Substrate specificities of recombinant mannan-binding lectin-associated serine proteases-1 and -2.

Mannan-binding lectin (MBL)-associated serine proteases-1 and 2 (MASP-1 and MASP-2) are homologous modular proteases that each interact with MBL, an oligomeric serum lectin involved in innate immunity. To precisely determine their substrate specificity, human MASP-1 and MASP-2, and fragments from their catalytic regions were expressed using a baculovirus/insect cells system. Recombinant MASP-2 displayed a rather wide, C1s-like esterolytic activity, and specifically cleaved complement proteins C2 and C4, with relative efficiencies 3- and 23-fold higher, respectively, than human C1s. MASP-2 also showed very weak C3 cleaving activity. Recombinant MASP-1 had a lower and more restricted esterolytic activity. It showed marginal activity toward C2 and C3, and no activity on C4. The enzymic activity of both MASP-1 and MASP-2 was specifically titrated by C1 inhibitor, and abolished at a 1:1 C1 inhibitor:protease ratio. Taken together with previous findings, these and other data strongly support the hypothesis that MASP-2 is the protease that, in association with MBL, triggers complement activation via the MBL pathway, through combined self-activation and proteolytic properties devoted to C1r and C1s in the C1 complex. In view of the very low activity of MASP-1 on C3 and C2, our data raise questions about the implication of this protease in complement activation.

Crystal structure of the catalytic domain of human complement c1s: a serine protease with a handle.

C1s is the highly specific modular serine protease that mediates the proteolytic activity of the C1 complex and thereby triggers activation of the complement cascade. The crystal structure of a catalytic fragment from human C1s comprising the second complement control protein (CCP2) module and the chymotrypsin-like serine protease (SP) domain has been determined and refined to 1.7 A resolution. In the areas surrounding the active site, the SP structure reveals a restricted access to subsidiary substrate binding sites that could be responsible for the narrow specificity of C1s. The ellipsoidal CCP2 module is oriented perpendicularly to the surface of the SP domain. This arrangement is maintained through a rigid module-domain interface involving intertwined proline- and tyrosine-rich polypeptide segments. The relative orientation of SP and CCP2 is consistent with the fact that the latter provides additional substrate recognition sites for the C4 substrate. This structure provides a first example of a CCP-SP assembly that is conserved in diverse extracellular proteins. Its implications in the activation mechanism of C1 are discussed.

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.

NMR structures of the C-terminal end of human complement serine protease C1s.

Synthetic peptides derived from the C-terminal end of the human complement serine protease C1s were analysed by circular dichroism and nuclear magnetic resonance (NMR) spectroscopy. Circular dichroism indicates that peptides 656-673 and 653-673 are essentially unstructured in water and undergo a coil-to-helix transition in the presence of increasing concentrations of trifluoroethanol. Two-dimensional NMR analyses performed in water/trifluoroethanol solutions provide evidence for the occurrence of a regular alpha-helix extending from Trp659 to Ser668 (peptide 656-673), and from Tyr656 to Ser668 (peptide 653-673), the C-terminal segment of both peptides remaining unstructured under the conditions used. Based on these and other observations, we propose that the serine protease domain of C1s ends in a 13-residue alpha-helix (656Tyr-Ser668) followed by a five-residue C-terminal extension. The latter appears to be flexible and is probably locked within C1s through a salt bridge involving Glu672.

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.

NMR structures of a mitochondrial transit peptide from the green alga Chlamydomonas reinhardtii.

The 26-amino-acid pre-sequence of the ATP synthase beta subunit that directs the protein from the cytosol to mitochondria in the unicellular green alga Chlamydomonas reinhardtii has been synthesised and analysed using NMR spectroscopy/circular dichroism and compared to a chloroplast transit peptide from the same organism. The results demonstrate that the peptide, though mainly unstructured in water, undergoes a strong conformational change in a 36% water/64% 2,2,2-trifluoroethanol mixture. In this solvent condition, an alpha-helix was characterised by NMR from residue 2 to 26. Structure calculations under NMR restraints lead to a population of models of which 60% are kinked at position 9-10. Structural analysis indicates two hydrophobic sectors on the models with a discontinuity at the 9-10 kink level. The structures suggest a different interaction mode with the mitochondrial membrane compared to the chloroplast transit peptide.

Localization of the N-terminal and C-terminal ends of triadin with respect to the sarcoplasmic reticulum membrane of rabbit skeletal muscle.

