Recombinant C-terminal Domain Research Articles

Cylindracin, a Cys-rich protein expressed in the fruiting body of Cyclocybe cylindracea, inhibits growth of filamentous fungi but not yeasts or bacteria.

Mushrooms are the fruiting bodies of fungi and are important reproductive structures that produce and disseminate spores. The Pri3 gene was originally reported to be specifically expressed in the primordia (a precursor to the mature fruiting body) of the edible mushroom Cyclocybe aegerita. Here, we cloned a Pri3-related cDNA from Cyclocybe cylindracea, another species in the same genus, and showed that the gene is specifically expressed at the pileus surface of the immature fruiting body but not in the primordia. Immunohistochemistry showed that the translated protein is secreted into a polysaccharide layer of the pileus surface. The recombinant C-terminal Cys-rich domain of the protein showed antifungal activity against three filamentous fungi and inhibited hyphal growth and conidiogenesis. These results suggest that the PRI3-related protein of C. cylindracea, named cylindracin, plays an important role in the defense against pathogens.

Recombinant C-Terminal Catalytic Domain of Rat L-Gulono Lactone Oxidase Produced in Bacterial Cells Is Enzymatically Active.

The L-gulonolactone oxidase enzyme (GULO) catalyzes the last step of L-ascorbic acid (vitamin C) biosynthesis. This enzymatic activity is lost in primates. The full-length rat GULO has been previously produced in plants and demonstrated to be active. In this study, we compared the activity of two variants of GULO produced in Escheriachia coli cells, full-length rat GULO (fGULO) and its C-terminal catalytic domain (cGULO). The expression and purification of the recombinant proteins were optimized, and their biological activity was confirmed by two methods, the GULO activity assay in the protein extracts and the 'in-gel' staining for GULO activity. Both variants of recombinant GULO were biologically active in both assays. However, cGULO is more promising than fGULO for ascorbic acid production because it is more efficiently produced by bacteria. Furthermore, the optimal activities of the fGULO and cGULO recombinant proteins were observed at pH 7 and 6.5, and at temperatures of 40 and 30 °C, respectively. Kinetic studies revealed that at low substrate concentrations, Km values for fGULO and cGULO were 53.5 ± 5 and 42 ± 6.3 µM, respectively.

Introduction of a new recombinant vaccine based on GRP78 for breast cancer immunotherapy and evaluation in a mouse model.

Breast cancer is one of the most prevalent malignancies in women. Several treatment options are available today, including surgery, chemotherapy, and radiotherapy. Immunotherapy, as a highly specific therapy, involves adaptive immune responses and immunological memory. In our present research, we used the recombinant C-terminal domain of the GRP78 (glucose- regulated protein 78) protein to induce an immune response and investigate its therapeutic impact in the 4T1 breast cancer model. BALB/c mice were immunized with the cGRP78 protein. The humoral immune response was assessed by ELISA. Then, BALB/c mice were injected subcutaneously with 1×106 4T1 tumor cells. Subsequently, tumor size and survival rate measurements, MTT, and cytokine assays were performed. The animals receiving the cGRP78 vaccine showed significantly more favorable survival and slower tumor growth rates compared with unvaccinated tumor-bearing mice as the negative control mice. Circulating levels of tumoricidal cytokines such as IFNγ were higher, whereas tolerogenic cytokines such as IL-2, 6, and 10 either did not increase or had a decreasing trend in mice receiving cGRP78. cGRP78 vaccines generated potent immunotherapeutic effects in a breast cancer mouse model. This novel strategy of targeting the GRP78 protein can promote the development of cancer vaccines and immunotherapies for breast cancer malignancies.

A Novel TLR4-Binding Domain of Peroxiredoxin From Entamoeba histolytica Triggers NLRP3 Inflammasome Activation in Macrophages.

