Partitioning of beta-galactosidase fusion proteins in PEG/potassium phosphate aqueous two-phase systems.

Four different beta-galactosidase fusion proteins have been partitioned in poly(ethylene glycol) (PEG) 4000/potassium phosphate aqueous two-phase systems. The partition coefficients (K) of staphylococcal protein A-beta-galactosidase (SpA beta gal) (K = 3.5) and staphylococcal protein A-streptococcal protein G-beta-galactosidase (AG beta gal) (K = 2.8) were compared with the partition coefficients of their constituent molecules, beta-galactosidase, SpA, and protein AG. It was found that by fusing beta-galactosidase to the smaller proteins SpA and protein AG, their partition coefficients were increased four to five times. Experimental data were fitted into, and found to agree with, the Albertsson partition model of interacting molecules. The compatibility with PEG and potassium phosphate of beta-galactosidase, SpA, and two different versions of the SpA beta gal protein, displayed as precipitation curves, showed a relationship to the protein partition coefficients in PEG/potassium phosphate systems. High solubility in one phase component was accompanied by preferential partitioning to the phase rich in the same component in the PEG/potassium phosphate system. Also, a changed linker region in SpA beta gal resulted in a more soluble protein. This, together with the improved K values of the target proteins by fusion, shows that it is possible to use beta-galactosidase as an affinity handle.     

Cellular and biochemical responses to environmental and experimentally induced stress in sea urchin coelomocytes

Coelomocytes are considered to be immune effectors of sea urchins. Subpopulations of coelomocytes can be purified from a total cell suspension. The proportion of each cell type can vary not only among species, but also between individuals of the same species, according to their size and physiological conditions. We tested the hypothesis that coelomocytes play a role in defense mechanisms activated by adverse external conditions. Total coelomocytes from control and stressed (temperature, pollution, and injuries) sea urchins were analyzed for their expression of the 70 kDa heat shock protein (hsp70), a well recognized stress marker. Further analysis was performed by separation of coelomocytes into subpopulations by step gradients. We demonstrated that sea urchin coelomocytes respond to temperature shock and to polluted seawater by the upregulation of hsp70. Among coelomocytes certain cells, known as red spherula cells, showed a great increase in number in animals collected from polluted seawaters or subjected to "accidental" injury. The present study confirms the immunological function of sea urchin coelomocytes, as indicated by the upregulation of the hsp70 molecular marker, and suggests that sea urchin coelomocytes can be utilized as sensitive bio-indicators of environmental stress.     

Mitogenic response of human and murine T lymphocytes to staphylococcal protein A: Not mediated by binding to cell surface immunoglobulins

Staphylococcal protein A (SpA) stimulates human lymphocytes more efficiently than murine lymphocytes. The subsets of lymphocytes responding to SpA were heterogeneous. In the subsets, cortisone resistant, low Thy 1,2, minor populations of murine thymocytes and mature peripheral T lymphocytes were predominantly stimulated by SpA. Though SpA stimulated cortisone-resistant thymocytes, further treatment of these cells with anti-mouse immunoglobulin sera and guinea pig complement failed to influence the mitogenic response to SpA. It seems likely that SpA contains a substance stimulating T lymphocytes, through binding to sites other than the surface immunoglobulins.     

Functional properties of proteins from the coelomic fluid of the wounded sea star Asterias rubens (L)

Impact on viability and adhesion of three protein fractions, separated by size, from the coelomic fluid of wounded Asterias rubens′, was tested on autologous coelomocytes. In addition antimicrobial property of the protein fractions was tested on the Gram-negative bacterium Vibrio parahaemolyticus. All fractions promoted viability and the larger proteins facilitated adhesion of the coelomocytes. The strongest antimicrobial effect was caused by the fraction with the smallest proteins.     

Human monoclonal IgG rheumatoid factor has structural homology with bacterial Fc receptor proteins

A cloned lymphoblast cell line, hRF-1, that secreted human monoclonal IgG4 rheumatoid factor autoantibody was produced by Epstein-Barr virus transformation of lymphocytes from rheumatoid arthritis synovium. The binding of hRF-1 rheumatoid factor to IgG globulins of different mammalian species was similar to the binding specificity of Staphylococcus aureus protein A (SpA) and to antibodies found in the sera from patients with rheumatoid arthritis. hRF-1 also had the same binding pattern to human IgG subclasses as SpA. Direct competition was observed between SpA and hRF-1 in binding IgG Fc. These results provide evidence for structural homology between a bacterial Fc receptor protein (SpA) and the monoclonal IgG rheumatoid factor.     

