Cleavage of human immunoglobulins by proteinase from Staphylococcus aureus.
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Bacterial Proteins↗
Biomedical subjects
J Zikán
Publications and source records attributed to J Zikán.
Cleavage of human immunoglobulins by serine proteinase from Staphylococcus aureus.
The serine proteinase (SP) released into the environment by most strains of S. aureus cleaves human IgG, IgM and IgA of both subclasses--IgA 1 and IgA 2. SP cleaves H chains of all immunoglobulin classes and the SC of S-IgA, the L chains are degraded partially. The SP-induced cleavage results in a large spectrum of fragments under reducing conditions within a broad range of Mr (approx. 41,000 to less than 12,400). This indicates that the enzyme does not affect the Ig molecule in the hinge region only. The degree of cleavage depends on the enzyme:substrate ratio and on the duration of incubation. The generation of small fragments is associated with the loss of antigenic determinants that results from the decreased binding of the cleaved material in the ELISA method. Partial cleavage of L chains suggests that the enzyme alters part of the molecule that is involved in antigen binding. Even if the ability of antigen binding remains preserved after cleaving Ig with SP, the antibody function is disturbed by splitting off the Fc region or by its degradation into small fragments. SP has to be considered as one of the virulence factors of S. aureus that may protect bacteria against the defence mechanisms of the host.
Electrophoresis, Polyacrylamide Gel↗
Role of saccharides in immunoglobulin--Fc receptor interactions.
The rosettes formed by mouse peritoneal macrophages or DCH-5 cells and TNP-erythrocytes coated with anti-TNP antibodies of different isotypes were inhibited to various extent by monosaccharides. The most effective inhibitors were N-acetylglucosamine, glucosamine, mannose and N-acetylneuraminic acid in 1-5 mmol/L concentrations. Even more efficient were glycopeptides isolated from IgG molecules. The Fc receptors (FcRs) released from DCH-5 cells during cultivation and gradually separated by affinity chromatography on immobilized IgG reacted with aggregated IgG and inhibited the rosette formation. The FcRs eluted by monosaccharides influenced mainly the number of rosettes mediated by IgA and IgE while those eluted with a glycine-HCl buffer inhibited preferentially IgG rosettes. As shown by SDS-PAGE the heterogeneity of the fraction eluted with a mixture of monosaccharides revealed one main component with an effective molar mass of 50 kg/mol. The glycine-HCl eluate contained two major components of 55 and 38 kg/mol. The IgG-Sepharose 4B bound all the fractions but only the binding of the 50 kg/mol molecule could be inhibited by monosaccharides.
Animals↗
The interaction of a protein from the coelomic fluid of earthworms with staphylococcal protein A.
A Staphylococcal protein A (SpA)-binding protein was isolated from coelomic fluids of the annelids, Lumbricus terrestris (LT) and Eisenia foetida (EF), by affinity chromatography on SpA-Sepharose. Analyses by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) under reducing and nonreducing conditions showed that SpA-binding activity is associated with a single chain protein, with a mol wt of 62 kD. The carbohydrate moiety of this protein consists of 21.4 g/100 g of O-linked and N-linked oligosaccharides. The amino acid composition of SpA-binding protein did not show structural homology with that of human IgG1 heavy chain (Daw), which also binds SpA.
Amino Acids↗
[The effect of increased production of androgens on the development of body build morphology. Case report of an androgen-active tumor in a monozygotic twin].
The authors present an account on monozygotic twins where in one girl an androgen-active adenoma of the adrenal cortex during early puberty led to the development of the Morphogram of Body Build (Case-report of throughout the period of investigation intact from the hormonal aspect. Four years after resection of the tumour endocrine and anthropometric characteristics of the two sisters were evaluated: the hormonal status of both is normal, the somatic differences are manifested by a different final height, biacromial width and chest circumference. The results are conceived as a precedent for evaluation of risks associated with administration of anabolic steroids in children.
Adenoma↗
Heterogeneity of binding of monoclonal IgA to staphylococcal protein A is related to the IgA polymerization state.
The human IgA2-lambda myeloma protein Fel consists of covalent dimers and monomers which are partially self-associated. Affinity chromatography of this protein on staphylococcal protein A-Sepharose revealed that approximately 8% of the protein was retained and eluted by acid buffer. Although retained protein Fel was highly aggregated, in the presence of dissociating agents mostly monomeric form was found. Affinity rechromatography and electrophoresis of both fractions from affinity chromatography revealed that the retained fraction possessed substantially higher affinity for SpA than did the nonretained one. This could be due to the multivalency of protein Fel aggregates.
