Ivanka Tsacheva

Mutational Analyses of the Recombinant Globular Regions of Human C1q A, B, and C Chains Suggest an Essential Role for Arginine and Histidine Residues in the C1q-IgG Interaction

The first step in the activation of the classical complement pathway by immune complexes involves the binding of the globular domain (gC1q) of C1q to the Fc regions of aggregated IgG or IgM. Each gC1q domain is a heterotrimer of the C-terminal halves of one A (ghA), one B (ghB), and one C (ghC) chain. Our recent studies have suggested a modular organization of gC1q, consistent with the view that ghA, ghB, and ghC are functionally autonomous modules and have distinct and differential ligand-binding properties. Although C1q binding sites on IgG have been previously identified, the complementary interacting sites on the gC1q domain have not been precisely defined. The availability of the recombinant constructs expressing ghA, ghB, and ghC has allowed us, for the first time, to engineer single-residue substitution mutations and identify residues on the gC1q domain, which are involved in the interaction between C1q and IgG. Because C1q is a charge pattern recognition molecule, we have sequentially targeted arginine and histidine residues in each chain. Consistent with previous chemical modification studies and the recent crystal structure of gC1q, our results support a central role for arginine and histidine residues, especially Arg114 and Arg129 of the ghB module, in the C1q-IgG interaction.

New insight into the autoimmunogenicity of the complement protein C1q

C1q along with its physiological role in maintenance of homeostasis and normal function of the immune system is involved in pathological conditions associated with repetitive generation of anti-C1q autoantibodies. The time and events that cause their first appearance are still unknown. We addressed this issue by analyzing the immunogenicity of C1q in two target groups-one of non-diseased humans and the other of lupus nephritis (LN) patients whose autoimmune disorder is associated with high titers of anti-C1q autoantibodies. The non-diseased humans were represented by pregnant women because the sex hormones are thought to be involved in triggering autoimmune pathologies by their ability to tip the balance of female adaptive immune response to production of antibodies. We screened, using ELISA, 31 sera from healthy pregnant women for the presence of IgM and IgG classes of autoantibodies, recognizing epitopes within the native C1q molecule, its collagen-like region (CLR) and globular head fragment (gC1q). The latter was represented by recombinant analogs of the three globular fragments of A, B and C chains, comprising C1q-ghA, ghB and ghC. We did not find IgM antibodies for all test-antigens which suggest that the natural IgM antibodies are not involved in triggering autoimmunity to C1q. Still more, we did not detect anti-CLR antibodies which have been proved pathogenic in already manifested LN. We completed the analysis with comparative epitope mapping of gC1q and we found similar immunogenic behavior in both target groups-ghA and ghC contained the immunodominant epitopes. This implies that the initial immune response to C1q might occur when the molecule has interacted with its ligands via ghB as part of gC1q. The presence of anti-gC1q in both healthy and diseased humans also implies that these antibodies, unlike anti-CLR, may have a contribution to an onset of autoimmunity.

Biochemical analysis of the epitope specificities of anti-C1q autoantibodies accompanying human lupus nephritis reveals them as a dynamic population in the course of the disease

We analyzed the epitope specificities of the polyclonal anti-C1q antibodies, present in human LN sera, searching to deduce the structural characteristics of C1q associated with its transition to an autoantigen. We screened 78 serum samples from LN patients distributed in three clinical groups - non-active, moderately active and severely active. We found three classes of C1q autoepitopes: (a) neo-epitopes, exposed upon immobilization due to conformational changes; (b) epitopes formed by sequences that are brought together by the conformation of the whole molecule; (c) cryptic epitopes that become exposed only after fragmentation of C1q. The latter suggest that the immunogen involved in the initiation of anti-C1q autoantibodies might be an extrinsic molecule that shares some degree of structural similarity to C1q. None of the tested epitope specificities was associated with active LN. We found a prevalence of anti-gC1q antibodies among the non-active LN patients suggesting that they might be the fraction of the polyclonal anti-C1q, preceding the initiation of autoimmunity to C1q, or alternatively, preceding LN flare.

Generation of Recombinant Antibodies against Orchardgrass Acidic nsLTP-Like Proteins

Embryogenic and non-embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.) secreted into the culture medium a set of proteins, among which low molecular mass (11/12 kDa) proteins were found. However, only the 11/12 kDa proteins from the embryogenic suspension cultures reacted specifically with an antiserum raised against the carrot EP2 non-specific lipid transfer protein (nsLTP). Two-dimensional (2-D) electrophoretic analysis revealed that the extracellular nsLTP-like proteins from the embryogenic lines were acidic proteins, with pI values ranging between 4.3 and 6.4, and the 11/12 kDa proteins of the nonembryogenic lines were basic ones (pI 8-9.3). This is only the second case to report on the accumulation of extracellular acidic nsLTP-like proteins in the culture medium during somatic embryogenesis. A naı¨ve phage display Griffin1. library was used to select single-chain phage antibodies, which specifically bind to acidic nsLTP-like proteins. Nine phage clones were selected after four rounds of biopanning of the target proteins blotted on a nitrocellulose membrane. Three soluble monoclonal single-chain phage antibodies, expressed in the non-suppressor E. coli strain HB2151, were purified by metal affinity chromatography and found to be highly specific for the acidic nsLTP-like proteins from the embryogenic suspension cultures. The application of the selected monoclonal antibodies for localization and elucidation of the role of the acidic nsLTP-like proteins in vivo is discussed.