Antibodies were raised against synthetic peptides corresponding to the N-terminal (residues 2-17) and C-terminal (residues 691-706) ends of rabbit skeletal muscle triadin, a 95 kDa protein located in the sarcoplasmic reticulum membrane at the triad junction. The specificity of the antibodies generated was tested by ELISA and Western blot analysis. These tests demonstrated the ability of the antibodies to react specifically with the proteins. The anti-N-terminus antibodies bound to sarcoplasmic reticulum vesicles, indicating that the N-terminal end of the membrane-embedded triadin is exposed on the cytoplasmic side of the vesicles. In contrast, the anti-C-terminus antibodies were able to react with sarcoplasmic reticulum vesicles only after permeabilization of the vesicles with a detergent, indicating that the C-terminal end is exposed on the luminal side of the vesicles. These immunological data were complemented by proteolysis experiments using carboxypeptidases and endoproteinase Arg C. A mixture of carboxypeptidases A, B and Y was used to induce degradation of the C-terminal end of triadin in sarcoplasmic reticulum vesicles. This degradation, and a concomitant loss of reactivity of the anti-C-terminus antibodies in Western blots, was observed only when the vesicles were permeabilized, providing further evidence for the luminal localization of the C-terminal end of triadin. Treatment of sarcoplasmic reticulum vesicles with endoproteinase Arg C resulted in the removal of the N-terminal end of triadin, probably due to cleavage after Arg-34. This is a further indication of the cytoplasmic localization of the N-terminal end of triadin (and of its first 34 amino acids). When the proteolysis with endoproteinase Arg C was carried out with permeabilized vesicles, the cleavage occurred after Arg-141 or Arg-157, indicating that at least one of these residues is luminal.

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.

NMR structures of ferredoxin chloroplastic transit peptide from Chlamydomonas reinhardtii promoted by trifluoroethanol in aqueous solution.

The 32-amino acid transit peptide of the unicellular green alga Chlamydomonas reinhardtii ferredoxin has been synthesized and analysed by NMR spectroscopy and circular dichroism. The results show that while the peptide is unstructured in water, it undergoes an alpha-helix formation from residue 3 to 13 in a 30:70 molar-ratio mixture of 2,2,2-trifluoroethanol. The remainder of the peptide is still unstructured in CF3CD2OD/H2O mixtures, but is distributed on a side opposite to a hydrophobic ridge formed by Met5, Phe9 and Val13 on the induced alpha-helix. The NMR structures driven by 2,2,2-trifluoroethanol in aqueous solution, are discussed in terms of potent interactions with the chloroplast envelope and its translocation molecular machinery.

Transmembrane orientation of the N-terminal and C-terminal ends of the ryanodine receptor in the sarcoplasmic reticulum of rabbit skeletal muscle.

Antibodies were raised against synthetic peptides corresponding to the N-terminal (residues 2-15) and the C-terminal (residues 5027-5037) parts of the rabbit skeletal muscle ryanodine receptor. The specificity of the antibodies generated was tested by e.l.i.s.a., Western blotting and immunofluorescence. All these tests demonstrated the specificity of the antibodies and their ability to react with both the native and the denaturated ryanodine receptor. Both the anti-N-terminus and the anti-C-terminus antibodies bound to sarcoplasmic reticulum vesicles, indicating that each end of the membrane-embedded ryanodine receptor is exposed to the cytoplasmic side of the vesicles. These immunological data were complemented with proteolysis experiments using carboxypeptidase A. Carboxypeptidase A induced degradation of the C-terminal end of the ryanodine receptor in sarcoplasmic reticulum vesicles and a concomitant loss of reactivity of the anti-C-terminus antibodies in Western blots, providing extra evidence for the cytoplasmic localization of the C-terminal end of the ryanodine receptor.

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.

Interaction of C1 with HIV-1.

In contrast to animal retroviruses such as murine leukemia virus, HIV-1 is not lysed by human complement. Nevertheless, HIV-1 activates complement via the classical pathway independently of antibody. Evidence is provided for activation of the reconstituted C1 complex by the virus, resulting from direct interaction between C1q and the external part of the viral transmembrane envelope protein (sgp41). Using C1q fragments and synthetic peptides covering the putative interaction regions in C1q and sgp41, we obtain evidence that the C1q/HIV-1 interaction involves: A site on C1q that appears to be located in the intermediary region between the collagen-like and the globular regions of C1q, and which may be conformational, involving two or more C1q chains. A site on gp41 located between residues 601 and 613 (gp160 nomenclature), i.e. within the immunodominant domain of HIV-1. This site shares homology with the corresponding region of HIV-2.

Localization of a putative magnesium-binding site within the cytoplasmic domain of the sarcoplasmic reticulum Ca(2+)-ATPase.

The amino acid sequences of several P-type ATPases share regions of high similarity. The functions of some of these regions, although several proposals have been made, have not yet been absolutely identified. In particular, one of these domains, located within the cytoplasmic loop in the area known as the 'hinge' domain, exhibits the highest degree of conservation. In the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA-1), this region is located at residues 700-712. Comparison of the sequence in this domain with calcium-binding proteins reveals similarities with the center of the helix-loop-helix EF-hand structure that is known to form divalent-cation-binding sites. A 38-residue polypeptide, corresponding to the domain 682-719 of the Ca(2+)-ATPase was synthesized and tested for its ability to bind divalent cations. Circular-dichroism, intrinsic-fluorescence and fluorescence-energy-transfer studies performed on this polypeptide in solution support the hypothesis that this domain has, in the protein, the ability to bind a divalent cation, presumably Mg2+, with an affinity of 10-15 mM. This property is observed for the isolated polypeptide in aqueous solvent and in the presence of low concentrations of the alpha-helix promoter 2,2,2-trifluoroethanol. Substitution of either one or two critical amino acids in the sequence induces a significant reduction of the binding properties. It is proposed that this sequence is involved in the co-ordination of a Mg2+ in the nucleotide-binding site and/or in the phosphorylation site of P-type ATPases.