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U Sjöbring

Publications and source records attributed to U Sjöbring.

At least 37 records · Page 2Linked to original sources
PubMed1995

Mapping of the immunoglobulin light chain-binding site of protein L.

Protein L is a cell surface protein expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus. The molecule binds specifically and with high affinity to immunoglobulins (Ig) of a wide range of animal species. The Ig-binding activity is mediated through five highly homologous domains, each 72 to 76 amino acid residues long, which interact with framework regions in the variable domain of Ig light chains. The interaction does not interfere with the antigen binding capacity of the antibody. The fold of the Ig light chain-binding domains of Protein L is comprised of an alpha-helix packed against a four stranded beta-sheet and is similar to the fold of the IgG heavy chain-binding domains of streptococcal protein G, despite the fact that the two proteins show no significant sequence homology. In the present work, heteronuclear NMR spectroscopy has been utilized to define the interaction between the N-terminal Ig-binding domain of Protein L and the variable domain of a human Ig kappa light chain. The Ig-binding region of the Protein L domain involves most of the residues in the second beta-strand, the C-terminal residues of the alpha-helix and the loop connecting the alpha-helix with the third beta-strand. The Ig light chain-binding surface of Protein L thus resembles the surface of Protein G which binds to the C gamma 1 domain of IgG, but is different from the portion of Protein G involved in the contact with the C gamma 2-C gamma 3 interface region. The data suggest that the global fold shared by the Ig-binding domains of Proteins L and G provide bacteria with a flexible template for the evolution of surface structures capable of interacting with different conserved parts of Ig molecules of the infected host.

Bacterial Proteins↗
PubMed1995

Diagnosis of tuberculous meningitis: a comparative analysis of 3 immunoassays, an immune complex assay and the polymerase chain reaction.

OBJECTIVE: To compare 3 immunoassays, an immune complex assay, and an application of the polymerase chain reaction (PCR) for the diagnosis of tuberculous meningitis (TBM). MATERIAL: Cerebrospinal fluid (CSF) from 33 patients with TBM and from 34 control patients with infectious and non-infectious CNS diseases was analysed. RESULTS: The antibody immunoassays were either nonspecific or insensitive. However, detection of mycobacterial IgG immune complexes correlated strongly with infection, as they were detected in the CSF from 64% of the patients with TBM compared to only 3 (9%) of the control samples. PCR analysis, using Mycobacterium tuberculosis-specific oligonucleotide primers, also strongly correlated with infection, as DNA was amplified from 54% of the samples from patients with TBM, but from only 2 (6%) of the control samples. Both 'false positive' samples were also positive in the immune complex assay and came from 2 patients with otogenic brain abscesses. It is conceivable that these patients suffered from otogenic tuberculosis with secondary non-mycobacterial meningitis. When combining the immune complex assay with DNA-detection by PCR, 100% of the culture positive and 74% of culture negative samples were found to be positive, while maintaining a high specificity. CONCLUSION: Parallel analysis to detect mycobacterial immune complexes and M. tuberculosis-specific DNA by PCR from the CSF of patients may offer a sensitive and specific tool for the diagnosis of TBM.

Adolescent↗
PubMed1994

Three-dimensional solution structure of an immunoglobulin light chain-binding domain of protein L. Comparison with the IgG-binding domains of protein G.

Protein L is a multidomain protein expressed at the surface of some strains of the anaerobic bacterial species Peptostreptococcus magnus. It has affinity for immunoglobulin (Ig) through interaction with framework structures in the variable Ig light chain domain. The Ig-binding activity is located to five homologous repeats called B1-B5 in the N-terminal part of the protein. We have determined the three-dimensional solution structure of the 76 amino acid residue long B1 domain using NMR spectroscopy and distance geometry-restrained simulated annealing. The domain is composed of a 15 amino acid residue long disordered N-terminus followed by a folded portion comprising an alpha-helix packed against a four-stranded beta-sheet. These secondary structural elements are well determined with a backbone atomic root mean square deviation from their mean of 0.54 A. The B domains of protein L show very limited sequence homology to the domains of streptococcal protein G interacting with the heavy chains of IgG. However, despite this fact, and their different binding properties, the fold of the B1 domain was found to be similar to the fold of the IgG-binding protein G domains [Wikström, M., Sjöbring, U., Kastern, W., Björck, L., Drakenberg, T., & Forsén, S. (1993) Biochemistry 32, 3381-3386]. In the present study, the solution structure of the B1 domain enabled a more detailed comparison which can explain the different Ig-binding specificities of these two bacterial surface proteins. Among the differences observed, the alpha-helix orientation is the most striking.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Streptokinase activates plasminogen bound to human group C and G streptococci through M-like proteins.