Macrophages promote early host responses to infection by releasing pro-inflammatory cytokines, and they are crucial to combat amoebiasis, a disease affecting millions of people worldwide. Macrophages elicit pro-inflammatory responses following direct cell/cell interaction of Entamoeba histolytica, inducing NLRP3 inflammasome activation with high-output IL-1β/IL-18 secretion. Here, we found that trophozoites could upregulate peroxiredoxins (Prx) expression and abundantly secrete Prxs when encountering host cells. The C-terminal of Prx was identified as the key functional domain in promoting NLRP3 inflammasome activation, and a recombinant C-terminal domain could act directly on macrophage. The Prxs derived from E. histolytica triggered toll-like receptor 4-dependent activation of NLRP3 inflammasome in a cell/cell contact-independent manner. Through genetic, immunoblotting or pharmacological inhibition methods, NLRP3 inflammasome activation was induced through caspase-1-dependent canonical pathway. Our data suggest that E. histolytica Prxs had stable and durable cell/cell contact-independent effects on macrophages following abundantly secretion during invasion, and the C-terminal of Prx was responsible for activating NLRP3 inflammasome in macrophages. This new alternative pathway may represent a potential novel therapeutic approach for amoebiasis, a global threat to millions.

Mechanism of Staphylococcus aureus peptidoglycan O-acetyltransferase A as an O-acyltransferase

The O-acetylation of exopolysaccharides, including the essential bacterial cell wall polymer peptidoglycan, confers resistance to their lysis by exogenous hydrolases. Like the enzymes catalyzing the O-acetylation of exopolysaccharides in the Golgi of animals and fungi, peptidoglycan O-acetyltransferase A (OatA) is predicted to be an integral membrane protein comprised of a membrane-spanning acyltransferase-3 (AT-3) domain and an extracytoplasmic domain; for OatA, these domains are located in the N- and C-terminal regions of the enzyme, respectively. The recombinant C-terminal domain (OatAC) has been characterized as an SGNH acetyltransferase, but nothing was known about the function of the N-terminal AT-3 domain (OatAN) or its homologs associated with other acyltransferases. We report herein the experimental determination of the topology of Staphylococcus aureus OatAN, which differs markedly from that predicted in silico. We present the biochemical characterization of OatAN as part of recombinant OatA and demonstrate that acetyl-CoA serves as the substrate for OatAN Using in situ and in vitro assays, we characterized 35 engineered OatA variants which identified a catalytic triad of Tyr-His-Glu residues. We trapped an acetyl group from acetyl-CoA on the catalytic Tyr residue that is located on an extracytoplasmic loop of OatAN Further enzymatic characterization revealed that O-acetyl-Tyr represents the substrate for OatAC We propose a model for OatA action involving the translocation of acetyl groups from acetyl-CoA across the cytoplasmic membrane by OatAN and their subsequent intramolecular transfer to OatAC for the O-acetylation of peptidoglycan via the concerted action of catalytic Tyr and Ser residues.

Recombinant C-Terminal Domains from Scorpine-like Peptides Inhibit the Plasmodium berghei Ookinete Development In Vitro

Malaria is a parasitic disease, caused by protozoa of the genus Plasmodium, and is transmitted to humans through the bites of infected Anopheles mosquitoes. More than 200 million cases of malaria are reported annually and about 400,000 deaths worldwide. Currently, the use of antimalarial drugs has been efficient in most cases, however, resistance to these drugs is increasing, making it necessary and essential to have a new range of possible drugs or medicines to combat this disease. Scorpion venom contains peptides whose functions are now intensively studied. Some of these peptides are known as Scorpine-like, which have anti-bacterial and antiplasmodial properties as they have been described to inhibit the development of parasites responsible for malaria. Scorpine-like peptides are composed of two structural domains: one α-helical N-terminal domain, and a C-terminal domain with the cysteine-stabilized α/β motif, which confers the peptide the function of blocking potassium channels and/or anti-bacteria activity. In this work, two C-terminal domains from Scorpine-like peptides were constructed and expressed in Escherichia coli, and their function was analyzed. We were able to demonstrate that the recombinant C-terminal domains rCterVm and rCterHg showed antiplasmodial activity producing 60% and 90% inhibition, respectively, of Plasmodium berghei development at 1 µM and 0.15 µM concentration, which makes these peptides promising candidates against Malaria.

