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N M Thielens

Publications and source records attributed to N M Thielens.

At least 19 recordsLinked to original sources
PubMed2001

Interaction properties of human mannan-binding lectin (MBL)-associated serine proteases-1 and -2, MBL-associated protein 19, and MBL.

The mannan-binding lectin (MBL) activation pathway of complement plays an important role in the innate immune defense against pathogenic microorganisms. In human serum, two MBL-associated serine proteases (MASP-1, MASP-2) and MBL-associated protein 19 (MAp19) were found to be associated with MBL. With a view to investigate the interaction properties of these proteins, human MASP-1, MASP-2, MAp19, as well as the N-terminal complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein-1-epidermal growth factor (CUB-EGF) segments of MASP-1 and MASP-2, were expressed in insect or human kidney cells, and MBL was isolated from human serum. Sedimentation velocity analysis indicated that the MASP-1 and MASP-2 CUB-EGF segments and the homologous protein MAp19 all behaved as homodimers (2.8-3.2 S) in the presence of Ca(2+). Although the latter two dimers were not dissociated by EDTA, their physical properties were affected. In contrast, the MASP-1 CUB-EGF homodimer was not sensitive to EDTA. The three proteins and full-length MASP-1 and MASP-2 showed no interaction with each other as judged by gel filtration and surface plasmon resonance spectroscopy. Using the latter technique, MASP-1, MASP-2, their CUB-EGF segments, and MAp19 were each shown to bind to immobilized MBL, with K:(D) values of 0.8 nM (MASP-2), 1.4 nM (MASP-1), 13.0 nM (MAp19 and MASP-2 CUB-EGF), and 25.7 nM (MASP-1 CUB-EGF). The binding was Ca(2+)-dependent and fully sensitive to EDTA in all cases. These data indicate that MASP-1, MASP-2, and MAp19 each associate as homodimers, and individually form Ca(2+)-dependent complexes with MBL through the CUB-EGF pair of each protein. This suggests that distinct MBL/MASP complexes may be involved in the activation or regulation of the MBL pathway.

Amino Acid Motifs↗
PubMed1999

Association of terminal complement proteins in solution and modulation by suramin.

The association of terminal complement proteins was investigated by analytical ultracentrifugation and multi-angle laser light scattering. Native C8 and C9 formed a heterodimer in solution of physiological ionic strength with a free-energy change DeltaG degrees of -8.3 kcal/mol and a dissociation constant Kd of 0.6 microM (at 20 degrees C) that was ionic strength- and temperature-dependent. A van't Hoff plot of the change in Kd was linear between 10 and 37 degrees C and yielded values of DeltaH degrees = -12.9 kcal/mol and DeltaS degrees = -15.9 cal mol-1 deg-1, suggesting that electrostatic forces play a prominent role in the interaction of C8 with C9. Native C8 also formed a heterodimer with C5, and low concentrations of polyionic ligands such as protamine and suramin inhibited the interaction. Suramin induced high-affinity trimerization of C8 (Kd = 0.10 microM at 20 degrees C) and dimerization of C9 (Kd = 0.86 microM at 20 degrees C). Suramin-induced C8 oligomerization may be the primary reason for the drug's ability to prevent complement-mediated hemolysis. Analysis of sedimentation equilibria and also of the fluorescence enhancement of suramin when bound to protein provided evidence for two suramin-binding sites on each C9 and three on each C8 in the oligomers. Oligomerization could be reversed by high suramin concentrations, but 8-aminonaphthalene-1,3,6- trisulfonate (ANTS2- ), which mimics half a suramin molecule, could not compete with suramin binding and oligomerization suggesting that the drug also binds nonionically to the proteins.

Calorimetry, Differential Scanning↗
PubMed1999

The N-terminal CUB-epidermal growth factor module pair of human complement protease C1r binds Ca2+ with high affinity and mediates Ca2+-dependent interaction with C1s.