Antibodies, Monoclonal↗
Protein aggregates in extracorporally perfused rabbit kidneys.
Eighteen rabbit kidneys were perfused ex vivo for 1 h with allogeneic blood, and in 16 a solution of xenogeneic aggregate-free, aggregated or antibody-complexed protein was added to the perfusate 5 min after the start (human immunoglobulins or serum albumin, partly cationized, were used). The kidneys were examined by light and electron microscopy and the human and rabbit immunoglobulin (or albumin) precipitates were detected by direct immunofluorescence and ultrastructural immunohistochemistry. In 13 kidneys the perfusion produced small segmental glomerular endocapillary aggregates of platelets, leukocytes, and granular precipitates reactive with both anti-rabbit and anti-human antibodies. No typical deposits were seen in mesangium or in periphery of glomerular capillaries but rabbit Ig penetrated to the inter- and subepithelial spaces of proximal convoluted tubules. Three kidneys perfused by cationized aggregated human Ig (or by cationized albumin-antialbumin complexes) exhibited a destructive lesion with rapid breakdown of blood flow and massive global endocapillary plugs of similar ultrastructure but with focal endothelial sloughing. Pericapillary granular precipitates of human and rabbit Ig were seen in these kidneys. When the blood with cationized Ig aggregates was used for perfusion of two further kidneys extensive endocapillary aggregates with endothelial damage reappeared but the extracapillary penetration and precipitation were lacking and the blood flow largely improved. Membrane polyanion of podocytes stained by colloidal iron was preserved even in close proximity of cationized complex precipitates. Thus, in the ex-vivo perfusion model the preformed neutral aggregates did not penetrate through the glomerular capillary wall and were not phagocytized by mesangial cells. The cationized aggregates induced rapid circulatory failure with massive platelet clumping and granular pericapillary "humps" ultrastructurally different from the deposits of human and experimental immune complex glomerulonephritis.
Animals↗
Interleukin 1-like factor produced by a hybrid of an adherent mouse spleen cell and a thymoma cell.
A hybrid cell line (DCH-5) constructed from an adherent cell of mouse spleen and the thymoma BW5147, and selected for adherence to hydrophobic surfaces, secretes a factor which augments the mitogenic response of thymocytes. Properties of the factor were compared with those of a P388D1 and J774.1 macrophage-derived interleukin 1 (IL-1). On polyacrylamide gel electrophoresis in Tris-glycinate buffer the IL-1-like factor of DCH-5 cells was heterogeneous and its components were more negatively charged than components of macrophage-derived IL-1. Charge differences between these factors were also confirmed by isoelectric focusing (IEF) (pI of IL-1 was 5.4, pI of the IL-1-like factor was 3.5). SDS-PAGE and gel filtration demonstrated that both factors consisted of several components of near molar masses (approximately 17 kg/mol). Gel filtration showed that in PBS the IL-1-like factor of DCH-5 cells was partially aggregated. Antibodies specific to IL-1 inhibited the activity of the IL-1-like factor of DCH-5 cells. Thus, mouse DCH-5 cells provide a new source of IL-1-like factor which might be useful for further elucidation of the heterogeneity of interleukins.
Animals↗
Antigenic properties of T cell antigen-specific receptors isolated from the surface of rabbit and mouse spleen and lymph node cells.
The presence of a allotypic determinants was tested in fractions obtained by gel filtration of antigen-specific receptors isolated by immunoadsorption from lymphoid cells of antigen-stimulated a3-3 rabbits. This technique, as well as the inhibition of the reaction of isolated receptors with anti-T cell receptor antisera (anti R) by anti-a3 antibodies failed to demonstrate the presence of a allotypic determinants. The inhibitory effect of antigen-specific receptors isolated from the lymphoid cells of stimulated A/J mice on the cytotoxic effect of anti-Ia antibodies on mouse spleen cells in the presence of rabbit complement was tested. All preparations inhibited the cytotoxic reaction with the average effectivity of 60%. In order to confirm the presence of Ia determinants on the rabbit and mouse T cell receptor molecules it was shown that the reactions of three anti-R antisera with 12 different receptor preparations were inhibited by anti-Ia antibodies. SDS-PAGE analyses of 125I-labelled mouse specific receptors and the precipitate obtained by anti-R antisera showed that T cell receptors were present in fractions with molar mass 100 and 85 kg/mol. The molar mass of the former fraction after reduction and alkylation was 45 kg/mol.