Identification, Molecular Cloning, and Recombinant Gene Expression of an Extracellular A-Amylase from Dactylis Glomerata L. Embryogenic Suspension Cultures

ABSTRACT Somatic embryogenesis is a unique phenomenon in the plant kingdom and involves the transition of somatic cells into cells that are capable of forming an embryo. Proteins secreted into the medium of suspension cell cultures play an important role by either promoting or inhibiting embryo development. A monoclonal antibody MAb 3G2 raised against extracellular proteins from orchardgrass (Dactylis glomerata L.) embryogenic suspension culture recognized specifically a 48 kD protein with pI 5.2 (designated EP48) from the cell wall and culture medium of microclusters, which could serve as an early marker for embryogenic potential in D. glomerata cultures [Tchorbadjieva M., Kalmukova R., Pantchev I., Kyurkchiev S. 2005. Planta, 222, 811–819]. In this study, in-gel trypsin digestion of EP48 separated by 2-D gel electrophoresis and LC-MS/MS mass spectrometry identified the protein as α-amylase. The full-length cDNA encoding D. glomerata L. α-amylase (designated DgAmy1, GenBank accession number GU 067465.1) was cloned by rapid amplification of cDNA ends (RACE) and was shown to contain an open reading frame of 1284 bp encoding α-amylase protein of 427 amino acids with a calculated molecular mass of the mature protein of 44.742 kDa and pI of 5.12. Comparative analysis showed that DgAmy1 shares an overall identity of 59–89% with α-amylases from other plant species, and is most closely related to that of barley (Hordeum vulgare). Homology modeling using barley α-amylase Amy2 as a template revealed that DgAmy1 contains three domains (A, B, C) and active sites that are common with other plant α-amylases. However, it lacks the substrate binding sites 1 and 2 typical for cereal α-amylases. The recombinant DgAmy1 expressed in E. coli BL21 (DE3) and purified by Ni2+ Sepharose was biologically active as revealed by activity zymography using soluble starch as a substrate. The high yield (200 mg protein from 1 L culture medium) and purity (99%) of the recombinant DgAmy1 will be helpful for further understanding of the structure/function correlations and especially the role that this extracellular α-amylase plays at early stages of somatic embryogenesis.

Chaperone-Like Effect of Polyzwitterions on the Interaction of C1q with IgG

The amphiphilic polyzwitterion (PZ) poly(ethylene oxide-b-N,N-dimethyl(methacryloyloxyethyl) ammonium propanesulfonate), zwitterionic surfactant (ZS) n-dodecyl- N,N-dimethyl-3-ammonium-1-propanesulfonate, and zwitterionic monomer (ZM) N,Ndimethyl( methacryloyloxyethyl)ammonium propanesulfonate were analyzed for their suggested chaperone-like effect on the interaction of C1q and IgG. Our results proved that the PZ retarded the C1q interaction with IgG, demonstrating a specific protein-folding helper effect. The ZS enhanced this interaction, when the ZS concentration was lower than the critical micelle concentration (CMC), and retarded it, when the ZS concentration was above the CMC. The ZM, with no self-assembling ability, did not influence this interaction. These results support the hypothesis of a hydrophobic interaction between Pts and hydrophobic domains of partly denatured protein molecules. The amphiphilic self-assemblies, formed by polyzwitterionic macromolecules or zwitterionic surfactants, have the ability to transform the hydrophobic domains of the protein molecules into hydrophilic ones, covering them with their hydrophilic parts.

ENHANCEMENT OF THE SENSITIVITY OF THE SOLID PHASE C1q BINDING ASSAY BY IMMOBILIZATION OF C1q VIA C1q-SPECIFIC RECOMBINANT ANTIBODY scFv9(G)

ABSTRACT A new and improved ELISA method for immune complex detection [scFv9(G)-Clq ELISA] was developed. The assay is based on the C1q-solid phase binding test. C1q was immobilized on microtiter ELISA plates, coated with the single-chain antibody scFv9(G), specific to the collagen-like region of C1q. The obtained results indicated a significantly reduced nonspecific background, when C1q was immobilized via the scFv9(G)-capture molecule. The comparative analysis between the two wildly used methods for IC detection (C1q-SP-RIA and QUIDEL CIC—C1q ELISA) and the new improved Fv9(G)-C1q ELISA shows a good agreement. The method is simple, reproducible and may be a good alternative for the routine C1q-SP-RIA.

Registration of the Interaction Between C1q Human Complement Derivatives and Immunoglobulins by Elisa—Role of the Solid Phase

ABSTRACT The interaction of C1q, the first subcomponent of the human complement, with its immunoglobulin ligands from immune complexes is the crucial step in the activation of the classical complement pathway. Thus the mechanism of these interaction and the factors, which influence them, are from high interest. In the present study the effect of immobilization of the interacting proteins on a solid support in ELISA was investigated. The obtained results lead us to the conclusion, that the immobilization process may have a significant influence on the binding activity of tested proteins, especially when hydrophobic interactions are involved.