteinases (clip-SPs). In this study, a cDNA encoding a proPO activating enzyme (PPAE) from the black tiger shrimp, Penaeus monodon, designated as PmPPAE1, was cloned and characterized. The full-length cDNA contains an open reading frame (ORF) of 1392 bp encoding a predicted protein of 463 amino acids including an 18 amino acid signal peptide. The PmPPAE1 protein exhibits a characteristic sequence structure of clip-SPs consisting of the clip domain at the N-terminus and a SP domain at the C-terminus. Sequence analysis showed that PmPPAE1 exhibited the highest amino acid sequence similarity (70%) to a PPAE of the crayfish, Pacifastacus leniusculus. PmPPAE1 mRNA is abundantly expressed in hemocytes, and this is regulated after systemic Vibrio harveyi infection supporting that it is an immune-responsive gene. RNA interference-mediated suppression of PmPPAE1, performed by injection of double-stranded RNA (dsRNA) corresponding to the PmPPAE1 gene into shrimp, resulted in a significant reduction of PmPPAE1 but not other clip-SP and related gene transcript levels of P. monodon, suggesting gene-specific knockdown. RNAi-mediated silencing of PmPPAE1 gene significantly decreased the total PO activity (36.7%) in shrimp and additionally increased the mortality of V. harveyi infected shrimp, the latter of which correlated with an increase in the number of viable bacteria in the hemolymph. These results indicate that PmPPAE1 functions in the proPO system and is an important component in the shrimp immune system.

Molecular cloning, structural analysis and expression of complement component Bf/C2 genes in the nurse shark, Ginglymostoma cirratum

Factor B and C2 are serine proteases that provide the catalytic subunits of C3 and C5 convertases of the alternative (AP) and classical (CP) complement pathways. Two Bf/C2 cDNAs, GcBf/C2-1 and -2 (previously referred to as nsBf/C2-A and nsBf/C2-B), were isolated from the nurse shark, Ginglymostoma cirratum. GcBf/C2-1 and -2 are 3364 and 3082 bp in length and encode a leader peptide, three CCPs, one VWFA, the serine protease domain and have a putative factor D/C1s/MASP cleavage site. Southern blots show that there might be up to two Bf/C2-like genes for each of the two GcBf/C2 isoforms. GcBf/C2-1 and -2 are constitutively expressed, albeit at different levels, in all nine tissues examined. Expression in erythrocytes is a novel finding. Structural analysis has revealed that the localization of glycosylation sites in the SP domain of both putative proteins indicates that the molecular organization of the shark molecules is more like C2 than factor B. Phylogenetic analysis indicates that GcBf/C2-1 and -2 and TrscBf of Triakis scyllia (another shark species) originated from a common ancestor and share a remote ancestor with Bf and C2 of mammals and bony fish.