An ability to interact with plasminogen or plasmin could provide micro-organisms with a mechanism for invasion. Thus, group A, C and G streptococci secrete streptokinase which binds and activates plasminogen. Some streptococci also express surface structures which bind plasminogen without causing its activation. Plasminogen-binding surface proteins were extracted from one group C and one group G streptococcal isolate. Both proteins were found to bind plasmin, fibrinogen and serum albumin in addition to plasminogen. Gene fragments encoding the streptococcal proteins were amplified by PCR and were subsequently cloned and expressed in Escherichia coli. DNA sequence determination revealed for both genes open reading frames encoding proteins which contained repetitive domains and a carboxyl-terminal unrepeated region that were typical of M and M-like proteins. Though the amino-terminal regions of the group C and G streptococcal proteins demonstrated a rather high overall similarity between themselves, they were not similar to the variable regions of other M-like proteins with one exception: there was a 46% identity between the first 22 amino acids of the group G streptococcal protein and the corresponding sequence of PAM, the plasminogen-binding M-like protein of type M53 group A streptococci. Like the proteins extracted from the streptococci, the recombinant proteins bound plasminogen, fibrinogen and albumin. The three plasma proteins bound to separate sites on the streptococcal M-like proteins. Plasminogen bound by the group C and G streptococcal proteins was readily activated by streptokinase, providing evidence for a functional link between the secreted plasminogen-activator and proteins exposed on the bacterial surface.

Amino Acid Sequence↗
PubMed1994

Protein H--a surface protein of Streptococcus pyogenes with separate binding sites for IgG and albumin.

Protein H, a molecule expressed at the surface of some strains of Streptococcus pyogenes, has affinity for the constant (IgGFc) region of immunoglobulin (Ig) G. In absorption experiments with human plasma, protein H-sepharose could absorb not only IgG but also albumin from plasma. The affinity constant for the reaction between albumin and protein H was 7.8 x 10(9) M-1, which is higher than the affinity between IgG and protein H (Ka = 1.6 x 10(9) M-1). Fragments of protein H were generated with deletion plasmids and polymerase chain reaction (PCR) technology. Using these fragments in various protein-protein interaction assays, the binding of albumin was mapped to three repeats (C1-C3) in the C-terminal half of protein H. On the albumin molecule, the binding site for protein H was found to overlap the site for protein G, another albumin- and IgGFc-binding bacterial surface protein. Also IgGFc-binding could be mapped with the protein H fragments and the region was found N-terminally of the C repeats. A synthetic peptide (25 amino acid residues long) based on a sequence in this region was shown to inhibit the binding of protein H to immobilized IgG or IgGFc. This sequence was not found in previously described IgGFc-binding proteins. However, two other cell surface proteins of S. pyogenes exhibited highly homologous regions. The results identify IgGFc- and albumin-binding regions of protein H and further define and emphasize the convergent evolution among bacterial surface proteins interacting with human plasma proteins.

Amino Acid Sequence↗
PubMed1994

Plasminogen, absorbed by Escherichia coli expressing curli or by Salmonella enteritidis expressing thin aggregative fimbriae, can be activated by simultaneously captured tissue-type plasminogen activator (t-PA).

Curli are fimbrial structures expressed by Escherichia coli that specifically interact with matrix proteins such as fibronectin and laminin. Similar structures are also expressed by Salmonella enteritidis and have been denoted thin aggregative fimbriae. Bacteria expressing curli and thin aggregative fimbriae were found to bind radiolabelled plasminogen as well as the tissue-type plasminogen activator (t-PA). By contrast, E. coli carrying a gene locus with an insertionally inactivated chromosomal curlin subunit were unable to bind the two human proteins. The purified subunit polypeptides of curli and thin aggregative fimbriae bound plasminogen and t-PA with high affinity (1 x 10(8) to 2 x 10(8) M-1). The binding of plasminogen and t-PA to curli-expressing E. coli was only partially inhibited by fibronectin and laminin. Plasminogen absorbed from human plasma by curli-expressing E. coli was readily converted to plasmin by t-PA; both plasmin and t-PA were functionally active when bound to the bacteria. A simultaneous binding of fibrinolytic proteins and matrix proteins to fimbriae of E. coli and S. enteritidis could provide these pathogens with both adhesive and invasive properties.