Enhanced recombinant C-terminal domain of gli2 gene expression can improve wound healing through promoting cdc25b and N-Myc genes expression

Abstract Wound is an important cause of morbidity and mortality in population because it can be caused by many factors like events, illness and surgery. Identification of wound healing factors and their role can be useful for effective treatment. GLI2 as an important activator of SHH signaling pathway should be further investigated. In this study we focused on the effect of a recombinant derivative of GLI2 gene on the progression of wound healing and expression of cdc25a and Myc genes as two transcription factors involved in wound healing pathways. A fragment containing coding sequence of functional domains of C-terminal of GLI2 protein was cloned in pcDNA3.1+ plasmid. A 1 cm diameter circular area wound up on the back of the one group of wistar albino rats. In the next step, purified recombinant plasmid containing GLI2 C-terminal domains coding sequence, was injected subcutaneously around the wound similar to what we do about the DNA vaccine strategy. Wound area was following during 7 days and all results was documented by digital pictures. Peripheral tissue of the wounds was dissected and studied for cdc25a and N-Myc genes expression real-time RT-PCR analysis. Total of wound scare was dissected after 7 days since injection. These biopsies were subjected to RNA extraction and Real-Time PCR analysis for expression analysis of Gli2, N-myc and cdc25b genes. In addition, a piece of every biopsy was analyzed by immunohistochemistry method for detection and analysis of gli2 protein. Our results showed a significant improvement in wound healing treatment by recombinant injected pcDNA3.1/GLI compared with the control group. Also, the affected tissue of rats that express recombinant gli2, had significant increasing of N-myc and cdc25b gene expression, which leads to acceleration of cell proliferation during wound healing. GLI2 protein is the major activator of SHH pathway and this signaling route has a vital role in renewing and repair of tissues.GLI2 protein can be consider as a potent factor in wound healing. Especially GLI2 as a transcription factor can regulates the expression of many proteins that play important role in healing process. Our results support the function of some of GLI motives in gene expression separately. Overexpression of N-Myc and cdc25b genes compared with the control group, can indirectly explain the function mechanism of GLI2 in the tissue repair.

C-Terminal Domain of Bacillus cereus Hemolysin II Is Able to Interact with Erythrocytes

Hemolysin II (HlyII) is one of the pathogenic factors of Bacillus cereus. With respect to the prototype of β-barrel toxins, the α-toxin of S. aureus, this pore-forming protein has a C-terminal domain (CTD) of 94 amino acids. The role of CTD in membrane pore formation and cell lysis is not clear, although removal of this portion of the protein is known to significantly reduce hemolytic activity. A representative panel of monoclonal antibodies against recombinant CTD recognizing full-length HlyII was obtained. Using the obtained monoclonal antibodies, CTD was shown to bind to rabbit red blood cells.

Purification, crystallization and preliminary X-ray crystallographic studies on the C-terminal domain of the flagellar protein FliL from Helicobacter pylori.

FliL is an inner membrane protein, occupying a position between the rotor and the stator of the bacterial flagellar motor. Its proximity to, and interactions with, the MS (membrane and supramembranous) ring, the switch complex and the stator proteins MotA/B suggests a role in recruitment and/or stabilization of the stator around the rotor, although the precise role of FliL in the flagellum remains to be established. In this study, recombinant C-terminal domain of Helicobacter pylori FliL (amino-acid residues 81-183) has been expressed in Escherichia coli and purified to > 98% homogeneity. Purified recombinant protein behaved as a monomer in solution. Crystals were obtained by the hanging-drop vapour-diffusion method using ammonium phosphate monobasic as a precipitant. These crystals belong to space group P1, with unit-cell parameters a = 62.5, b = 82.6, c = 97.8 Å, α = 67.7, ꞵ = 83.4, γ = 72.8°. A complete data set has been collected to 2.8 Å resolution using synchrotron radiation. This is an important step towards elucidation of the function of FliL in the bacterial flagellar motor.

