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T S Jokiranta

Publications and source records attributed to T S Jokiranta.

At least 19 recordsLinked to original sources
PubMed2001

Multiple ligand binding sites on domain seven of human complement factor H.

Foreign particles and damaged host cells can activate the complement system leading to their destruction by the host defense system. Factor H (fH) plays a vital role in restricting complement activation on host cells through interactions with polyanions such as heparin, while allowing activation to proceed on foreign surfaces. Complement activation by damaged host cells is also down regulated by fH, which is localized to injured areas through interactions with C-reactive protein (CRP). A number of pathogens have developed mechanisms by which they can also bind fH and thus exploit its protective properties. One such organism is Group A Streptococcus (GAS) which mediates fH binding via its surface expressed M-protein. fH consists of 20 conserved short consensus repeat (SCR) units and mutagenesis studies indicate that the seventh repeat is responsible for interactions with heparin, CRP and M-protein. We recently performed molecular modelling of fH SCR 7 and identified a cluster of positively charged residues on one face of the domain. By alanine replacement mutagenesis, we demonstrated that these residues are involved in heparin, CRP and M protein binding, which indicates that there is a common site within fH SCR 7 responsible for multiple ligand recognition.

Amino Acid Sequence↗
PubMed2001

Complement C3b interactions studied with surface plasmon resonance technique.

The surface plasmon resonance (SPR) phenomenon is utilized in a number of new real time biosensors. In this study, we have used this technique to study interactions between the central complement component C3b and its multiple ligands by using the Biacore equipment. The SPR technique is particularly suitable for analysis of the alternative complement pathway (AP) because the inherent nature of the latter is to amplify deposition of C3b on various surfaces. C3b was coupled onto the sensor surface and the coupling efficiency was compared under various conditions on both polystyrene and carboxymethylated dextran surfaces. After enzymatic C3b coupling or standard amine C3b coupling, we analyzed and compared the binding of four C3b ligands to the surface: factor B, factor H, C5 and the soluble complement receptor 1 (sCR1, CD35). Binding of each ligand to C3b was detected when C3b had been coupled either enzymatically or using the amine coupling, but the half-lives of the interactions were found to vary depending on the coupling procedure. Factor H binds to C3b via three interaction sites. The target sites are exposed on the C3b, C3c and C3d fragments of C3, respectively. Therefore, we also tested by using the Biacore whether factor B, C5 and sCR1 bind to C3c and/or C3d. It was found that factor B bound to C3d, but not to C3c. On the other hand, both C5 and sCR1 bound to C3c, but not to C3d. In conclusion, this study shows that SPR is a powerful tool in analyzing and mapping the interactions of C3b with its multiple ligands.

Binding Sites↗
PubMed2001

Complement evasion by Borrelia burgdorferi: serum-resistant strains promote C3b inactivation.

The most characteristic features of the Lyme disease pathogens, the Borrelia burgdorferi sensu lato (s.l.) group, are their ability to invade tissues and to circumvent the immune defenses of the host for extended periods of time, despite elevated levels of borrelia-specific antibodies in serum and other body fluids. Our aim in the present study was to determine whether B. burgdorferi is able to interfere with complement (C) at the level of C3 by accelerating C3b inactivation and thus to inhibit the amplification of the C cascade. Strains belonging to different genospecies (Borrelia garinii, B. burgdorferi sensu stricto, and Borrelia afzelii) were compared for their sensitivities to normal human serum and abilities to promote factor I-mediated C3b degradation. B. burgdorferi sensu stricto and B. afzelii strains were found to be serum resistant. When the spirochetes were incubated with radiolabeled C3b, factor I-mediated degradation of C3b was observed in the presence of C-resistant B. afzelii (n = 3) and B. burgdorferi sensu stricto (n = 1) strains but not in the presence of C-sensitive B. garinii (n = 7) strains or control bacteria (Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis). Immunoblotting and radioligand binding analyses showed that the C-resistant strains had the capacity to acquire the C inhibitors factor H and factor H-like protein 1 (FHL-1) from growth medium and human serum. A novel surface protein with an apparent molecular mass of 35 kDa was found to preferentially bind to the N terminus region of factor H. Thus, the serum-resistant B. burgdorferi s.l. strains can circumvent C attack by binding the C inhibitors factor H and FHL-1 to their surfaces and promoting factor I-mediated C3b degradation.