Staphylococcus aureus isolated from Kawasaki disease patients hyper-releases extracellular protein A.

S. aureus isolates from patients with Kawasaki disease (KD) release high levels of extracellular protein A (SpA), as compared to S. aureus in other diseases. The molecular weight of this released protein A is about 70 kDa. Extracellular KD SpA purified by affinity chromatography possessed the same amino acid sequence at the NH2-terminal IgG binding region and the same antigenic specificity as recombinant and cell-wall-bound SpA preparations. The size of DNA fragments containing the spa gene from S. aureus KD strains was 160-165 kb. All of these DNA fragments contained the igb portion encoding the IgG-binding region of KD SpA. Significantly higher molecular size of the SpA molecules hyper-released in the stationary-phase culture and the lack of production of other exo-proteins allow us to speculate that S. aureus isolated from patients with KD have mutations occurring in the agr locus.     

Infiltration of the synovial membrane with macrophage subsets and polymorphonuclear cells reflects global disease activity in spondyloarthropathy

Considering the relation between synovial inflammation and global disease activity in rheumatoid arthritis (RA) and the distinct but heterogeneous histology of spondyloarthropathy (SpA) synovitis, the present study analyzed whether histopathological features of synovium reflect specific phenotypes and/or global disease activity in SpA. Synovial biopsies obtained from 99 SpA and 86 RA patients with active knee synovitis were analyzed for 15 histological and immunohistochemical markers. Correlations with swollen joint count, serum C-reactive protein concentrations, and erythrocyte sedimentation rate were analyzed using classical and multiparameter statistics. SpA synovitis was characterized by higher vascularity and infiltration with CD163⁺ macrophages and polymorphonuclear leukocytes (PMNs) and by lower values for lining-layer hyperplasia, lymphoid aggregates, CD1a⁺ cells, intracellular citrullinated proteins, and MHC–HC gp39 complexes than RA synovitis. Unsupervised clustering of the SpA samples based on synovial features identified two separate clusters that both contained different SpA subtypes but were significantly differentiated by concentration of C-reactive protein and erythrocyte sedimentation rate. Global disease activity in SpA correlated significantly with lining-layer hyperplasia as well as with inflammatory infiltration with macrophages, especially the CD163⁺ subset, and with PMNs. Accordingly, supervised clustering using these synovial parameters identified a cluster of 20 SpA patients with significantly higher disease activity, and this finding was confirmed in an independent SpA cohort. However, multiparameter models based on synovial histopathology were relatively poor predictors of disease activity in individual patients. In conclusion, these data indicate that inflammatory infiltration of the synovium with CD163⁺ macrophages and PMNs as well as lining-layer hyperplasia reflect global disease activity in SpA, independently of the SpA subtype. These data support a prominent role for innate immune cells in SpA synovitis and warrant further evaluation of synovial histopathology as a surrogate marker in early-phase therapeutic trials in SpA.     

Characterization of an IL-1 receptor from Asterias forbesi coelomocytes

The tremendous importance of cytokines to immune defensive systems suggests that they have been conserved through evolution. The existence of interleukin (IL)-1-like molecules in several invertebrate groups substantiates this hypothesis. To characterize further the relationship of invertebrate IL-1-like molecules, we have used competitive binding assays to show that invertebrate coelomocytes of the starfish Asterias forbesi possess an IL-1-specific binding protein. Competitive binding experiments used radiolabeled human IL-1alpha. IL-1 bound specifically to the coelomocytes by a single high-affinity binding site (K(d) = 8.72 x 10(-10)/M). There are approximately 6000 binding sites per cell. The specificity of the receptor was confirmed by demonstrating that, among a group of cytokines and lymphokines tested, only vertebrate IL-1- or echinoderm IL-1-like molecules and the vertebrate IL-1 receptor antagonist inhibit IL-1 binding. Treatment of coelomocytes (labeled with IL-1alpha) with bivalent water-soluble crosslinkers identified a membrane protein of approximately 70 kDa to which IL-1 is specifically crosslinked.     