Carboxy-terminal glycosyl hydrolase 18 domain of a carbohydrate active protein of Chitinophaga pinensis is a non-processive exochitinase

The recombinant C-terminal domain of chitinase C of Chitinophaga pinensis (CpChiC-GH18C) exhibits the highest activity at pH 6.0 and 35 °C, with a Km of 76.13 (mg-1 ml), a kcat of 10.16 (s-1), and a kcat/Km of 0.133 (mg-1 ml s-1) on colloidal chitin. Analysis of degradation of (GlcNAc)3-6 oligomers shows that CpChiC-GH18C releases (GlcNAc)2 as the main product, indicating an exo-type cleavage pattern. CpChiC-GH18C hydrolyzes the chitin polymers yielding GlcNAc, (GlcNAc)2, and (GlcNAc)3 as end products with no sign of processivity. Circular dichroism spectra indicate that the secondary and tertiary structures of CpChiC-GH18C are unaltered up to 45 °C and the protein denatures without an intermediate state. The urea-induced unfolding is a two-state process and the unfolding of native CpChiC-GH18C occurs in a single step. Among the metal ions tested, Hg2+ completely inhibits the enzyme activity. The chemical modulators, p-hydroxymercuribenzoic acid and N-bromosuccinimide considerably decrease the enzyme activity. Sequence analysis and homology modeling suggest that CpChiC-GH18C lacks a tryptophan residue at the aglycon site. Further, the CpChiC-GH18C has a shallow and open groove, suggesting that CpChiC-GH18C is non-processive exo-type chitinase with properties suitable for the bioconversion of chitin waste.

Abstract WMP117: Perlecan Regulates Pericyte Dynamics in the Repair Process of the Blood-Brain Barrier Against Ischemic Stroke

Introduction: The blood-brain barrier (BBB) breakdown occurs when the integrity of BBB components is lost as a consequence of ischemic stroke. Perlecan, a major heparan sulfate proteoglycan of basement membranes, is expressed by endothelial cells (ECs) and is adjacent to pericytes (PCs), suggesting supportive functions in the BBB. Recent studies highlight the importance of PCs in the process of repairing BBB functions, which are triggered by the upregulation of platelet-derived growth factor receptor β (PDGFRβ). We hypothesized that perlecan may play a protective role in BBB maintenance through the interaction with PCs during the repair process of the BBB disruption. Methods: We induced a 60-minute middle cerebral artery occlusion (MCAO) in adult conditional perlecan -deficient ( Perlecan -/- -Tg) mice in a C57BL/6 background, which express the perlecan transgene only in cartilage to rescue the perinatal lethality of Perlecan -/- mice. Recombinant perlecan C-terminal domain V (DV) was used for in vitro assays. Results: Perlecan -/- -Tg mice exhibited larger infarct volumes and more BBB leakage than the control mice on post-surgery day (PSD) 2 after MCAO. While the control mice showed increased numbers of PDGFRβ-positive PCs around the ischemic lesion on PSD 3, such upregulation was not observed in Perlecan -/- -Tg mice, suggesting that perlecan may participate in PC activation against the BBB breakdown. In wild-type mice, the expression of integrin α5, a potential receptor for perlecan, was upregulated both in PCs and in ECs in the ischemic lesion. In culture, PCs showed attachment activity to DV through integrin α5β1. DV induced well-assembled actin stress fibers and focal adhesions in PCs and promoted the PC migration induced by PDGF-BB. At molecular level, DV enhanced the PDGF-BB-induced phosphorylation of PDGFRβ, in addition to that of SHP-2 and FAK, suggesting that DV enhanced both PDGFRβ and integrin signaling. Conclusions: These results revealed that perlecan regulates the activation of PCs through the cooperative function of PDGFRβ and integrin α5β1, contributing to the repair process of the BBB. Perlecan DV may provide a potential therapeutic effect, based on a new approach from extracellular matrix to the disrupted BBB in ischemic stroke.

