Véronique Rossi

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 Ca2+. 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 KD 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 Ca2+-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 Ca2+-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.

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 Å 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.

Identification of the C1q-binding Sites of Human C1r and C1s A REFINED THREE-DIMENSIONAL MODEL OF THE C1 COMPLEX OF COMPLEMENT

The C1 complex of complement is assembled from a recognition protein C1q and C1s-C1r-C1r-C1s, a Ca2+-dependent tetramer of two modular proteases C1r and C1s. Resolution of the x-ray structure of the N-terminal CUB1-epidermal growth factor (EGF) C1s segment has led to a model of the C1q/C1s-C1r-C1r-C1s interaction where the C1q collagen stem binds at the C1r/C1s interface through ionic bonds involving acidic residues contributed by the C1r EGF module (Gregory, L. A., Thielens, N. M., Arlaud, G. J., Fontecilla-Camps, J. C., and Gaboriaud, C. (2003) J. Biol. Chem. 278, 32157–32164). To identify the C1q-binding sites of C1s-C1r-C1r-C1s, a series of C1r and C1s mutants was expressed, and the C1q binding ability of the resulting tetramer variants was assessed by surface plasmon resonance. Mutations targeting the Glu137-Glu-Asp139 stretch in the C1r EGF module had no effect on C1 assembly, ruling out our previous interaction model. Additional mutations targeting residues expected to participate in the Ca2+-binding sites of the C1r and C1s CUB modules provided evidence for high affinity C1q-binding sites contributed by the C1r CUB1 and CUB2 modules and lower affinity sites contributed by C1s CUB1. All of the sites implicate acidic residues also contributing Ca2+ ligands. C1s-C1r-C1r-C1s thus contributes six C1q-binding sites, one per C1q stem. Based on the location of these sites and available structural information, we propose a refined model of C1 assembly where the CUB1-EGF-CUB2 interaction domains of C1r and C1s are entirely clustered inside C1q and interact through six binding sites with reactive lysines of the C1q stems. This mechanism is similar to that demonstrated for mannan-binding lectin (MBL)-MBL-associated serine protease and ficolin-MBL-associated serine protease complexes.

Structural biology of C1: dissection of a complex molecular machinery

The classical pathway of complement is initiated by the C1 complex, a multimolecular protease comprising a recognition subunit (C1q) and two modular serine proteases (C1r and C1s) associated as a Ca2+‐dependent tetramer (C1s‐C1r‐C1r‐C1s). Early studies have allowed identification of specialized functional domains in these proteins and have led to low‐resolution models of the C1 complex. The objective of current studies is to gain deeper insights into the structure of C1, and the strategy used for this purpose mainly consists of dissecting the C1 components into modular fragments, in order to solve their three‐dimensional structure and establish the structural correlates of their function. The aim of this article is to provide an overview of the structural and functional information generated by this approach, with particular emphasis on the domains involved in the assembly, the recognition function, and the highly specific proteolytic properties of C1.

Functional Characterization of Complement Proteases C1s/Mannan-binding Lectin-associated Serine Protease-2 (MASP-2) Chimeras Reveals the Higher C4 Recognition Efficacy of the MASP-2 Complement Control Protein Modules

C1s and mannan-binding lectin-associated serine protease-2 (MASP-2) are the proteases that trigger the classical and lectin pathways of complement, respectively. They have identical modular architectures and cleave the same substrates, C2 and C4, but show markedly different efficiencies toward C4. Multisite-directed mutagenesis was used to engineer hybrid C1s/MASP-2 molecules where either the complement control protein (CCP) modules or the serine protease (SP) domain of C1s were swapped for their MASP-2 counterparts. The resulting chimeras (C1s(MASP-2 CCP1/2) and C1s(MASP-2 SP), respectively) were expressed and characterized chemically and functionally. Whereas C1s(MASP-2 SP) was recovered as an active enzyme, C1s(MASP-2 CCP1/2) was produced in a proenzyme form and was susceptible to activation by C1r, indicating that the activation properties of the chimeras were dictated by the nature of their SP domain. Similarly, each activated chimera had an esterolytic activity characteristic of its own SP domain and cleaved C2 with an efficiency comparable with that of their parent C1s and MASP-2 proteases. Both chimeras cleaved C4, but whereas C1s(MASP-2 SP) and C1s had Km values in the micromolar range, C1s(MASP-2 CCP1/2) and MASP-2 had Km values in the nanomolar range, resulting in 21–27-fold higher kcat/Km ratios. Thus, the higher C4 cleavage efficiency of MASP-2 arises from a higher substrate recognition efficacy of its CCP modules. Remarkably, C1s(MASP-2 CCP1/2) retained C1s ability to associate with C1r and C1q to form a pseudo-C1 complex and to undergo activation within this complex, indicating that the C1s-CCP modules have no direct implication in either function.

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 C s 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 triantennary, trisialylated species NeuAc3 Ga13 GIcNAc5 Man3 Fucl (molecular mass = 3007 ± 1), in relative proportions of approximately 1:1:1. The carbohydrate heterogeneity at Asn391 gives rise to three major types of C s 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.

