A Recombinant Vaccine Effectively Induces C5a-Specific Neutralizing Antibodies and Prevents Arthritis

OBJECTIVES: To develop and validate a recombinant vaccine to attenuate inflammation in arthritis by sustained neutralization of the anaphylatoxin C5a. METHODS: We constructed and expressed fusion protein of C5a and maltose binding protein. Efficacy of specific C5a neutralization was tested using the fusion protein as vaccine in three different arthritis mouse models: collagen induced arthritis (CIA), chronic relapsing CIA and collagen antibody induced arthritis (CAIA). Levels of anti-C5a antibodies and anti-collagen type II were measured by ELISA. C5a neutralization assay was done using a rat basophilic leukemia cell-line transfected with the human C5aR. Complement activity was determined using a hemolytic assay and joint morphology was assessed by histology. RESULTS: Vaccination of mice with MBP-C5a led to significant reduction of arthritis incidence and severity but not anti-collagen antibody synthesis. Histology of the MBP-C5a and control (MBP or PBS) vaccinated mice paws confirmed the vaccination effect. Sera from the vaccinated mice developed C5a-specific neutralizing antibodies, however C5 activation and formation of the membrane attack complex by C5b were not significantly altered. CONCLUSIONS: Exploitation of host immune response to generate sustained C5a neutralizing antibodies without significantly compromising C5/C5b activity is a useful strategy for developing an effective vaccine for antibody mediated and C5a dependent inflammatory diseases. Further developing of such a therapeutic vaccine would be more optimal and cost effective to attenuate inflammation without affecting host immunity

Endoglucanase and Mannanase from Blue Mussel, Mytilus edulis: Purification, Characterization, Gene and Three Dimensional Structure

Two polysaccharide-degrading enzymes (endo-1,4-D-glucanase and β-mannanase) from blue mussel, Mytilus edulis, have been purified to homogeneity using a combination of several chromatographic steps. Each enzyme has been characterized with regard to its molecular weight, isoelectric point, pH and temperature stability, pH and temperature optimum and substrate specificity. The amino acid sequence of the endoglucanase has been determined at the protein level. The two enzymes are true blue mussel proteins as confirmed at the DNA level. The nucleotide sequences of synthesized cDNA from digestive gland and of genomic DNA from gill tissue were compared. Both genes contain introns, a property typical of eucaryotic organisms. Amino acid sequence based classification has revealed that the endoglucanase belongs to the glycoside hydrolase family 45, subfamily 2 while β-mannanase is a member of family 5. Both enzymes form insoluble inclusion bodies when expressed in Escherichia coli. Refolding attempts were unsuccessful. However, the β-mannanase was successfully expressed in the methylotropic yeast Pichia pastoris with an expression level above 100 mg/l in shaking culture. Crystals of the endoglucanase were made from the native protein and a dataset was collected to 1.85 Å resolution using an in-house rotating anode x-ray source. Crystals were also produced using recombinant β-mannanase and a dataset was collected to 1.4 Å resolution at the ESRF synchrotron beamline ID14-EH1. The three dimensional structure of the endoglucanase was solved by X-ray crystallography

Glucomannan and beta-glucan degradation by Mytilus edulis Cel45A : Crystal structure and activity comparison with GH45 subfamily A, B and C

The enzymatic hydrolysis of barley beta-glucan, konjac glucomannan and carboxymethyl cellulose by a beta-1,4-D-endoglucanase MeCel45A from blue mussel, Mytilus edulis, which belongs to subfamily B of glycoside hydrolase family 45 (GH45), was compared with GH45 members of subfamilies A (Humicola insolens HiCel45A), B (Trichoderma reesei TrCel45A) and C (Phanerochaete chrysosporium PcCel45A). Furthermore, the crystal structure of MeCel45A is reported. Initial rates and hydrolysis yields were determined by reducing sugar assays and product formation was characterized using NMR spectroscopy. The subfamily B and C enzymes exhibited mannanase activity, whereas the subfamily A member was uniquely able to produce monomeric glucose. All enzymes were confirmed to be inverting glycoside hydrolases. MeCel45A appears to be cold adapted by evolution, as it maintained 70% activity on cellohexaose at 4 degrees C relative to 30 degrees C, compared to 35% for TrCel45A. Both enzymes produced cellobiose and cellotetraose from cellohexaose, but TrCel45A additionally produced cellotriose

Chromatographic methods for the isolation of, and refolding of proteins from, Escherichia coli inclusion bodies

New methods for the chromatographic isolation of inclusion bodies directly from crude Escherichia coli homogenates and for the refolding of denatured protein are presented. The traditional method of differential centrifugation for the isolation of purified inclusion bodies is replaced by a single gel-filtration step. The principle is that the exclusion limit of the gel particles is chosen such that only the inclusion bodies are excluded, i.e., all other components of the crude homogenate penetrate the gel under the conditions selected. In the novel column refolding process, a decreasing gradient of denaturant (urea or Gu-HCl), combined with an increasing pH gradient, is introduced into a gel-filtration column packed with a gel medium that has an exclusion limit lower than the molecular mass of the protein to be refolded. A limited sample volume of the protein, dissolved in the highest denaturant concentration at the lowest pH of the selected gradient combination, is applied to the column. During the course of elution, the zone of denatured protein moves down the column at a speed approximately threefold higher than that of the denaturant. This means that the protein sample will gradually pass through areas of increasingly lower denaturant concentrations and higher pH, which promotes refolding into the native conformation. The shape and slope of the gradients, as well as the flow rate, will influence the refolding rate and can be adjusted for different protein samples. The principle is illustrated using a denatured recombinant scFv fusion protein obtained from E. coli inclusion bodies