Development of a novel anti-human aspartyl-(asparaginyl) β-hydroxylase monoclonal antibody with diagnostic and therapeutic potential.

Human aspartyl-(asparaginyl)-β-hydroxylase (HAAH) has recently been the subject of several studies, as it was previously observed to be overexpressed in numerous types of carcinoma cells and tissues in patient tumor samples. HAAH has been implicated in tumor invasion and metastasis, indicating that it may be an important target and biomarker for tumor diagnosis and treatment. However, the immunological tools currently available for the study of this protein, including monoclonal antibodies, are limited, as is the present knowledge regarding the role of HAAH in tumor therapy and diagnosis. In the present study, a recombinant C-terminal domain of HAAH was expressed in Pichia pastoris and a novel monoclonal antibody (mAb) targeting HAAH (HAAH-C) was constructed. Immunofluorescence and antibody-dependent cellular cytotoxicity (ADCC) assays were used to demonstrate the specificity and ADCC activity of this antibody. The results demonstrated that this anti-C-terminal HAAH mAB, in combination with an existing anti-N terminal HAAH mAb, exhibited a high response to native HAAH from carcinoma cell culture supernatant, as measured with a double antibody sandwich enzyme-linked immunosorbent assay. This validated novel mAB-HAAH-C may prompt further studies into the underlying mechanisms of HAAH, and the exploration of its potential in tumor diagnosis and therapy.

Abstract A20: Globo H ceramide acts as immune checkpoint and angiogenic factor in tumor microenvironment

Abstract Since aberrant glycosylation is a feature of cancer cells, targeting tumor associated carbohydrate antigen (TACA) for cancer immunotherapy is gaining international attention. Globo H, the most prevalent TACA over-expressed in cancers of breast, lung, pancreas, stomach, colon, etc., is a hotly pursued target for cancer vaccine. Both Globo H and its pentasaccharide precursor, Gb5, are expressed in breast cancer stem cells. Notably, Globo H ceramide (GHCer) acts as an immune checkpoint molecule, as reflected by uptake of GHCer shed from cancer cells by tumor infiltrating lymphocytes Addition of GHCer to human PBMC, mouse splenocytes or purified CD4+ T cells inhibited their proliferative response to anti-CD3/CD28, and reduced the secretion of IL-2, IFN-γ and IL-4. GHCer also significantly suppressed the proliferation of splenocytes or CD19+ B cells in response to LPS or LPS + IL4 +CD40 ligand and their production of IgG and IgM, with negligible generation of plasma cells. Ceramide displayed no such inhibitory effects. On the other hand, GHCer failed to raise the number of regulatory T cells, or their expression of FOXP3/CTLA4, nor did it increase apoptosis. Molecularly, GHCer significantly suppressed the Notch1 signaling during activation of human PBMC and murine T and B cells. This was linked to GHCer-induced upregulation of expression of ID3 and itch, leading to ID3-dependent downregulation of Notch1 and itch-mediated Notch1 degradation. Furthermore, GHCer increased the expression of egr2 and egr3 at 2hrs after activation which preceded itch upregulation. These results provide the first evidence for GHCer to facilitate the escape of cancer cells from immune surveillance via down-regulation of Notch1 signaling at transcriptional level by ID3, and protein level by egr2/3 controlled itch expression. In addition, GHCer is also incorporated into endothelial cells, through the absorption of microvesicles shed from tumor cells. In endothelial cells, GHCer addition induces migration, tube formation, and intracellular Ca2+ mobilization in vitro and angiogenesis in vivo. Clinically, Globo H+ breast cancer specimens contained Globo H+ tumor infiltrating lymphocytes, and higher vessel density than Globo-H- tumors. Mechanistic investigations linked its angiogenic effects to its binding to TRAX, GHCer is also incorporated into endothelial cells, enhancing angiogenesis. Clinically, Globo H+ breast cancer specimens contained Globo H+ tumor infiltrating lymphocytes, and higher vessel density than Globo-H- tumors. Mechanistic investigations linked the angiogenic effects of GHCer to its binding to TRAX, with consequent release of PLCb1 from TRAX to trigger Ca2+ mobilization. The binding of GHCer to TRAX was validated by Biacore system with immobilized TRAX protein on sensor chip CM5. At concentrations ranging from 9.77 nM to 5 μM, GHCer binds TRAX with a fast association rate, followed by slow dissociation. The KD of the GHCer binding with TRAX was estimated to be about 4.09 x10-8 M, by fitting the binding curves with BIAevaluation 4.1. On the other hand, galactosyl-ceramide, lactosyl-ceramide or Gb4-ceramide showed negligible binding to TRAX. Preliminary data showed that recombinant C-terminal domain of PLCB1 bound to TRAX and was competed by GHCer in a concentration-dependent manner. Taken together, these data support the notion that GHCer plays triple roles in serving as a cancer antigen (including breast cancer stem cells), as an immune checkpoint and as an angiogenic factor, thereby propelling the ongoing multi-national phase II/III clinical trial of globo H vaccine in breast cancer and ovarian cancer. Citation Format: Yu J, Cheng JY, Wang SH, Tsai YC, Wu JC, Hung JT, Lin J, Yeh KT, Yu AL. Globo H ceramide acts as immune checkpoint and angiogenic factor in tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A20.