Animals↗
Ontogeny of J chain expression in pig lymphocytes.
The ontogeny of lymphocytes expressing J chain in the cytoplasm (J+) was studied in pig foetuses by the immunofluorescent technique. Peripheral blood lymphocytes were the first J+ cells in prenatal life. The spleen and lymph nodes contained J+ cells in the last days of gestation. J+ cells were found in the lamina propria of the gut and some glands of conventional but not of germ-free piglets. J chain was not detected on or in cell membranes at any developmental stage.
Animals↗
The gamma 1 heavy-chain disease FOR protein is present in two molecular forms.
Heavy chain disease proteins (FOR) were isolated from human plasma. These proteins were also detected immunochemically in the urine of the patient. The proteins were disulphide-linked Fc-like dimers with molar mass 64.2 kg/mol and sedimentation rate S020,W = 0.356 ps (3.56 S). Similar amounts of aspartic acid and pyroglutamic acid were found at the N-terminus. After cyanogen bromide cleavage of the FOR proteins, three peptides were isolated and their amino acid composition and partial amino acid sequence was determined. We suggest that two Fc-like proteins of similar sizes are present in the plasma: (1) the first with N-terminal aspartic acid corresponding to position 221 of gamma 1 EU chain and (2) the second with N-terminal pyroglutamic acid. The first protein and small amounts of related low-molar mass fragments found also in the plasma could be degradation products of the second protein. Evidence is given on structural differences between the FOR proteins and the corresponding portion of the gamma 1 EU chain.
Amino Acid Sequence↗
Heterogeneity of human polyclonal IgE reacting with staphylococcal protein A.
A small part of polyclonal IgE (6%) was bound to protein A-Sepharose from the serum of M.P., containing a high concentration of IgE. No monoclonal IgE isolated from the serum of V.L. was bound to this sorbent. This binding of polyclonal IgE appears to be heterogeneous since a multiphasic pattern was observed with discontinuous pH gradient elution from protein A-Sepharose. Also, like IgE from the whole serum, monomeric IgE isolated from the serum of M.P. on Sepharose 6B showed this binding heterogeneity. It is suggested that IgE molecules with different affinities for protein A could belong to different isotypic or allotypic variants.
Binding Sites, Antibody↗
Cross-reactivity of human and a putative pig IgD. Pig IgD-like molecules as serum and lymphocyte components.
Proteins of pig serum and splenocytes which were isolated on immobilized guinea pig antibodies against human IgD were characterized by SDS-PAGE. In both cases the main isolated components possessed several properties nearly identical with those of human IgD: molar mass of the components and their chains, and susceptibility of the heavy chains, light chains, the chymotryptic peptide maps of the chains, and susceptibility of the heavy chains, the chymotryptic peptide maps of the chains, and susceptibility of the heavy chains in the native molecule to trypsin digestion. These IgD-like components not adsorbed on immobilized normal guinea-pig IgG. We suggest that the components of pig serum and splenocytes cross-reacting with highly specific antibodies against human IgD represent candidates for pig IgD. The percentage of the IgD-like molecules on the surface of pig splenocytes labeled with the antibody against human IgD was similar to the percentage of splenocytes bearing all immunoglobulins. Therefore, we suggest that IgD-like molecules were occurring on the surface of cells mostly together with molecules of another immunoglobulin class.
Animals↗
The appearance of an IgD-like molecule on pig lymphocytes during ontogeny.
An IgD-like molecule was found on the surface of pig fetal lymphocytes using guinea pig anti-human IgD antisera and indirect immunofluorescence. The binding of antiIgD antisera could be specifically inhibited with human IgD. Surface IgD-like molecules were found later in ontogeny than surface IgM (after about 70 d of gestation in the spleen). There is evidence for their endogenous origin. The number of lymphocytes bearing IgD-like molecules rapidly increased during ontogeny.
Animals↗
Influence of proteolysis inhibitors and immunoadsorption technique on the composition of rabbit antigen-specific receptor preparations. Presence of Fc receptors.
