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| 3. |
- Berggård, Karin, et al.
(författare)
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Binding of human C4BP to the hypervariable region of M protein: a molecular mechanism of phagocytosis resistance in Streptococcus pyogenes
- 2001
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 42:2, s. 539-551
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Tidskriftsartikel (refereegranskat)abstract
- The amino-terminal hypervariable region (HVR) of streptococcal M protein is required for the ability of this virulence factor to confer phagocytosis resistance. The function of the HVR has remained unknown, but the finding that many HVRs with extremely divergent sequences bind the human complement regulator C4b-binding protein (C4BP) has suggested that this ligand may play a role in phagocytosis resistance. We used the M22 system to study the function of bound C4BP and provide several lines of evidence that C4BP indeed contributes to phagocytosis resistance. First, the ability of anti-HVR antibodies to cause opsonization correlated with their ability to inhibit binding of C4BP. Secondly, a short deletion in the HVR eliminated C4BP binding and also reduced the ability of M22 to confer phagocytosis resistance. Thirdly, the addition of an excess of pure C4BP to a phagocytosis system almost completely blocked the effect of opsonizing anti-HVR antibodies. Together, our data indicate that binding of C4BP to the HVR of M22 plays an important role in phagocytosis resistance, but other properties of M22 also contribute. This study provides the first molecular insight into the mechanisms by which the HVR of an M protein confers phagocytosis resistance.
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| 4. |
- Berggård, Karin, et al.
(författare)
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Bordetella pertussis binds to human C4b-binding protein (C4BP) at a site similar to that used by the natural ligand C4b
- 2001
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Ingår i: European Journal of Immunology. - 1521-4141. ; 31:9, s. 2771-2780
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Tidskriftsartikel (refereegranskat)abstract
- Human complement regulators are important targets for pathogenic microorganisms. In one such interaction, Bordetella pertussis binds human C4b-binding protein (C4BP), a high-molecular-weight plasma protein that acts as inhibitor of the classical pathway of complement activation. At least two different B. pertussis surface components, one of which is the virulence factor filamentous hemagglutinin (FHA), contribute to the binding. We used a set of C4BP mutants and monoclonal antibodies to characterize the region in C4BP that binds B. pertussis and analyzed the salt sensitivity of the interaction. These studies indicated that positively charged residues at the interface between complement control protein modules 1-2 in the C4BP alpha-chain are important for binding, and that the site in C4BP that binds B. pertussis is very similar, but not identical, to the C4b-binding site. Bacteria-bound C4BP retained its complement regulatory function and B. pertussis selectively bound C4BP in human plasma, indicating that binding occurs also in vivo. Together, these findings indicate that B. pertussis exploits a site in C4BP, resembling that used by the natural ligand C4b.
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| 5. |
- Berggård, Karin
(författare)
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Interactions of human C4BP with Bordetella pertussis and Streptococcus pyogenes
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Many microorganisms have developed mechanisms to protect themselves against attack from the complement system of the host. One possible mechanism for a microorganism to evade complement attack is to bind a human complement regulator, which may allow the microorganism to down-regulate complement activation. This thesis describes studies of such interactions between human C4b-binding protein (C4BP), a complement regulator present in plasma, and two bacteria pathogenic for humans: Bordetella pertussis and Streptococcus pyogenes. All clinical isolates of B. pertussis, the etiologic agent of whooping cough, were shown to bind C4BP. The binding was found to be dependent on at least two different surface components, one of which is the virulence factor filamentous hemagglutinin (FHA). The region in C4BP that binds B. pertussis is very similar, but not identical, to the region used by the natural ligand C4b. Many, but not all, strains of S. pyogenes bind C4BP. The binding is due to M proteins, a group of surface proteins important for virulence. The binding site in C4BP for M proteins was studied and found to overlap with the binding site for C4b. Thus, two very different pathogens, B. pertussis and S. pyogenes, bind to the same region in C4BP as the natural ligand C4b. The M proteins of S. pyogenes are characterized by the presence of a hypervariable region (HVR), which allows the bacteria to evade host immunity due to antigenic variation. The HVR is required for the ability of S. pyogenes to resist phagocytosis, but the mechanism of action of the region has remained unknown. Previously, it has been demonstrated that C4BP binds to the HVR, but evidence has been lacking that bacteria-bound C4BP plays a role in phagocytosis resistance. A functional study presented in this thesis provided several lines of evidence that C4BP indeed contributes to phagocytosis resistance. Our data provide the first molecular explanation for the ability of an HVR to confer resistance to phagocytosis. We found that isolated HVRs retain the ability to bind C4BP, allowing direct characterization of the C4BP-binding HVRs. Synthetic peptides/HVRs with very little residue identity were found to bind C4BP with high specificity and computational modeling suggested that they have similar folds. However, the C4BP-binding HVRs were immunologically completely unrelated. These data show that a bacterial protein domain can exhibit extreme variability with regard to sequence and immunological properties, while retaining the ability to bind a ligand with high specificity.