Adsorption↗
PubMed1993

PAM, a novel plasminogen-binding protein from Streptococcus pyogenes.

The ability of group A streptococci to bind human plasminogen and plasmin has attracted interest, because it could provide the bacteria with a mechanism for invasion. M or M-like proteins account for the binding of several plasma proteins to group A streptococci. To investigate whether M or M-like proteins were responsible for the binding of plasminogen to group A streptococci, acid-extracted material from a type M53 streptococcal isolate was tested for its ability to bind plasminogen. Indeed, a 42-kDa plasminogen-binding protein was solubilized. Two oligonucleotides homologous with conserved sequences in known M protein genes were used as primers in the polymerase chain reaction, with chromosomal DNA from the M53 isolate. When cloned and expressed in Escherichia coli, a resulting fragment encoded a 43-kDa plasminogen-binding protein. Nucleotide sequence determination of the gene fragment revealed an open reading frame encoding a polypeptide of 43,580 Da, which matched the amino-terminal amino acid sequence of the plasminogen-binding protein extracted from M53 streptococci. The DNA sequence data also proved the relationship of the encoded protein, named PAM, to the M proteins. The plasminogen-binding domain was mapped to the amino-terminal third of PAM. Plasminogen absorbed by M53 streptococci or by immobilized PAM could be activated by streptokinase. The results provide further evidence of the diversity of the M protein family and suggest a new mechanism whereby these proteins contribute to the virulence of group A streptococci.

Amino Acid Sequence↗
PubMed1993

Proton nuclear magnetic resonance sequential assignments and secondary structure of an immunoglobulin light chain-binding domain of protein L.

The 1H NMR assignments have been made for the immunoglobulin (Ig) light chain-binding B1 domain of protein L from Peptostreptococcus magnus. The secondary structure elements and the global folding pattern were determined from nuclear Overhauser effects, backbone coupling constants, and slowly exchanging amide protons. The B1 domain was found to be folded into a globular unit of 61 amino acid residues, preceded by a 15 amino acid long disordered N-terminus. The folded portion of the molecule contains a four-stranded beta-sheet spanned by a central alpha-helix. The fold is similar to the IgG-binding domains of streptococcal protein G, despite the fact that the binding sites on immunoglobulins for the two proteins are different; protein G binds IgG through the constant (Fc) part of the heavy chain, whereas protein L has affinity for the variable domain of Ig light chains.

Amino Acid Sequence↗
PubMed1992

Protein LG: a hybrid molecule with unique immunoglobulin binding properties.

Immunoglobulin (Ig)-binding bacterial proteins have attracted theoretical interest for their role in molecular host-parasite interactions, and they are widely used as tools in immunology, biochemistry, medicine, and biotechnology. Protein L of the anaerobic bacterial species Peptostreptococcus magnus binds Ig light chains, whereas streptococcal protein G has affinity for the constant (Fc) region of IgG. In this report, Ig binding parts of protein L and protein G were combined to form a hybrid molecule, protein LG, which was found to bind a large majority of intact human Igs as well as Fc and Fab fragments, and Ig light chains. Binding to Ig was specific, and the affinity constants of the reactions between protein LG and human IgG, IgGFc fragments, and kappa light chains, determined by Scatchard plots, were 5.9 x 10(9), 2.2 x 10(9), and 2.0 x 10(9) M-1, respectively. The binding properties of protein LG were more complete as compared with previously described Ig-binding proteins when also tested against mouse and rat Igs. This hybrid protein thus represents a powerful tool for the binding, detection, and purification of antibodies and antibody fragments.

Amino Acid Sequence↗
PubMed1992

Convergent evolution among immunoglobulin G-binding bacterial proteins.

Protein G, a bacterial cell-wall protein with high affinity for the constant region of IgG (IgGFc) antibodies, contains homologous repeats responsible for the interaction with IgGFc. A synthetic peptide corresponding to an 11-amino acid-long sequence in the COOH-terminal region of the repeats was found to bind to IgGFc and block the interaction with protein G. Moreover, two other IgGFc-binding bacterial proteins (proteins A and H), which do not contain any sequences homologous to the peptide, were also inhibited in their interactions with IgGFc by the peptide. Finally, a decapeptide based on a sequence in IgGFc blocked the binding of all three proteins to IgGFc. This unusually clear example of convergent evolution emphasizes the complexity of protein-protein interactions and suggests that bacterial surface-protein interaction with host protein adds selective advantages to the microorganism.

Amino Acid Sequence↗
PubMed1992

Structure of peptostreptococcal protein L and identification of a repeated immunoglobulin light chain-binding domain.