Animals↗
PubMed2000

Each of the three binding sites on complement factor H interacts with a distinct site on C3b.

Factor H (fH) restricts activation of the alternative pathway of complement at the level of C3, both in the fluid phase and on self-structures, but allows the activation to proceed on foreign structures. To study the interactions between fH and C3b we used surface plasmon resonance analysis (Biacore(R)) and eight recombinantly expressed fH constructs containing fragments of the 20 short consensus repeat domains (SCRs) of fH. We analyzed the binding of these constructs to C3b and its cleavage products C3c and C3d. Three binding sites for C3b were found on fH. Site 1 was localized to the five amino-terminal SCRs (SCR1-5), and its reciprocal binding site on C3b was found to be lost upon the cleavage of C3b to C3c and C3d. Site 2 on fH was localized by exclusion probably within or near SCRs 12-14 (fragment SCR8-20 bound to C3b, C3c, and C3d; SCR8-11 did not bind to C3b at all; and SCR15-20 bound only to the C3d part of C3b). Site 3 on fH for C3b was localized to the carboxyl-terminal SCRs 19-20, and its reciprocal binding site was mapped to the C3d part of C3b. In conclusion, we confirmed and mapped three binding sites on fH for C3b and demonstrated that the three binding sites on fH interact with distinct sites on C3b. Multiple reciprocal interactions between C3b and fH can provide a basis for the different reactivity of the alternative pathway with different target structures.

Binding Sites↗
PubMed2000

Exceptional resistance of human H2 glioblastoma cells to complement-mediated killing by expression and utilization of factor H and factor H-like protein 1.

Of over 20 nucleated cell lines we have examined to date, human H2 glioblastoma cells have turned out to be the most resistant to complement-mediated cytolysis in vitro. H2 cells expressed strongly the membrane attack complex inhibitor protectin (CD59), moderately CD46 (membrane cofactor protein) and CD55 (decay-accelerating factor), but no CD35 (complement receptor 1). When treated with a polyclonal anti-H2 Ab, anti-CD59 mAb, and normal human serum, only 5% of H2 cells became killed. Under the same conditions, 70% of endothelial-like EA.hy 926 cells and 40% of U251 control glioma cells were killed. A combined neutralization of CD46, CD55, and CD59 increased H2 lysis only minimally, demonstrating that these complement regulators are not enough to account for the resistance of H2 cells. After treatment with Abs and serum, less C5b-9 was deposited on H2 than on U251 and EA.hy 926 cell lines. A reason for the exceptional resistance of H2 cells was revealed when RT-PCR and protein biochemical methods showed that the H2 cells, unlike the other cell lines tested, actively produced the soluble complement inhibitors factor H and factor H-like protein 1. H2 cells were also capable of binding human factor H from the fluid phase to their cell surface and promoted the cleavage of C3b to its inactive form iC3b more efficiently than U251 and EA.hy 926 cells. In accordance, anti-factor H mAbs enhanced killing of H2 glioblastoma cells. Taken together, our results show that production and binding of factor H and factor H-like protein 1 is a novel mechanism that these malignant cells utilize to escape complement-mediated killing.

Antibodies, Monoclonal↗
PubMed2000

Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner.