The Ca2+-dependent interaction between complement serine proteases C1r and C1s is mediated by their alpha regions, encompassing the major part of their N-terminal CUB-EGF-CUB (where EGF is epidermal growth factor) module array. In order to define the boundaries of the C1r domain(s) responsible for Ca2+ binding and Ca2+-dependent interaction with C1s and to assess the contribution of individual modules to these functions, the CUB, EGF, and CUB-EGF fragments were expressed in eucaryotic systems or synthesized chemically. Gel filtration studies, as well as measurements of intrinsic Tyr fluorescence, provided evidence that the CUB-EGF pair adopts a more compact conformation in the presence of Ca2+. Ca2+-dependent interaction of intact C1r with C1s was studied using surface plasmon resonance spectroscopy, yielding KD values of 10.9-29.7 nM. The C1r CUB-EGF pair bound immobilized C1s with a higher KD (1.5-1.8 microM), which decreased to 31.4 nM when CUB-EGF was used as the immobilized ligand and C1s was free. Half-maximal binding was obtained at comparable Ca2+ concentrations ranging from 5 microM with intact C1r to 10-16 microM for C1ralpha and CUB-EGF. The isolated CUB and EGF fragments or a CUB + EGF mixture did not bind C1s. These data demonstrate that the C1r CUB-EGF module pair (residues 1-175) is the minimal segment required for high affinity Ca2+ binding and Ca2+-dependent interaction with C1s and indicate that Ca2+ binding induces a more compact folding of the CUB-EGF pair.

Animals↗
PubMed1999

In vitro analysis of complement-dependent HIV-1 cell infection using a model system.

Previous studies based on the use of human serum as a source of C have provided evidence for the C-dependent enhancement of cell infection by HIV-1. The present study was undertaken to distinguish C from other serum factors and to identify the proteins and the mechanisms involved in C-dependent cell infection by HIV-1. The classical C activation pathway was reconstituted from the proteins C1q, C1r, C1s, C4, C2, C3, factor H, and factor I; each were purified to homogeneity. A mixture of these proteins at physiological concentrations was shown to reproduce the ability of normal human serum to enhance the infection of MT2 cells by HIV-1 at low doses of virus. This enhancing effect was abolished when heat-inactivated serum and C2- or C3-depleted serum were used, and was restored upon addition of the corresponding purified proteins. A mixture of two synthetic peptides corresponding to positions 10-15 and 90-97 of human C receptor type 2 (CD21) as well as soluble CD4 both inhibited the C-dependent infection process. These data provide unambiguous evidence that HIV-1 triggers a direct activation of the classical C pathway in vitro and thereby facilitates the infection of MT2 cells at low doses of virus. These findings are consistent with a mechanism involving increased interaction between the virus opsonized by C3b-derived fragment(s) and the CD21 cell receptors and subsequent virus entry through CD4 receptors.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Structure and functions of the interaction domains of C1r and C1s: keystones of the architecture of the C1 complex.

C1r and C1s, the proteases responsible for activation and proteolytic activity of the C1 complex of complement, share similar overall structural organizations featuring five nonenzymic protein modules (two CUB modules surrounding a single EGF module, and a pair of CCP modules) followed by a serine protease domain. Besides highly specific proteolytic activities, both proteases exhibit interaction properties associated with their N-terminal regions. These properties include the ability to bind Ca2+ ions with high affinity, to associate with each other within a Ca2+-dependent C1s-C1r-C1r-C1s tetramer, and to interact with C1q upon C1 assembly. Precise functional mapping of these regions has been achieved recently, allowing identification of the domains responsible for these interactions, and providing a comprehensive picture of their structure and function. The objective of this article is to provide a detailed and up-to-date overview of the information available on these domains, which are keystones of the assembly of C1, and appear to play an essential role at the interface between the recognition function of C1 and its proteolytic activity.

Complement C1r↗
PubMed1998

Baculovirus-mediated expression of truncated modular fragments from the catalytic region of human complement serine protease C1s. Evidence for the involvement of both complement control protein modules in the recognition of the C4 protein substrate.

C1s is the modular serine protease responsible for cleavage of C4 and C2, the protein substrates of the first component of complement. Its catalytic region (gamma-B) comprises two complement control protein (CCP) modules, a short activation peptide (ap), and a serine protease domain (SP). A baculovirus-mediated expression system was used to produce recombinant truncated fragments from this region, deleted either from the first CCP module (CCP2-ap-SP) or from both CCP modules (ap-SP). The aglycosylated fragment CCP2-ap-SPag was also expressed by using tunicamycin. The fragments were produced at yields of 0.6-3 mg/liter of culture, isolated, and characterized chemically and then tested functionally by comparison with intact C1s and its proteolytic gamma-B fragment. All recombinant fragments were expressed in a proenzyme form and cleaved by C1r to generate active enzymes expressing esterolytic activity and reactivity toward C1 inhibitor comparable to those of intact C1s. Likewise, the activated fragments gamma-B, CCP2-ap-SP, and ap-SP retained C1s ability to cleave C2 in the fluid phase. In contrast, whereas fragment gamma-B cleaved C4 as efficiently as C1s, the C4-cleaving activity of CCP2-ap-SP was greatly reduced (about 70-fold) and that of ap-SP was abolished. It is concluded that C4 cleavage involves substrate recognition sites located in both CCP modules of C1s, whereas C2 cleavage is affected mainly by the serine protease domain. Evidence is also provided that the carbohydrate moiety linked to the second CCP module of C1s has no significant effect on catalytic activity.