The gene for protein L, an immunoglobulin (Ig) light chain-binding protein expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus, was cloned and sequenced. The gene translates into a protein of 719 amino acid residues. Following a signal sequence of 18 amino acids and a NH2-terminal region ("A") of 79 residues, the molecule contains five homologous "B" repeats of 72-76 amino acids each. Further, toward the COOH terminus, two additional repeats ("C") were found. These are not related to the "B" repeats, but are highly homologous to each other. After the C repeats (52 amino acids each), a hydrophilic, proline-rich putative cell wall-spanning region ("W") was found, followed at the COOH-terminal end by a hydrophobic membrane anchor ("M"). Fragments of the gene were expressed, and the corresponding peptides were analyzed for Ig-binding activity. The B repeats were found to be responsible for the interaction with Ig light chains. An Escherichia coli high level expression system was adapted for the production of large amounts of two Ig-binding protein L fragments comprising one and four B repeats, respectively.

Amino Acid Sequence↗
PubMed1992

Evaluation of polymerase chain reaction, tuberculostearic acid analysis, and direct microscopy for the detection of Mycobacterium tuberculosis in sputum.

Tuberculosis remains a major global cause of morbidity and mortality. There is an urgent need for improved bacteriologic diagnosis of Mycobacterium tuberculosis infection. Three methods for rapid identification of M. tuberculosis in sputum samples (direct microscopy, gas chromatography-mass spectrometry [GC-MS], and polymerase chain reaction [PCR]), were compared with culture on Lowenstein-Jensen medium. Growth of M. tuberculosis was observed in 38 of 145 sputum samples. Detection of acid-fast bacilli by direct microscopy gave a sensitivity of 66% and a specificity of 100%. Detection of tuberculostearic acid by GC-MS gave a sensitivity of 55% and a specificity of 87%. Amplification by PCR of a fragment of the insertion sequence IS6110 gave a sensitivity of 95% and a specificity of 93% compared with culture and a corrected specificity of 99% compared with both culture and clinical data. This study indicates that PCR can be adapted for clinical use and is the method of choice for rapid diagnosis of pulmonary tuberculosis.

Base Sequence↗
PubMed1992

Isolation and molecular characterization of a novel albumin-binding protein from group G streptococci.

Many streptococcal strains are known to bind the two most abundant plasma proteins, namely, immunoglobulin G and albumin. Protein G isolated from group C and G streptococci has been demonstrated to have separate binding regions for each of these proteins. However, some group G streptococcal strains bind only serum albumin. This report describes the isolation of a 48-kDa albumin-binding protein from such a strain (DG12). The affinity constant of this protein for human serum albumin was determined to be 5 x 10(9) M-1, and the protein interacted strongly also with serum albumin from several other mammalian species. The gene encoding the albumin-binding protein was cloned and expressed in Escherichia coli. DNA sequence analysis of this gene revealed a unique NH2-terminal sequence and three types of repeats in the encoded protein. One of these repeated sequences has significant homology with the albumin-binding domains of protein G, and it was demonstrated that the albumin binding of the DG12 protein was localized within these domains. Another type of repeat is localized in the putative wall-spanning region of the molecule. This repeat sequence, which has the length of only 4 amino acids (LysProGluVal), is repeated 14 times. The relationship of the albumin-binding protein to other cell-wall-associated proteins of pathogenic streptococci is discussed.

Amino Acid Sequence↗
PubMed1991

Streptococcal protein G. Gene structure and protein binding properties.

Protein G was solubilized from 31 human group C and G streptococcal strains with the muralytic enzyme mutanolysin. As judged by the mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the binding patterns of the solubilized protein G molecules in Western blot experiments, the strains could be divided into three groups, represented by the group G streptococcal strains G148 and G43 and the group C streptococcal strain C40. The 65-kDa G148 protein G and the 58-kDa C40 protein G showed affinity for both immunoglobulin G (IgG) and human serum albumin (HSA), whereas the 40-kDa G43 protein G bound only IgG. Despite the different molecular patterns, the three protein G species had identical NH2-terminal amino acid sequences. Apart from the 65-kDa peptide, digestion of G148 streptococci with mutanolysin also produced a 52-kDa IgG- and HSA-binding peptide and a 14-kDa HSA-binding peptide. It was demonstrated that these peptides resulted from cleavage of 65-kDa protein G by proteolytic components in the mutanolysin preparation. The protein G genes of the C40 and G43 strains were cloned and sequenced, and their structure was compared to the previously published sequence of the G148 protein G gene. As compared to G148, both the C40 and G43 genes lacked a 210-base pair fragment in the IgG-binding region, accounting for the 10-fold lower affinity of these proteins for IgG. The G43 gene also lacked a 450-base pair fragment in the 5'-end of the gene, explaining why the G43 protein G did not bind HSA. The differences in protein G structure did not correlate with the clinical origin of the strains used in this study. The IgG-binding region of protein G was further mapped. Thus, a peptide corresponding to a single IgG-binding unit was obtained by the cloning and expression of a 303-base pair polymerase chain reaction-generated DNA fragment. The affinity of this 11.5-kDa peptide for human IgG was 8.0 x 10(7) M-1, as determined by Scatchard plots. Finally, a 55-amino acid-long synthetic peptide, corresponding to one of the three repeated domains in the COOH-terminal half of strain G148 protein G, effectively blocked binding of protein G to IgG.