Multiple ovulations are uncommon in humans, cattle and many breeds of sheep. Pituitary gonadotrophins and as yet unidentified ovarian factors precisely regulate follicular development so that, normally, only one follicle is selected to ovulate. The Inverdale (FecXI) sheep, however, carries a naturally occurring X-linked mutation that causes increased ovulation rate and twin and triplet births in heterozygotes (FecXI/FecX+; ref. 1), but primary ovarian failure in homozygotes (FecXI/FecXI; ref. 2). Germ-cell development, formation of the follicle and the earliest stages of follicular growth are normal in FecXI/FecXI sheep, but follicular development beyond the primary stage is impaired. A second family unrelated to the Inverdale sheep also has the same X-linked phenotype (Hanna, FecXH). Crossing FecXI with FecXH animals produces FecXI/FecXH infertile females phenotypically indistinguishable from FecXI/FecXI females. We report here that the FecXI locus maps to an orthologous chromosomal region syntenic to human Xp11.2-11.4, which contains BMP15, encoding bone morphogenetic protein 15 (also known as growth differentiation factor 9B (GDF9B)). Whereas BMP15 is a member of the transforming growth factor beta (TGFbeta) superfamily and is specifically expressed in oocytes, its function is unknown. We show that independent germline point mutations exist in FecXI and FecXH carriers. These findings establish that BMP15 is essential for female fertility and that natural mutations in an ovary-derived factor can cause both increased ovulation rate and infertility phenotypes in a dosage-sensitive manner.

Amino Acid Sequence↗
PubMed2000

Different regulation of factor H and FHL-1/reconectin by inflammatory mediators and expression of the two proteins in rheumatoid arthritis (RA).

Factor H and the FHL-1/reconectin protein are two human plasma proteins that act as important regulators of the alternative complement pathway. Each protein is encoded by a unique transcript, but both mRNAs are derived from the factor H gene by means of alternative processing. In order to address potential functional differences between the two proteins we analysed their expression in hepatic and non-hepatic cells and studied their regulation by inflammatory mediators. We demonstrate that factor H and FHL-1/reconectin transcripts which are regulated by the same gene promoter and are initiated at the same transcription start site are differently expressed. Expression of the molecules is induced and regulated by the inflammatory mediators interferon-gamma (IFN-gamma) and the anti-inflammatory glucocorticoid dexamethasone. Both factor H and FHL-1/reconectin are expressed and secreted by synovial fibroblasts and are present in synovial fluid derived from patients suffering from rheumatoid or reactive arthritis. The local synthesis in synovial fibroblasts and their induction by IFN-gamma and dexamethasone, but not by tumour necrosis factor-alpha, suggests for each of the two complement regulators a protective role in RA.

Alternative Splicing↗
PubMed2000

Resistance of Trichomonas vaginalis to metronidazole: report of the first three cases from Finland and optimization of in vitro susceptibility testing under various oxygen concentrations.

Trichomonas vaginalis is a globally common sexually transmitted human parasite. Many strains of T. vaginalis from around the world have been described to be resistant to the current drug of choice, metronidazole. However, only a few cases of metronidazole resistance have been reported from Europe. The resistant strains cause prolonged infections which are difficult to treat. T. vaginalis infection also increases the risk for human immunodeficiency virus transmission. We present a practical method for determining the resistance of T. vaginalis to 5-nitroimidazoles. The suggested method was developed by determining the MICs and minimal lethal concentrations (MLCs) of metronidazole and ornidazole for T. vaginalis under various aerobic and anaerobic conditions. Using this assay we have found the first three metronidazole-resistant strains from Finland, although the origin of at least one of the strains seems to be Russia. Analysis of the patient-derived and previously characterized isolates showed that metronidazole-resistant strains were also resistant to ornidazole, and MLCs for all strains tested correlated well with the MICs. The suggested MICs of metronidazole for differentiation of sensitive and resistant isolates are >75 microg/ml in an aerobic 24-h assay and >15 microg/ml in an anaerobic 48-h assay.

Aerobiosis↗
PubMed1999

Functional properties of complement factor H-related proteins FHR-3 and FHR-4: binding to the C3d region of C3b and differential regulation by heparin.

The human factor H-related proteins FHR-3 and FHR4 are members of a family of proteins related to the complement factor H. Here, we report that the two proteins bind to the C3d region of complement C3b. The apparent K(A) values for the interactions of FHR-3 and FHR-4 with C3b are 7.5 x 10(6) M(-1) and 2.9 x 10(6) M(-1), respectively. Binding studies performed with C3b-coated pneumococci confirmed the results obtained with the biosensor system. A C-terminal construct of factor H showed similar binding characteristics. The interaction of FHR-3, but not of FHR4, with opsonised pneumococci was inhibited by heparin.