Glycan Activation of Clec4b Induces Reactive Oxygen Species Protecting against Neutrophilia and Arthritis

Animal models for complex diseases are needed to position and analyze the function of interacting genes. Previous positional cloning identified Ncf1 and Clec4b to be major regulators of arthritis models in rats. Here, we investigate epistasis between Ncf1 and Clec4b, two major regulators of arthritis in rats. We find that Clec4b and Ncf1 exert an additive effect on arthritis given by their joint ability to regulate neutrophils. Both genes are highly expressed in neutrophils, together regulating neutrophil availability and their capacity to generate reactive oxygen species. Using a glycan array, we identify key ligands of Clec4b and demonstrate that Clec4b-specific stimulation triggers neutrophils into oxidative burst. Our observations highlight Clec4b as an important regulator of neutrophils and demonstrate how epistatic interactions affect the susceptibility to, and severity of, autoimmune arthritis

Three-dimensional crystal structure and enzymic characterization of β-mannanase Man5A from blue mussel Mytilus edulis

Endo-β-1,4-d-mannanase is the key depolymerizing enzyme for β-1,4-mannan polymers present in the cell walls of plants and some algae, as well as in some types of plant seeds. Endo-1,4-β-mannanase from blue mussel Mytilus edulis (MeMan5A) belongs to the glycoside hydrolase (GH) family 5 enzymes. The MeMan5A structure has been determined to 1.6 Å resolution using the multiple-wavelength anomalous dispersion method at the selenium K edge with selenomethionyl MeMan5A expressed in the yeast Pichia pastoris. As expected for GH 5 enzymes, the structure showed a (βα) 8-barrel fold. An unusually large number of histidine side-chains are exposed on the surface, which may relate to its location within the crystalline style of the digestive tract of the mussel. Kinetic analysis of MeMan5A revealed that the enzyme requires at least six subsites for efficient hydrolysis. Mannotetraose (M4) and mannopentaose (M5) were shown to interact with subsites -3 to +1, and -3 to +2, respectively. A clear kinetic threshold was observed when going from M4 to M5, indicating that the +2 subsite provides important interaction in the hydrolysis of short oligomeric mannose substrates. The catalytic centre motif at subsite -1 found in superfamily GH clan A is, as expected, conserved in MeMan5A, but the architecture of the catalytic cleft differs significantly from other GH 5 enzyme structures. We therefore suggest that MeMan5A represents a new subfamily in GH 5. © 2006 Elsevier Ltd. All rights reserved.This work was supported by Swedish Science Research Council (VR) grantPeer Reviewe

Cartilage-binding antibodies initiate joint inflammation and promote chronic erosive arthritis

Background: Antibodies binding to cartilage proteins are present in the blood and synovial fluid of early rheumatoid arthritis patients. In order to develop animal models mimicking the human disease, we have characterized the arthritogenic capacity of monoclonal antibodies directed towards different joint proteins in the cartilage. Methods: Purified antibodies specific to unmodified or citrullinated collagen type II (CII), collagen type XI (CXI), and cartilage oligomeric matrix protein (COMP) were produced as culture supernatant, affinity purified, pooled as antibody cocktails (Cab3 and Cab4), and injected intravenously into mice to induce arthritis. An adjuvant (lipopolysaccharide or mannan) was subsequently injected intraperitoneally on either day 5 or day 60 to enhance arthritis. Antibody binding and complement activation on the cartilage surface were analyzed by immunohistochemical methods. Bone erosions and joint deformations were analyzed by histological assessments, enzyme-linked immunosorbent assays, and micro-CT. Luminex was used to detect CII-triple helical epitope-specific antibody responses. Results: The new cartilage antibody cocktails induced an earlier and more severe disease than anti-CII antibody cocktail. Many of the mouse strains used developed severe arthritis with 3 antibodies, binding to collagen II, collagen XI, and cartilage oligomeric matrix protein (the Cab3 cocktail). Two new models of arthritis including Cab3-induced LPS-enhanced arthritis (lpsCAIA) and Cab3-induced mannan-enhanced arthritis (mCAIA) were established, causing severe bone erosions and bone loss, as well as epitope spreading of the B cell response. Cab4, with addition of an antibody to citrullinated collagen II, induced arthritis more efficiently in moderately susceptible C57BL/6 J mice. Conclusions: The new mouse model for RA induced with cartilage antibodies allows studies of chronic development of arthritis and epitope spreading of the autoimmune response and bone erosion

Effect of MBP-C5a vaccination in CAIA.

<p>Four-month-old male QB mice were injected intravenously with a cocktail of three complement activating monoclonal antibodies (6 mg in total/mouse) binding to CII on day 0, followed by LPS (25 µg/mouse intraperitoneally) on day +5. MBP-C5a or PBS emulsified in CFA/IFA was injected at the back of the mice on days −21, −10 and −2 as indicated by arrows. The mice were scored every day and the graphs present (A) incidence (maximal possible value is 100) and (B) severity of arthritis (maximal possible value is 60). Serum samples were collected from these mice on days −1 and +21 as indicated in the figure. Error bars indicate SEM and n denotes number of mice in each group. The severity of arthritis was analyzed by Mann–Whitney U ranking test and the incidence by χ² test. C5a vaccinated animals had less frequent (38% versus 95%; p<0.0005) as well as severity of arthritis (mean maximum score, p<0.0001) compared to control (PBS with CFA or IFA) vaccinated animals. n, indicates number of mice in each group.</p