The recombinant C-terminal fragment of tetanus toxin protects against cholinotoxicity by intraseptal injection of β-amyloid peptide (25–35) in rats

The recombinant C-terminal domain of tetanus toxin (Hc-TeTx) is a new non-toxic peptide of the tetanus toxin that exerts a protective action against glutamate excitotoxicity in motoneurons. Moreover, its efficacy as a neuroprotective agent has been demonstrated in several animal models of neurodegeneration. The eleven amino acids in the β amyloid peptide (Aβ25-35) mimic the toxic effects of the full β amyloid peptide (Aβ1-42), causing the impairment of the cholinergic system in the medial septum (MS) which, in turn, alters the septo-hippocampal pathway and leads to learning and memory impairments. The aim of this study was to examine the neuroprotective effects of the Hc-TeTx fragment against cholinotoxicity. The Hc-TeTx fragment (100 ng) was injected into the rats intercranially, with the Aβ(25-35) (2 μg) then injected into their MS. The animals were tested for spatial learning and memory in the eight-arm radial maze. The brains were removed to assess cholinergic markers, such as choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), and to explore neurodegeneration in the MS and hippocampus, using amino-cupric silver and H&E staining. Finally, capase-3, a marker of apoptosis, was examined in the MS. Our results clearly demonstrate that the application of Hc-TeTx prevents the loss of cholinergic markers (ChAT and AChE), the activation of capase-3, and neurodegeneration in the MS and the CA1 and CA3 subfields of the hippocampus. All these improvements were reflected in spatial learning and memory performance, and were significantly higher compared with animals treated with Aβ(25-35). Interestingly, the single administration of Hc-TeTx into the MS modified the ChAT and AChE expression that affect cognitive processes, without inducing neurodegeneration or an increase in capase-3 expression in the MS and hippocampus. In summary, our findings suggest that the recombinant Hc-TeTx fragment offers effective protection for the septo-hippocampal pathway, given that it reduces the neurodegeneration caused by Aβ(25-35) and improves learning and memory processes.

Dual Thermosensitive Hydrogels Assembled from the Conserved C-Terminal Domain of Spider Dragline Silk.