Animals↗
PubMed1998

Structural and functional studies on C1r and C1s: new insights into the mechanisms involved in C1 activity and assembly.

C1r and C1s, the enzymes responsible for the activation and proteolytic activity of the C1 complex of complement, are modular serine proteases featuring similar overall structural organizations, yet expressing very distinct functional properties within C1. This review will initially summarize available information on the structure and function of the protein modules and serine protease domains of C1r and C1s. It will then focus on the regions of both proteases involved in: (i) assembly of C1s-C1r-C1r-C1s, the Ca(2+)-dependent tetrameric catalytic subunit of C1; (ii) expression of C1 catalytic activities. Particular emphasis will be aid on recent structural and functional studies that provide new insights into the complex mechanisms involved in the assembly, activation, and proteolytic activity of C1.

Calcium↗
PubMed1997

Structure and assembly of the catalytic region of human complement protease C1r: a three-dimensional model based on chemical cross-linking and homology modeling.

C1r is the modular serine protease responsible for autocatalytic activation of C1, the first component of the complement classical pathway. Its catalytic region is a noncovalent homodimer of two gamma-B monomers, each comprising two contiguous complement control protein (CCP) modules, IV and V [also known as short consensus repeats (SCRs)], a 15-residue intermediary segment, and the serine protease B domain. With a view to gain insight into domain-domain interactions within this region, fragment C1r (gamma-B)2, obtained by autolytic proteolysis of the active protease, was cross-linked with the water-soluble reagent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Cross-linked species gamma-B intra and gamma-B inter, containing intra- and intermonomer cross-links, respectively, were isolated and then fragmented by CNBr cleavage and trypsin digestion. N-Terminal sequence and mass spectrometry analyses of the resulting cross-linked peptides allowed us to identify one intramonomer cross-link between Lys426 of module V and the C-terminal Asp688 of the serine protease B domain and one intermonomer cross-link between the N-terminal Gly280 of fragment gamma and Glu493 of the B domain. Three-dimensional homology modeling of the CCP modules IV and V and of the B domain was also performed. The complementary information provided by chemical cross-linking and homology modeling studies was used to construct a three-dimensional model of the gamma-B monomer, in which module V interacts with the serine protease on the side opposite to both the active site and the Arg446-Ile447 activation site. Also, a tentative three-dimensional model of the (gamma-B)2 dimer was built, indicating a loose "head to tail" association of the monomers, with the active sites facing opposite directions toward the outside of the dimer. The latter model is compared with available low-resolution structural data, and its functional implications are discussed in terms of the conformational changes occurring during C1r activation.

Amino Acid Sequence↗
PubMed1996

Identification of a cryptic protein kinase CK2 phosphorylation site in human complement protease Clr, and its use to probe intramolecular interaction.

Treatment of human (activated)C1r by CK2 resulted in the incorporation of [32P]phosphate into the N-terminal alpha region of its non-catalytic A chain. Fragmentation of 32P-labelled (activated)C1r followed by N-terminal sequence and mass spectrometry analyses allowed identification of Ser189 as the phosphorylation site. Accessibility of Ser189 was low in intact C1r, due in part to the presence of one of the oligosaccharides borne by the alpha region, further reduced in the presence of calcium, and abolished when C1r was incorporated into the C1s-C1r-C1r-C1s tetramer or the C1 complex. In contrast, phosphorylation was enhanced in the isolated alpha fragment and insensitive to calcium. Taken together, these data provide support for the occurrence of a (Ca2+)-dependent interaction between the alpha region and the remainder of the C1r molecule.

Amino Acid Sequence↗
PubMed1995

Analysis of the N-linked oligosaccharides of human C1s using electrospray ionisation mass spectrometry.