Apolipoproteins↗
PubMed1999

Nephritogenic lambda light chain dimer: a unique human miniautoantibody against complement factor H.

A unique monoclonal Ig lambda light chain dimer (protein LOI) was isolated from the serum and urine of a patient with hypocomplementemic membranoproliferative glomerulonephritis. In vitro the lambda light chain dimer efficiently activated the alternative pathway of complement (AP). When added to normal human serum, LOI temporarily enhanced AP hemolytic activity, but during a prolonged incubation the hemolytic activity was depleted. Protein LOI was found to bind to factor H, the main regulator molecule of AP. By binding to the short consensus repeat domain 3 of factor H, the dimer LOI blocked one of three interaction sites between H and C3b and thus inhibited the activity of H and induced an uncontrolled activation of the AP. Structural analysis showed that LOI belonged to the Vlambda3a subgroup of lambda light chains. The variable (V) region of LOI was most closely related to the predicted product of the Vlambda3 germline gene Iglv3s2, although it contained several unique residues that in a tertiary homology model structure form an unusual ring of charged residues around a hydrophobic groove in the putative Ag binding site. This site fitted considerably well with a putative binding site in the molecular model of domain 3 of factor H containing a reciprocal ring of charged amino acids around a hydrophobic area. Apparently, functional blocking of factor H by the Ab fragment-like lambda light chain dimer had initiated the development of a severe form of membranoproliferative glomerulonephritis. Thus, the lambda light chain dimer LOI represents the first described pathogenic miniautoantibody in human disease.

Amino Acid Sequence↗
PubMed1999

Regulation of complement activation by C-reactive protein: targeting the complement inhibitory activity of factor H by an interaction with short consensus repeat domains 7 and 8-11.

C-reactive protein (CRP) is a major acute phase protein whose functions are not totally clear. In this study, we examined the interaction of CRP with factor H (FH), a key regulator of the alternative pathway (AP) of complement. Using the surface plasmon resonance technique and a panel of recombinantly expressed FH constructs, we observed that CRP binds to two closely located regions on short consensus repeat (SCR) domains 7 and 8-11 of FH. Also FH-like protein 1 (FHL-1), an alternatively spliced product of the FH gene, bound to CRP with its most C-terminal domain (SCR 7). The binding reactions were calcium-dependent and partially inhibited by heparin. In accordance with the finding that CRP binding sites on FH were distinct from the C3b binding sites, CRP preserved the ability of FH to promote factor I-mediated cleavage of C3b. We propose that the function of CRP is to target functionally active FH and FHL-1 to injured self tissues. Thereby, CRP could restrict excessive complement attack in tissues while allowing a temporarily enhanced AP activity against invading microbes in blood.

Binding Sites↗
PubMed1999

The factor H protein family.

The factor H gene family provides a prime example of a multidomain multifunctional protein family whose individual members are defined by conserved common structural elements and display diverse but often overlapping functions. The six identified members of this protein family represent secreted plasma proteins that are primarily synthesized in the liver. Here, we summarize the current understanding of the function of these proteins and suggest a common role in complement control. Factor H is the best characterized member and acts as a complement regulator. The protein displays cofactor activity for factor I in the degradation of the central complement component C3b, acts as a decay accelerating factor for the C3 convertase, C3bBb and is a competitor for factor B binding to C3b. Factor H is a multifunctional protein and displays functions outside the complement system: it binds to the cellular integrin receptor (CD11b/CD18), interacts with cell surface glycosaminoglycans and also binds to the surface of certain pathogenic microorganisms. In addition, factor H has several binding sites for the C3 protein. The factor H-like protein 1 (FHL-1) or reconectin shares the complement regulatory functions with factor H and interacts with heparin. The protein displays cell spreading activity and binds to the N-terminus of the streptococcal M protein. The function of the factor H-related proteins (FHR-1 to FHR-4) is currently under investigation. These proteins are differently distributed. Three proteins (FHR-1, FHR-2 and FHR-4) are constituents of lipoproteins, while FHR-3 interacts with heparin. Binding to C3b and C3d has been demonstrated for FHR-3 and FHR-4 and the two proteins display a cofactor related activity.