Stimuli-responsive hydrogels have great potentials in biomedical and biotechnological applications. Due to the advantages of precise control over molecular weight and being biodegradable, protein-based hydrogels and their applications have been extensively studied. However, protein hydrogels with dual thermosensitive properties are rarely reported. Here we present the first report of dual thermosensitive hydrogels assembled from the conserved C-terminal domain of spider dragline silk. First, we found that recombinant C-terminal domain of major ampullate spidroin 1 (MaSp1) of the spider Nephila clavipes formed hydrogels when cooled to approximately 2 °C or heated to 65 °C. The conformational changes and self-assembly of the recombinant protein were studied to understand the mechanism of the gelation processes using multiple methods. It was proposed that the gelation in the low-temperature regime was dominated by hydrogen bonding and hydrophobic interaction between folded protein molecules, whereas the gelation in the high-temperature regime was due to cross-linking of the exposed hydrophobic patches resulting from partial unfolding of the protein upon heating. More interestingly, genetic fusion of the C-terminal domain to a short repetitive region of N. clavipes MaSp1 resulted in a chimeric protein that formed a hydrogel with significantly improved mechanical properties at low temperatures between 2 and 10 °C. Furthermore, the formation of similar hydrogels was observed for the recombinant C-terminal domains of dragline silk of different spider species, thus demonstrating the conserved ability to form dual thermosensitive hydrogels. These findings may be useful in the design and construction of novel protein hydrogels with tunable multiple thermosensitivity for applications in the future.

The C-terminal domains of two homologous Oleaceae β-1,3-glucanases recognise carbohydrates differently: Laminarin binding by NMR

Ole e 9 and Fra e 9 are two allergenic β-1,3-glucanases from olive and ash tree pollens, respectively. Both proteins present a modular structure with a catalytic N-terminal domain and a carbohydrate-binding module (CBM) at the C-terminus. Despite their significant sequence resemblance, they differ in some functional properties, such as their catalytic activity and the carbohydrate-binding ability. Here, we have studied the different capability of the recombinant C-terminal domain of both allergens to bind laminarin by NMR titrations, binding assays and ultracentrifugation. We show that rCtD-Ole e 9 has a higher affinity for laminarin than rCtD-Fra e 9. The complexes have different exchange regimes on the NMR time scale in agreement with the different affinity for laminarin observed in the biochemical experiments. Utilising NMR chemical shift perturbation data, we show that only one side of the protein surface is affected by the interaction and that the binding site is located in the inter-helical region between α1 and α2, which is buttressed by aromatic side chains. The binding surface is larger in rCtD-Ole e 9 which may account for its higher affinity for laminarin relative to rCtD-Fra e 9.

Factor H binds to Trypanosoma cruzi trypomastigotes via its C-terminus and contributes to parasite resistance against the alternative pathway of complement (MPF2P.809)

Abstract Trypanosoma cruzi (T. cruzi), the causal agent of devastating Chagas disease, possesses multifactorial resistance to the alternative pathway (AP) of complement. AP regulatory protein factor H (fH) uses its C-terminal 19-20 domains to recognize polyanions (i.e. sialic acid) and C3b on host cells, facilitating decay of C3 convertases and inactivation of C3b via the N-terminus of fH. We have shown that recombinant C-terminal fH domains 19-20 (rH19-20) compete with fH for binding to C3b and host cell polyanions, inhibiting protection from the AP. T. cruzi tripomastigotes resist the AP by capturing sialic acid from host cells. The specific role that fH plays in protecting T. cruzi from the AP and the fH domains involved remain unknown. Here we assessed the susceptibility of T. cruzi (MF, Y and Brenner strains) to AP-mediated killing, and the ability of fH to bind directly to the parasite by FACS. For determining the fH domains needed for the interaction with T. cruzi, trypomastigotes were incubated with normal human sera in the presence or absence of equimolar amounts of overlapping recombinant fH proteins (2 to 3 domains each) spanning domains 7 to 20. The ability of each protein to increase AP-mediated killing of T. cruzi was assessed. Only rH19-20 effectively competed with serum fH, inducing a significant decrease in parasite survival (p<0.0001), indicating that the C-terminus of fH is essential for fH-mediated recognition and protection of T. cruzi trypomastigotes from the AP.