Amino Acid Sequence↗
PubMed1990

Polymerase chain reaction for detection of Mycobacterium tuberculosis.

A polymerase chain reaction for the specific detection of mycobacteria belonging to the Mycobacterium tuberculosis complex was developed. Using a single primer pair derived from the nucleotide sequence of protein antigen b of M. tuberculosis, we achieved specific amplification of a 419-base-pair DNA fragment in M. tuberculosis and M. bovis. After DNA was extracted from mycobacteria by using a simple, safe lysis procedure, we detected the 419-base-pair sequence in samples containing few mycobacteria. Preliminary data suggested that this technique could be applied to clinical specimens for early and specific diagnosis of tuberculosis.

Base Sequence↗
PubMed1989

Ig-binding bacterial proteins also bind proteinase inhibitors.

Protein G is a streptococcal cell wall protein with separate binding sites for IgG and human serum albumin (HSA). In the present work it was demonstrated that alpha 2-macroglobulin (alpha 2M) and kininogen, two proteinase inhibitors of human plasma, bound to protein G, whereas 23 other human proteins showed no affinity. alpha 2M was found to interact with the IgG-binding domains of protein G, and in excess alpha 2M inhibited IgG binding and vice versa. A synthetic peptide, corresponding to one of the homologous IgG-binding domains of protein G, blocked binding of protein G to alpha 2M. Protein G showed affinity for both native and proteinase complexed alpha 2M but did not bind to the reduced form of alpha 2M, or to the C-terminal domain of the protein known to interact with alpha 2M receptors on macrophages. Binding of protein G to alpha 2M and kininogen did not interfere with their inhibitory activity on proteinases, and the interaction between protein G and the two proteinase inhibitors was not due to proteolytic activity of protein G. The finding that protein G has affinity for proteinase inhibitors was generalized to comprise also other Ig binding bacterial proteins. Thus, alpha 2M and kininogen, were shown to bind both protein A of Staphylococcus aureus and protein L of Peptococcus magnus. The results described above suggest that Ig-binding proteins are involved in proteolytic events, which adds a new and perhaps functional aspect to these molecules.

Bacterial Proteins↗
PubMed1989

The Fc binding site for streptococcal protein G is in the C gamma 2-C gamma 3 interface region of IgG and is related to the sites that bind staphylococcal protein A and human rheumatoid factors.

The isolated 35 kDa fragment of protein G obtained by papain digestion of group G streptococci was found to bind solid phase intact IgG, Fc (2C gamma 2 + 2C gamma 3 domains), F(ab')2 and F(acb)2 (F(ab')2 + 2C gamma 2 domains) fragments but not pFc' (2C gamma 3 domains) fragments. The level of binding to rabbit F(acb)2 and rabbit F(ab')2 fragments was similar. Protein G binding to solid phase Fc fragments was inhibited by IgG, Fc, staphylococcal protein A and its monovalent fragment D, but was enhanced by F(ab')2 fragments. Chemical modification of tyrosine but not histidine residues of IgG abrogated its ability to inhibit the binding of protein G to solid phase Fc fragments. Protein G was found to strongly inhibit the binding of a monoclonal and a polyclonal human rheumatoid factor to IgG. These findings indicate that protein G binds with separate sites to the Fc and F(ab')2 fragments of IgG, that the interaction with the Fc fragment occurs at the C gamma 2-C gamma 3 domain interface region and that tyrosine but not histidine residues in this area are likely involved. The relationship of the Fc fragment-binding site specificity of protein G to that of other microbial IgG binding proteins and human rheumatoid factors is discussed.

Animals↗