Genetic analysis of endocytosis in Caenorhabditis elegans: coelomocyte uptake defective mutants

The coelomocytes of Caenorhabditis elegans are scavenger cells that continuously and nonspecifically endocytose fluid from the pseudocoelom (body cavity). Green fluorescent protein (GFP) secreted into the pseudocoelom from body wall muscle cells is endocytosed and degraded by coelomocytes. We show that toxin-mediated ablation of coelomocytes results in viable animals that fail to endocytose pseudocoelomic GFP, indicating that endocytosis by coelomocytes is not essential for growth or survival of C. elegans under normal laboratory conditions. We examined known viable endocytosis mutants, and performed RNAi for other known endocytosis genes, for coelomocyte uptake defective (Cup) phenotypes. We also screened for new genes involved in endocytosis by isolating viable mutants with Cup defects; this screen identified 14 different genes, many with multiple alleles. A variety of Cup terminal phenotypes were observed, consistent with defects at various steps in the endocytic pathway. Available molecular information indicates that the Cup mutant screen has identified novel components of the endocytosis machinery that are conserved in mammals but not in Saccharomyces cerevisiae, the only other organism for which large-scale genetic screens for endocytosis mutants have been performed.     

Amassin,an olfactomedin protein,mediates the massive intercellular adhesion of sea urchin coelomocytes

Sea urchins have a fluid-filled body cavity, the coelom, containing four types of immunocytes called coelomocytes. Within minutes after coelomic fluid is removed from the body cavity, a massive cell-cell adhesion of coelomocytes occurs. This event is referred to as clotting. Clotting is thought to be a defense mechanism against loss of coelomic fluid if the body wall is punctured, and it may also function in the cellular encapsulation of foreign material and microbes. Here we show that this intercoelomocyte adhesion is mediated by amassin, a coelomic plasma protein with a relative molecular mass (Mr) of 75 kD. Amassin forms large disulfide-bonded aggregates that adhere coelomocytes to each other. One half of the amassin protein comprises an olfactomedin (OLF) domain. Structural predictions show that amassin and other OLF domain-containing vertebrate proteins share a common architecture. This suggests that other proteins of the OLF family may function in intercellular adhesion. These findings are the first to demonstrate a function for a protein of the OLF family.     

Electron microscopic and hydrodynamic studies of protein A-immunoglobulin G soluble complexes

The soluble complexes formed by reacting staphylococcal protein A (SpA) with rabbit immunoglobulin G (IgG) antibodies were characterized by hydrodynamic and electron microscopic methods. In moderate SpA excess, equilibrium mixtures of SpA and rabbit IgG formed four discrete complexes that sedimented at approximately 7, 10, 13, and 15S. The putative complexes were visible by electron microscopy and appeared to contain one, two, three, and approximately five molecules of IgG. Probably because of its elongated shape, SpA was not clearly visible in these mixtures or in control preparations of SpA alone. Both native IgG and IgG modified by cleavage of its single-hinge disulfide bond formed similar complexes on interaction with SpA. It was possible to resolve heterogeneous mixtures of IgG-SpA complexes by using an analytical ultracentrifuge equipped with a photoelectric scanner interfaced to a small computer. The relative concentrations and sedimentation velocities of different complexes in a mixture were determined from computer-generated integral and derivative plots. Both hydrodynamic and electron microscopic methods revealed that the distribution of complexes was sensitive to the IgG to SpA molar ratio. The relative amounts of faster complexes increased as the IgG to SpA molar ratio was increased. Surprisingly, when the IgG to SpA molar ratio was greater than or equal to 2, the complexes were converted into a unique 17S complex. This rather unprecedented transformation was reversible: the addition of excess SpA caused the dissociation of the 17S complex into a mixture of the 7, 10, 13, and 15S structures. The average translational diffusion coefficient of the 17S complex was 2.62 +/- 0.13 Ficks. In the electron microscope, the complex appeared to be exceptionally compact with an average diameter of 287 A. The stoichiometry of the 17S complex, together with sedimentation equilibrium, diffusion, and electron microscopic measurements, indicated that it is composed of four molecules of IgG and two molecules of SpA.     