Information on the structures of the oligosaccharides linked to Asn residues 159 and 391 of the human complement protease C1s was obtained using mass spectrometric and monosaccharide analyses. Asn159 is linked to a complex-type biantennary, bisialylated oligosaccharide NeuAc2 Gal2 GlcNAc4 Man3 (molecular mass = 2206 +/- 1). Asn391 is occupied by either a biantennary, bisialylated oligosaccharide, or a triantennary, trisialylated species NeuAc3 Gal3 GlcNAc5 Man3 (molecular mass = 2861 +/- 1), or a fucosylated triatennary, trisialylated species NeuAc3 Gal3 GlcNAc5 Man3 Fuc1 (molecular mass = 3007 +/- 1), in relative proportions of approximately 1:1:1. The carbohydrate heterogeneity at Asn391 gives rise to three major types of C1s molecules of molecular masses 79,318 +/- 8 (A), 79,971 +/- 8 (B), and 80,131 +/- 8 (C), with an average mass of 79,807 +/- 8. A minor modification, yielding an extra mass of 132 +/- 2, is also detected within positions 1-153.

Asparagine↗
PubMed1995

HIV-1 rsgp41 depends on calcium for binding of human c1q but not for binding of gp120.

Human immunodeficiency virus type 1 activates the complement cascade via the classical pathway by direct binding of C1q through specific sites in the TM surface protein, gp41. In this paper we investigated the divalent cation dependence of the interaction between HIV-1 gp41 and C1q or gp120. A solid phase radioimmunoassay was used to investigate the interaction between a recombinant soluble form of HIV-1 gp41 (rsgp41) and C1q and an enzyme linked immunoassay was used to investigate the interaction between rsgp41 and gp120. The interaction between C1q and rsgp41, but not between C1q and immune complexes, was dependent upon the presence of calcium. Calcium could not be replaced by larger cations such as strontium, barium, lead or smaller ions such as magnesium and manganese. Zinc increased binding to 22% of binding achieved with calcium. The interaction between rsgp41 and gp120 was not dependent upon the presence of divalent ions. Thus, calcium is required for the interaction between rsgp41 and C1q, whereas the interaction between rsgp41 and gp120 is independent of divalent cations.

Calcium↗
PubMed1994

Activation of human complement serine-proteinase C1r is down-regulated by a Ca(2+)-dependent intramolecular control that is released in the C1 complex through a signal transmitted by C1q.

The activation of human C1, a Ca(2+)-dependent complex proteinase comprising a non-enzymic protein, C1q, and two serine proteinases, C1r and C1s, is based primarily on the intrinsic property of C1r to autoactivate. The aim of the present study was to investigate the mechanisms involved in the regulation of C1r autoactivation, with particular attention to the role of Ca2+ ions. Spontaneous activation of proenzyme C1r was observed upon incubation in the presence of EDTA, whereas Ca2+ ions reduced markedly the activation process. Several lines of evidence indicated that Ca2+ inhibited the intramolecular activation reaction but had little or no effect on the intermolecular activation reaction. C1q caused partial release of this inhibitory effect of Ca2+. Complete stabilization of C1r in its proenzyme form was obtained upon incorporation within the Ca(2+)-dependent C1s-C1r-C1r-C1s tetramer, and a comparable effect was observed when C1s was replaced by its Ca(2+)-binding alpha-fragment. Both tetramers, C1s-C1r-C1r-C1s and C1s alpha-C1r-C1r-C1s alpha, readily associated with C1q to form 16.0 S and 14.7 S complexes respectively in which C1r fully recovered its activation potential. Both complexes showed indistinguishable activation kinetics, indicating that the gamma B catalytic region of C1s plays no role in the mechanism that triggers C1r activation in C1. The collagen-like fragments of C1q retained the ability to bind to C1s-C1r-C1r-C1s, but, in contrast with intact C1q, failed to induce C1r activation in the resulting complex at temperatures above 25 degrees C. On the basis of these observations it is proposed that activation of the serine-proteinase domain of C1r is controlled by a Ca(2+)-dependent intramolecular mechanism involving the Ca(2+)-binding alpha-region, and that this control is released in C1 by a signal originating in C1q and transmitted through the C1q/C1r interface.