Complement Factor H↗
PubMed1996

Analysis of the recognition mechanism of the alternative pathway of complement by monoclonal anti-factor H antibodies: evidence for multiple interactions between H and surface bound C3b.

The ability of the alternative pathway of complement to discriminate targets as either activators or non-activators is mediated by different binding properties of factor H to surface-associated C3b molecules. In the present study we have probed the interaction between H and C3b using five anti-H mAb. The binding sites of the mAb were mapped by Western blotting using both recombinant and trypsin-generated H fragments. Two mAb bound to CCP1 (90X, 196X), two to CCP5 (MRC OX24, 86X) and one to CCP8-15a (131X). At a molar ratio 2:1 of 125I-H:mAb all tested mAb enhanced binding of H to both activator- and non-activator-bound C3b. At higher concentrations two mAb had an inhibitory effect on H binding to surface-associated C3b (OX24, 131X). Thus the mAb 131X inhibits H binding to surface-bound C3b but unlike OX24 it does not bind to the previously described C3b binding site within or near CCP4-5. These results indicate that there is an additional interaction site on factor H for surface-bound C3b.

Animals↗
PubMed1995

Extracellular domain of type I receptor for transforming growth factor-beta: molecular modelling using protectin (CD59) as a template.

We have observed that the extracellular domain of T beta RI and protectin (CD59), an inhibitor of the membrane attack complex of complement, share structural features, a distinct spacing of ten cysteines and a C-terminal 'Cys-box'. Based on these common features and the recently determined NMR-structure of protectin, a three-dimensional model for the extracellular domain of T beta RI was constructed. After energy minimization and molecular dynamics simulation, a structure with four extending fingers (pes quattvordigitorum) and two clusters of charged residues was obtained. This model provides a view to the understanding of interactions between T beta RI, T beta RII and TGF beta during ligand recognition and signal transduction.

Amino Acid Sequence↗
PubMed1995

Complement resistance of parasites.

The complement system is a first-line defence mechanism against parasites. All parasites causing deep infections and getting into contact with human plasma must, in one way or another, avoid the destructive effect of this powerful defence system. Several specific strategies of complement resistance of parasites have been reported, and this rather large spectrum of regulatory mechanisms covers the whole cascade of complement activation. Analysis of the known and elucidation of the yet unknown mechanisms will probably help in the development of new therapeutic and preventive approaches to control the different parasitic diseases. This paper will review the complement resistance mechanisms reported and their utilization by various parasites.

Animals↗
PubMed1993

Biotinylation of monoclonal antibodies prevents their ability to activate the classical pathway of complement.

Biotinylation of mAb has become a standard procedure for a variety of applications that exploit the specific high affinity interaction between biotin and avidin. In the present study, we investigated how biotinylation of mAb affects their ability to sensitize target cells to C-dependent lysis in vitro. mAb were biotinylated by cross-linking biotin covalently with an N-succinimidyl ester to the epsilon-amino groups of lysine residues. Human RBC were treated with two rat mAb, either alone or together: one against glycophorin A (YTH89.1), another against CD59 (protectin; YTH53.1), an inhibitor of the membrane attack complex of C. Melanoma cells (G361) were attacked by a mouse mAb (27A) against an O-acetylated GD3 ganglioside. As compared with the nonbiotinylated mAb, the biotinylated forms of all the investigated mAb were much weaker in causing classical C pathway-mediated lysis of the target cells. Biotinylation did not reduce the ability of the mAb to bind to their Ag, nor of the anti-CD59 mAb to neutralize the C lysis-restrictive effect of CD59. In binding assays using 125I-labeled C1q, significantly less C1q bound to the biotinylated anti-glycophorin-A and anti-CD59 mAb than to the nonbiotinylated mAb. These data show that biotinylated antibodies do not activate the classical C pathway because binding of C1q to the antibody Fc-regions is blocked.

Animals↗