Addition of proteolysis inhibitors during the isolation procedure of rabbit arsanylated bovine IgG specific receptors decreased significantly the amount of low-molar-mass proteins in all receptor preparations. Rabbit ARS-BGG-specific receptor preparations isolated by immunoadsorption technique contain molecules which do not react with antigen and antibodies against immunoglobulins and have identical molar mass and chymotryptic peptide composition as those of isolated Fc receptors. It is suggested that during isolation of antigen-specific receptors from the surface of lymphoid cells, Fc receptors react with complexes composed of antigen and Ig+ receptors on the surface of immunoadsorbent and are isolated together with antigen-specific receptors.
Animals↗
Production of monoclonal antibodies with haemagglutination-inhibition activity to the Skalica strain from the tick-borne encephalitis complex.
Hybridomas secreting monoclonal antibodies with haemagglutination-inhibition (HI) activity to the Skalica strain of tick-borne encephalitis (TBE) complex were prepared by the fusion of P3-NS1-Ag4-1 myeloma cell line with spleen cells of BALB/c mice immunized with the purified Skalica strain. The highest titres of monoclonal antibodies obtained from the hybridomas S-9, S-15 and S-16 ranged from 512 to 10,240, respectively; the ascitic fluid contained as many as 4.6 mg/ml of monoclonal antibodies. Its analysis by Ouchterlony's double immunodiffusion, agarose electrophoresis, and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of monoclonal antibodies with mu isotype of the heavy and kappa isotype of the light chain. The specificity of the monoclonal antibodies was proved using 11 different antigens from family Togaviridae in the HI test.
Animals↗
Characterization of antigen-specific receptors from pig lymphocytes.
Antigen-specific receptors were isolated from the surface of spleen and lymph node cells of ARS-BGG-stimulated miniature pigs using an immunoadsorption technique. The yields of the receptors can be increased by 20-hr cultivation of the cells protein-free medium prior to the isolation procedure. In this case antigen-specific molecules can be isolated not only from the surface of the cells but also from the culture medium. 125I-labelled isolated receptors were separated by SDS-PAGE into six fractions. The first corresponded to aggregated molecules and IgG, the mol. wts of the other fractions were 56,000, 35,000, 26,000 and 10,000. It has been shown that they all were able to bind antigen but only the first two reacted with anti-Ig antibodies and Staphylococcus aureus protein A-Sepharose 4B. Gel filtration of the same sample of 125I receptors on Sephacryl S-200 revealed the presence of four peaks. Three of them were able to bind antigen as detected by radioimmunoassay. Fractions of all three peaks reacted also with anti-Id and anti-R (anti-Ig- receptors) antisera, but only fractions of the first two peaks reacted with anti-Ig antisera. The finding that Ig- receptors and more heterogeneous than Ig+ receptors is discussed. No reaction of isolated 125I receptors with anti-SLA and anti-SLB antisera used was detected in the solid-phase radioimmunoassay.
Animals↗
Monoclonal IgG3 (kappa) antibodies against murine Thy-1.2 antigen produced by murine hybridomas. Differences in the specificity of the antigen binding site and in the structure of the hinge region.
Two monoclonal IgG3 (kappa) antibodies against murine Thy-1.2 antigen produced by murine 1B5 and 1aG4/C5 hybridomas were partially characterized. The 1aG4/C5 antibody has slightly higher affinity for the Thy-1.2 antigen in binding tests and more efficiently kills the Thy-1.2+ thymocytes in cytotoxicity assays as compared to the 1B5 antibody. The latter, in addition, reacts significantly with the Thy-1.1 antigen (the allelic form of Thy-1 antigen expressed on the cells of the donor of the immune cells. Both monoclonal antibodies exhibit some characteristic properties of IgG3 of myeloma origin, e.g. a tendency to aggregation, high pI and interaction with protein A. Our monoclonal antibodies are sensitive to pepsin digestion, resistant to trypsin, their disulphide bonds are rapidly cleaved by sulphitolysis and reduction by dithiothreitol. They possess characteristic acidic peptides bearing the disulphide bonds between the heavy chains. These antibodies, however, differ to some extent from each other in some properties (precipitation with staphylococcal protein A, solubility, pI, electrophoretic behaviour of the light chains). They possess different heavy chain peptides bearing the interchain disulphide bonds and thus they probably differ in the hinge region. This structural difference may be associated with different sensitivity of these two antibodies to sulphitolysis and proteolysis.
Amino Acids↗