Functional Characterization of the Recombinant Human C1 Inhibitor Serpin Domain: Insights into Heparin Binding

Variants of the human C1 inhibitor serpin domain containing three N-linked carbohydrates at positions 216, 231, and 330 (C1inhΔ97), a single carbohydrate at position 330 (C1inhΔ97DM), or no carbohydrate were produced in a baculovirus/insect cells system. An N-terminally His-tagged C1inhΔ97 variant was also produced. Removal of the oligosaccharide at position 330 dramatically decreased expression, precluding further analysis. All other variants were characterized chemically and shown to inhibit C1s activity and C1 activation in the same way as native C1 inhibitor. Likewise, they formed covalent complexes with C1s as shown by SDS-PAGE analysis. C1 inhibitor and its variants inhibited the ability of C1r-like protease to activate C1s, but did not form covalent complexes with this protease. The interaction of C1 inhibitor and its variants with heparin was investigated by surface plasmon resonance, yielding KD values of 16.7 × 10−8 M (C1 inhibitor), 2.3 × 10−8 M (C1inhΔ97), and 3.6 × 10−8 M (C1inhΔ97DM). C1s also bound to heparin, with lower affinity (KD = 108 × 10−8 M). Using the same technique, 50% inhibition of the binding of C1 inhibitor and C1s to heparin was achieved using heparin oligomers containing eight and six saccharide units, respectively. These values roughly correlate with the size of 10 saccharide units yielding half-maximal potentiation of the inhibition of C1s activity by C1 inhibitor, consistent with a “sandwich” mechanism. Using a thermal shift assay, heparin was shown to interact with the C1s serine protease domain and the C1 inhibitor serpin domain, increasing and decreasing their thermal stability, respectively.

Deciphering Complement Receptor Type 1 Interactions with Recognition Proteins of the Lectin Complement Pathway

Complement receptor type 1 (CR1) is a membrane receptor expressed on a wide range of cells. It is involved in immune complex clearance, phagocytosis, and complement regulation. Its ectodomain is composed of 30 complement control protein (CCP) modules, organized into four long homologous repeats (A–D). In addition to its main ligands C3b and C4b, CR1 was reported to interact with C1q and mannan-binding lectin (MBL) likely through its C-terminal region (CCP22–30). To decipher the interaction of human CR1 with the recognition proteins of the lectin complement pathway, a recombinant fragment encompassing CCP22–30 was expressed in eukaryotic cells, and its interaction with human MBL and ficolins was investigated using surface plasmon resonance spectroscopy. MBL and L-ficolin were shown to interact with immobilized soluble CR1 and CR1 CCP22–30 with apparent dissociation constants in the nanomolar range, indicative of high affinity. The binding site for CR1 was located at or near the MBL-associated serine protease (MASP) binding site in the collagen stalks of MBL and L-ficolin, as shown by competition experiments with MASP-3. Accordingly, the mutation of an MBL conserved lysine residue essential for MASP binding (K55) abolished binding to soluble CR1 and CCP22–30. The CR1 binding site for MBL/ficolins was mapped to CCP24–25 of long homologous repeat D using deletion mutants. In conclusion, we show that ficolins are new CR1 ligands and propose that MBL/L-ficolin binding involves major ionic interactions between conserved lysine residues of their collagen stalks and surface exposed acidic residues located in CR1 CCP24 and/or CCP25.

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.

Functional Role of the Linker between the Complement Control Protein Modules of Complement Protease C1s

C1s is the modular serine protease responsible for cleavage of C4 and C2, the protein substrates of the first component of C (C1). Its catalytic domain comprises two complement control protein (CCP) modules connected by a four-residue linker Gln340-Pro-Val-Asp343 and a serine protease domain. To assess the functional role of the linker, a series of mutations were performed at positions 340–343 of human C1s, and the resulting mutants were produced using a baculovirus-mediated expression system and characterized functionally. All mutants were secreted in a proenzyme form and had a mass of 77,203–77,716 Da comparable to that of wild-type C1s, except Q340E, which had a mass of 82,008 Da, due to overglycosylation at Asn391. None of the mutations significantly altered C1s ability to assemble with C1r and C1q within C1. Whereas the other mutations had no effect on C1s activation, the Q340E mutant was totally resistant to C1r-mediated activation, both in the fluid phase and within the C1 complex. Once activated, all mutants cleaved C2 with an efficiency comparable to that of wild-type C1s. In contrast, most of the mutations resulted in a decreased C4-cleaving activity, with particularly pronounced inhibitory effects for point mutants Q340K, P341I, V342K, and D343N. Comparable effects were observed when the C4-cleaving activity of the mutants was measured inside C1. Thus, flexibility of the C1s CCP1-CCP2 linker plays no significant role in C1 assembly or C1s activation by C1r inside C1 but plays a critical role in C4 cleavage by adjusting positioning of this substrate for optimal cleavage by the C1s active site.