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- Berggård, Tord, et al.
(författare)
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Calbindin D28k exhibits properties characteristic of a Ca2+ sensor.
- 2002
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 277:19, s. 16662-16672
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Tidskriftsartikel (refereegranskat)abstract
- Calbindin D28k is a member of the calmodulin super-family of Ca2+ -binding proteins and contains six EF-hands. The protein is generally believed to function as a Ca2+ buffer, but the studies presented in this work indicate that it may also act as a Ca2+ sensor. The results show that Mg2+ binds to the same sites as Ca2+ with an association constant of approximately 1.4 x 10(3) M-1 in 0.15 M KCl. The four high-affinity sites in calbindin D28k bind Ca2+ in a non-sequential, parallel manner. In the presence of physiological concentrations of Mg2+, the Ca2+ -affinity is reduced by a factor of two and the cooperativity, which otherwise is modest, increases. Based on the binding constants determined in the presence of physiological salt concentrations, we estimate that at the Ca2+ concentration in a resting cell calbindin D28k is saturated to 40-75% with Mg2+, but to less than 9 % with Ca2+. In contrast, the protein is expected to be nearly fully saturated with Ca2+ at the Ca2+ level of an activated cell. A substantial conformational change is observed upon Ca2+ binding, but only minor structural changes take place upon Mg2+-binding. This suggests that calbindin D28k undergoes Ca2+ -induced structural changes upon Ca2+ activation of a cell. Thus, calbindin D28k displays several properties that would be expected for a protein involved in Ca2+ -induced signal transmission and hence may function not only as a Ca2+ buffer, but also as a Ca2+ sensor. Digestion patterns resulting from limited proteolysis of the protein suggest that the loop of EF-hand 2, a variant site that does not bind Ca2+, becomes exposed upon Ca2+ binding.
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| 7. |
- Carlsson, Fredric, et al.
(författare)
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Evasion of phagocytosis through cooperation between two ligand-binding regions in Streptococcus pyogenes M protein.
- 2003
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Ingår i: Journal of Experimental Medicine. - : Rockefeller University Press. - 1540-9538 .- 0022-1007. ; 198:7, s. 1057-1068
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Tidskriftsartikel (refereegranskat)abstract
- The M protein of Streptococcus pyogenes is a major bacterial virulence factor that confers resistance to phagocytosis. To analyze how M protein allows evasion of phagocytosis, we used the M22 protein, which has features typical of many M proteins and has two well-characterized regions binding human plasma proteins: the hypervariable NH2-terminal region binds C4b-binding protein (C4BP), which inhibits the classical pathway of complement activation; and an adjacent semivariable region binds IgA-Fc. Characterization of chromosomal S. pyogenes mutants demonstrated that each of the ligand-binding regions contributed to phagocytosis resistance, which could be fully explained as cooperation between the two regions. Deposition of complement on S. pyogenes occurred almost exclusively via the classical pathway, even under nonimmune conditions, but was down-regulated by bacteria-bound C4BP, providing an explanation for the ability of bound C4BP to inhibit phagocytosis. Different opsonizing antisera shared the ability to block binding of both C4BP and IgA, suggesting that the two regions in M22 play important roles also under immune conditions, as targets for protective antibodies. These data indicate that M22 and similar M proteins confer resistance to phagocytosis through ability to bind two components of the human immune system.