Activity and immunodetection of lysozyme in earthworm Dendrobaena veneta (Annelida)

In the present study, lysozyme-like activity against Micrococcus luteus was detected in the coelomic fluid, the extract from coelomocytes, intestine and in the homogenates from cocoons of Dendrobaena veneta. Four hours after immunization with Escherichia coli, the lysozyme activity in the coelomic fluid increased about three times and in the extract of coelomocytes - four times, in comparison to the control. In three cases: of the coelomic fluid, the homogenates from cocoons and the extract from coelomocytes, the antibody against HEWL (hen egg white lysozyme) recognized only one protein with a molecular mass of about 14.4 kDa. In the coelomic fluid, apart from the protein with molecular mass of 14.4 kDa the antibody directed against human lysozyme recognized an additional protein of 22 kDa. Using the bioautography technique after electrophoretic resolution of native proteins in acidic polyacrylamide gels, two lytic zones of M. luteus were observed in the case of the coelomic fluid and three after the analysis of the extract of coelomocytes and the egg homogenates. The results indicated the existence of several forms of lysozyme with a different electric charge in the analyzed D. veneta samples. The highest lysozyme activity in the intestine of D. veneta was observed in the midgut. The antibody directed against human lysozyme indicated a strong positive signal in epidermal and midgut cells of earthworm.     

Insights into the pathogenesis of axial spondyloarthropathy from network and pathway analysis

Background

Complex chronic diseases are usually not caused by changes in a single causal gene but by an unbalanced regulating network resulting from the dysfunctions of multiple genes or their products. Therefore, network based systems approach can be helpful for the identification of candidate genes related to complex diseases and their relationships. Axial spondyloarthropathy (SpA) is a group of chronic inflammatory joint diseases that mainly affect the spine and the sacroiliac joints. The pathogenesis of SpA remains largely unknown.

Results

In this paper, we conducted a network study of the pathogenesis of SpA. We integrated data related to SpA, from the OMIM database, proteomics and microarray experiments of SpA, to prioritize SpA candidate disease genes in the context of human protein interactome. Based on the top ranked SpA related genes, we constructed a SpA specific PPI network, identified potential pathways associated with SpA, and finally sketched an overview of biological processes involved in the development of SpA.

Conclusions

The protein-protein interaction (PPI) network and pathways reflect the link between the two pathological processes of SpA, i.e., immune mediated inflammation, as well as imbalanced bone modelling caused new boneformation and bone loss. We found that some known disease causative genes, such as TNFand ILs, play pivotal roles in this interaction.

     

Some improved versions of methods for the indirect detection of staphylococcal protein A gene expressed in Escherichia coli.

Several versions of methods for the indirect detection of expression of staphylococcal protein A gene (spa) in Escherichia coli (E. coli) were devised by making use of biological properties of staphylococcal protein A (SpA). i) Hemagglutination of sheep red blood cells (SRBC) sensitized with anti-SRBC-antibodies using heat-treated spa-transformed E. coli organisms; Native spa-transformed E. coli organisms did not agglutinate the sensitized SRBC. The heat-treatment (60 C, 4 hr) of the transformants, however, caused positive hemagglutination like SpA-positive Staphylococcus aureus (S. aureus) organisms. ii) Halo formation around colonies on agar plates containing normal dog serum, which is originally used for the detection of SpA of S. aureus. A mutant strain NMJ was isolated, which showed formation of the halo of precipitate due to interaction between immunoglobulin and SpA. iii) A new version of immunodetection; After lysis of the transformants grown on a nitrocellulose membrane by alkali, SpA could be directly detected by immuno-detection procedures after inactivation of endogenous peroxidase in bacteria by phenylhydrazine and hydrogen peroxide.     

Coelomocytes and post-traumatic response in the common sea star Asterias rubens

Coelomocytes are recognized as the main cellular component of the echinoderm immune system. They are the first line of defense and their number and type can vary dramatically during infections or following injury. Sea stars have been used as a model system to study the regeneration process after autotomy or predation. In the present study we examined the cellular and biochemical responses of coelomocytes from the European sea star Asterias rubens to traumatic stress using immunochemical and biochemical approaches. In terms of trauma and post-traumatic stress period, here we consider the experimental arm amputation and the repair phase involved in the first 24 hours post-amputation, which mimicked a natural predation event. Four cell morphotypes were distinguishable in the coelomic fluid of both control and post-traumatic-stressed animals (phagocytes, amoebocytes, vibratile cells, hemocytes), but phagocytes were the major components, accounting for about 95% of the total population. Thus, the effects measured relate to the overall population of coelomocytes. A modest increase in the total number of freely circulating coelomocytes was observed 6 hours post-amputation. Interestingly, a monoclonal antibody (McAb) to a sea urchin embryo adhesion protein (toposome) cross-reacted with isolated sea star coelomocytes and stained the coelomic epithelium of control animals with an increase in trauma-stressed arms. In addition, coelomocytes from trauma-stressed animals showed a time-dependent increase in Hsp70 levels, as detected by both immunocytochemistry and immunoblotting within 24 hours after arm tip amputation, with a peak at 6 hours after amputation. Our findings indicate a clear role for coelomocytes and classic stress molecules in the post-traumatic stress associated with the early repair phase of regeneration.     