Agonist-induced Ca2+ Sensitization in Smooth Muscle: REDUNDANCY OF RHO GUANINE NUCLEOTIDE EXCHANGE FACTORS (RhoGEFs) AND RESPONSE KINETICS, A CAGED COMPOUND STUDY

Many agonists, acting through G-protein-coupled receptors and Gα subunits of the heterotrimeric G-proteins, induce contraction of smooth muscle through an increase of [Ca(2+)]i as well as activation of the RhoA/RhoA-activated kinase pathway that amplifies the contractile force, a phenomenon known as Ca(2+) sensitization. Gα12/13 subunits are known to activate the regulator of G-protein signaling-like family of guanine nucleotide exchange factors (RhoGEFs), which includes PDZ-RhoGEF (PRG) and leukemia-associated RhoGEF (LARG). However, their contributions to Ca(2+)-sensitized force are not well understood. Using permeabilized blood vessels from PRG(-/-) mice and a new method to silence LARG in organ-cultured blood vessels, we show that both RhoGEFs are activated by the physiologically and pathophysiologically important thromboxane A2 and endothelin-1 receptors. The co-activation is the result of direct and independent activation of both RhoGEFs as well as their co-recruitment due to heterodimerization. The isolated recombinant C-terminal domain of PRG, which is responsible for heterodimerization with LARG, strongly inhibited Ca(2+)-sensitized force. We used photolysis of caged phenylephrine, caged guanosine 5'-O-(thiotriphosphate) (GTPγS) in solution, and caged GTPγS or caged GTP loaded on the RhoA·RhoGDI complex to show that the recruitment and activation of RhoGEFs is the cause of a significant time lag between the initial Ca(2+) transient and phasic force components and the onset of Ca(2+)-sensitized force.

Structure basis 1/2SLPI and porcine pancreas trypsin interaction.

SLPI (secretory leukocyte protease inhibitor) is a 107-residue protease inhibitor which inhibits various serine proteases, including elastase, cathepsin G, chymotrypsin and trypsin. SLPI is obtained as a multiple inhibitor in lung defense and in chronic airway infection. X-ray crystal structures have so far reported that they are full-length SLPIs with bovine α-chymotrypsin and 1/2SLPI (recombinant C-terminal domain of SLPI; Arg58-Ala107) with HNE (human neutrophil elastase). To understand the role of this multiple inhibitory mechanism, the crystal structure of 1/2SLPI with porcine pancreas trypsin was solved and the binding modes of two other complexes compared. The Leu residue surprisingly interacts with the S1 site of trypsin, as with chymotrypsin and elastase. The inhibitory mechanism of 1/2SLPI using the wide primary binding site contacts (from P2' to P5) with various serine proteases is discussed. These inhibitory mechanisms have been acquired in the evolution of the protection system for acute inflammatory diseases.

Cysteine endoprotease activity of human ribosomal protein S4 is entirely due to the C-terminal domain, and is consistent with Michaelis–Menten mechanism

It is known that tandem domains of enzymes can carry out catalysis independently or by collaboration. In the case of cysteine proteases, domain sequestration abolishes catalysis because the active site residues are distributed in both domains. The validity of this argument is tested here by using isolated human ribosomal protein S4, which has been recently identified as an unorthodox cysteine protease. Cleavage of the peptide substrate Z-FR↓-AMC catalyzed by recombinant C-terminal domain of human S4 (CHS4) is studied by fluorescence-monitored steady-state and stopped-flow kinetic methods. Proteolysis and autoproteolysis were analyzed by electrophoresis. The CHS4 domain comprised of sequence residues 116-263 has been cloned and ovreexpressed in Escherichia coli. The purified domain is enzymatically active. Barring minor differences, steady-state kinetic parameters for catalysis by CHS4 are very similar to those for full-length human S4. Further, stopped-flow transient kinetics of pre-steady-state substrate binding shows that the catalytic mechanism for both full-length S4 and CHS4 obeys the Michaelis-Menten model adequately. Consideration of the evolutionary domain organization of the S4e family of ribosomal proteins indicates that the central domain (residues 94-170) within CHS4 is indispensable. The C-terminal domain can carry out catalysis independently and as efficiently as the full-length human S4 does. Localization of the enzyme function in the C-terminal domain of human S4 provides the only example of a cysteine endoprotease where substrate-mediated intramolecular domain interaction is irrelevant for catalytic activity.