Calcium↗
PubMed1994

The envelope glycoprotein of HIV-1 gp120 and human complement protein C1q bind to the same peptides derived from three different regions of gp41, the transmembrane glycoprotein of HIV-1, and share antigenic homology.

gp41, the transmembrane glycoprotein of HIV-1, has been shown to be non-covalently associated with gp120. We have shown that it also binds human C1q. To analyze the interaction site(s) of gp41 with these two molecules, we established an enzyme-linked immunosorbent assay (ELISA) system using recombinant soluble gp41 [amino acids (aa) 539-684] and peptides thereof. In the cell-external part of gp41 three sites (aa 526-538, aa 590-613 and aa 625-655) were found to bind both gp120 and C1q. That gp120 and C1q use the same sites was evidenced by the fact that these proteins competed with each other for the same sites in recombinant soluble gp41 and gp41 peptides. It could be demonstrated by ELISA, that rabbit antibodies against human C1q recognized gp120, and rabbit antibodies against gp120 cross-reacted with C1q. Rabbit anti-gp120, HIV-1-positive human sera and anti-gp120 obtained from such sera agglutinated sensitized sheep erythrocytes with human C1q (EAC1q). These data suggest that in addition to functional homology between C1q and gp120 structural homology between these two molecules exists. This molecular mimicry might become the basis for immunologically relevant autoimmune phenomena.

Binding Sites↗
PubMed1993

Further characterization of the interaction between the C1q subcomponent of human C1 and the transmembrane envelope glycoprotein gp41 of HIV-1.

Previous studies have provided evidence for activation of the human C1 complex by HIV-1, resulting from direct interaction between C1q and the external portion of the viral transmembrane envelope protein, rsgp41. The present study was undertaken to locate more precisely, within C1q and rsgp41, the sites involved in the C1/HIV-1 interaction. Using a solid phase binding assay, we showed that 125I-labeled C1q binding to rsgp41 was dose dependent, saturable, and comparable with binding of C1q to IgG-OVA immune complexes. The globular and, to a lesser extent, the collagen-like regions of C1q both bound to rsgp41. In contrast, the globular region of C1q inhibited the C1q/rsgp41 interaction, whereas the collagen-like region of C1q did not. A series of peptides covering the putative C1q-binding site on gp41 (positions 590-613 of gp160) were synthesized and used as competitors in the C1q-rsgp41-binding assay. Peptide 601-613 (GIWGCSGKLICT) inhibited C1q binding the most efficiently, with 50% inhibition at a concentration of 100 microM. This peptide also inhibited binding of C1q to rsgp36, the protein of HIV-2 homologous to rsgp41. The inhibitory effect of this peptide was dependent in part on the presence of the S-S bridge normally connecting Cys 605 to Cys 611 because reduction of this bond significantly reduced its efficiency. These data suggest that the C1q/HIV-1 interaction involves a site on C1q located within the globular regions, and a major site located within the immunodominant domain of HIV-1, which shares homology with the corresponding region of HIV-2.

Amino Acid Sequence↗
PubMed1993

Chemical characterization and location of ionic interactions involved in the assembly of the C1 complex of human complement.

The C1 complex of human complement comprises two loosely interacting subunits, C1q and the Ca(2+)-dependent C1s-C1r-C1r-C1s tetramer. With a view to gain information on the nature of the ionic interactions involved in C1 assembly, we have studied the effects of the chemical modifications of charged residues of C1q or the tetramer on their ability to reconstitute the C1 complex. Treatment of C1q with pyridoxal-5'-phosphate, acetic anhydride, and citraconic anhydride, as well as with cyclohexanedione and diethylpyrocarbonate, inhibited its ability to associate with C1s-C1r-C1r-C1s. Treatment of the collagen-like fragments of C1q with the same reagents yielded the same effects. Treatment of C1s-C1r-C1r-C1s with 1-ethyl-3-[-3-(dimethylamino) propyl] carbodiimide also prevented C1 assembly, through modification of acidic amino acids which were shown to be located in C1r. Further studies on the location of the interaction sites within C1q, using ligand-blotting and competition experiments with synthetic peptides, were unsuccessful, suggesting that these sites are contributed to by two or three of the C1q chains. It is concluded that C1 assembly involves interactions between acidic amino acids of C1r and lysine (hydroxylysine) and arginine residues located within the collagen-like region of C1q. Sequence comparison with mannan binding protein, another collagen-like molecule which binds the C1s-C1r-C1r-C1s tetramer, suggests Arg A38, and HyL B32, B65, and C29 of C1q as possible interaction sites.

Acetic Anhydrides↗
PubMed1993

HIV and human complement: mechanisms of interaction and biological implication.

Human complement, although not lytic for HIV-1, interacts with the virus and is closely involved in the infectious process. It enhances infection in the absence of antibody, and turns neutralizing antibodies into agents which increase viral infectivity. In this review M.P. Dierich et al. summarize available information and discuss possible biological implications.

Animals↗