Is there evidence for a common amino acid sequence in proteins with membrane attaching ability?

A comparison between the primary sequence of staphylococcal protein A (SpA) and a wide range of published protein sequences revealed a limited, but striking homology in approximately two-thirds of them. The region in the SpA sequence with the common homology was identified as the octapeptide repeats comprising the cell wall peptidoglycan-binding domain. Available structural information and the known location of the proteins within their host cells suggests that this common octapeptide may be important in interaction of the protein with the cell surface (either membrane or wall).     

Simple method for quantitation of cell-bound protein A on Staphylococcus aureus cells by means of hemagglutination with sheep erythrocytes differentially sensitized with rabbit antibody and its clinical application

A simple method for quantitation of cell-bound protein A (SpA) on organisms of Staphylococcus aureus was successfully devised by using hemagglutination between staphylococcal organisms and a series of sheep erythrocyte suspensions sensitized with different amounts of anti sheep erythrocyte rabbit antiserum. The validity of the principle and the reproducibility of the method presented here were precisely analyzed and the details of the method are presented. The hemagglutination was quantitatively inhibited both by normal rabbit serum and by soluble SpA. Using the method presented here, 376 strains of S. aureus freshly isolated from clinical materials were subjected to SpA quantitation in cell-bound form. According to the results, there was an unexpected distribution profile in the amounts of cell-bound SpA among the clinical isolates, which showed a two-peak pattern. A possible usefulness of the method presented here in clinical investigations is briefly discussed also.     

Dynamics of proteolysis and its influence on the accumulation of intracellular recombinant proteins

A method to quantify the impact of proteolysis on accumulation of recombinant proteins in E. coli is described. A much smaller intracellular concentration of staphylococcal protein A (SpA) (14.7 mg · g⁻¹) compared to the fusion protein SpA-βgalactosidase (138 mg · g⁻¹) is explained by a very high proteolysis rate constant of SpA. The SpA synthesis rate reached a maximum one hour after induction and gradually decreased to half of this value at the end of the cultivation. The decrease of the synthesis rate and the 1st order kinetics of proteolysis lead to an equilibrium between synthesis and degradation of SpA from 2 h after induction. This resulted in no further SpA accumulation in cells, though synthesis continued for at least 10 h. Similar experiments with recombinant protein ZZT2 also revealed that most of the synthesized product was degraded. The order of proteolysis kinetics depended on the concentration of the recombinant protein: at low concentrations both SpA and ZZT2 were degraded according to first order kinetics, while at high concentrations ZZT2 was degraded according to zero order kinetics. In a protease Clp mutant the degradation rate decreased and intracellular concentration of ZZT2 increased from 50 mg · g⁻¹ to 120 mg · g⁻¹. The measurements of proteolysis rate throughout the cultivation enabled calculation of a hypothetical accumulation of the product assuming complete stabilization. In this case the concentration would have increased from 50 to 280 mg · g⁻¹ in 11 h. Thus, this method reveals the potential to increase the productivity by eliminating proteolysis.     

Caenorhabditis elegans SAND-1 is essential for RAB-7 function in endosomal traffic

The small rab-GTPase RAB-7 acts in endosome and endosome to lysosome traffic. We identified SAND-1 as a protein required for RAB-7 function based on similarities between SAND-1 and RAB-7 RNAi phenotypes. Although the initial uptake of yolk protein in oocytes, or of soluble secreted (ss) GFP in coelomocytes, appeared normal, further transport along the endocytic traffic route was delayed in the absence of SAND-1 function, and yolk proteins failed to reach yolk granules efficiently. Moreover, in coelomocytes, ssGFP and BSA-Texas-Red were endocytosed but not transported to lysosomes. We show that SAND-1 is essential for RAB-7 function at the transition from early to late endosomes, but not for RAB-7 function at lysosomes.