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| 9. |
- Larsson, Jörgen, et al.
(författare)
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Distribution of iodine 125-labeled alpha1-microglobulin in rats after intravenous injection
- 2001
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Ingår i: Journal of Laboratory and Clinical Medicine. - : Elsevier BV. - 0022-2143 .- 1532-6543. ; 137:3, s. 165-175
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Tidskriftsartikel (refereegranskat)abstract
- The 28-kd plasma protein alpha(1)-microglobulin is found in the blood of mammals and fish in a free, monomeric form and as high-molecular-weight complexes with molecular masses above 200 kd. In this study, iodine 125-labeled free and high-molecular weight rat alpha(1)-microglobulin (a mixture of alpha(1)-microglobulin/alpha(1)-inhibitor-3 and alpha(1)-microglobulin/fibronectin complexes) were injected intravenously into rats. The distribution of the proteins was measured by using scintillation camera imaging. Both forms of (125)I-labeled alpha(1)-microglobulin were rapidly cleared from the blood, with a half-life of 2 and 16 minutes for the initial and late phase, respectively, for free alpha(1)-microglobulin; and a half-life of 3 and 130 minutes for the initial and late phase, respectively, for the complexes. After 45 minutes, 6%, 16%, 27%, 13%, and 34% of the free (125)I-labeled alpha(1)-microglobulin and 18%, 21%, 6%, 10%, and 42% of the (125)I-labeled alpha(1)-microglobulin complexes were found in the blood, gastrointestinal tract, kidneys, liver, and the remainder of the body, respectively. The local distribution of injected (125)I-labeled alpha(1)-microglobulin in intestines and kidneys was investigated by microscopy and autoradiography. In the intestine, both forms were distributed in the basal layers, villi, and luminal contents. The results also suggested intracellular labeling of epithelial cells. Well-defined local regions containing higher concentrations of injected protein could be seen in the intestine. In the kidneys, both forms were found mostly in the cortex. Free (125)I-labeled alpha(1)-microglobulin was found predominantly in epithelial cells of a subset of the tubules, whereas the (125)I-labeled complexes were more evenly distributed. Intracellular labeling was indicated for both alpha(1)-microglobulin forms. The results thus indicate a rapid transport of (125)I-labeled alpha(1)-microglobulin from the blood to most tissues.
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| 10. |
- Matheu, Victor, et al.
(författare)
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Impact on allergic immune response after treatment with vitamin A
- 2009
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Ingår i: Nutrition & Metabolism. - : Springer Science and Business Media LLC. - 1743-7075. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Background: Vitamin A may have some influence on the immune system, but the role in allergy modulation is still unclear. Objective: To clarify whether high levels of retinoic acid (RA) affects allergic response in vivo, we used a murine experimental model of airway allergic disease. Methods: Ovalbumin (OVA)-immunization/OVA-challenge (OVA/OVA) and house dust mite (HDM)-immunization/HDM-challenge (HDM/HDM) experimental murine models of allergic airway disease, using C57Bl.10/Q groups of mice (n = 10) treated subcutaneously with different concentrations of all-trans RA (0, 50, 500 and 2,500 ug) every 2-days were used to assess the allergic immune response. Results: Levels of total and specific-IgE in sera were increased in all groups of RA treated OVA/OVA and HDM/HDM mice. Percentage and total amount of recruited eosinophil in airways by bronchoalveolar lavage fluid (BALF) were significantly enhanced in groups treated with 50, 500 and 2,500 ug of RA compared to non-treated mice. However, the group of mice treated with 2,500 ug had less eosinophil recruitment than the other two groups (50 and 500 ug). In parallel, levels of IL-5 and total IgE in BALF were also significantly diminished in the group treated with 2,500 ug compared to the other 2 groups (50 and 500 ug). Finally, total lung resistance was decreased in group treated with 2,500 ug compared to non-treated mice. Conclusion: Our results suggest that retinoic acid directly enhances allergic response in vivo, but in higher doses may produce of immune suppression.
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