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,N-dimethyl(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.

Localization of ligand-binding sites on human C1q globular head region using recombinant globular head fragments and single-chain antibodies.

As a charge pattern recognition molecule, human C1q can bind a range of immunoglobulin and non-immunoglobulin ligands via its carboxy-terminal globular domain and activate the classical complement pathway. Each globular domain has a heterotrimeric organization, composed of the carboxy-terminal halves of one A (ghA), one B (ghB), and one C (ghC) chain. Recently, we have found that the recombinant forms of individual ghA, ghB and ghC bind differentially to IgG, IgM, gp41 peptide 601-613 of human immunodeficiency virus-1 (HIV-1), gp21 peptide 400-429 of human T cell lymphotrophic virus-I (HTLV-I), beta-amyloid peptide, and apoptotic cells, suggesting a modular organization of the globular domain. This paper examines the interaction of ghA, ghB and ghC with two known C1q ligands: Klebsiella pneumoniae porin OmpK36 and salivary agglutinin. In addition, we have used a panel of recombinant single-chain antibodies (scFv) specific for ghA, ghB and ghC in order to map sites on the heterotrimeric globular domain which are likely to interact with IgG1, IgG3, IgM, OmpK36, salivary agglutinin and gp41 loop peptide. The combined use of recombinant ghA, ghB, ghC and single-chain antibodies has revealed at least three ligand-binding sites on the globular domain of C1q: one is IgG- and OmpK36-specific, the second (IgM-binding site) is most likely overlapping with IgG/OmpK36 binding site, and the third (the gp41-binding site) seems to be located at the junction between the collagen and globular domains.

Monoclonal antibody against a cell wall marker protein for embryogenic potential of Dactylis glomerata L. suspension cultures.

We identified and isolated a monoclonal antibody (MAb 3G2) raised against extracellular proteins from microcluster cells of orchard grass (Dactylis glomerata L.) embryogenic suspension culture. MAb 3G2 recognized with high specificity an antigen ionically bound within the primary cell wall and in the culture medium of microcluster cells. Two-dimensional polyacrylamide gel analysis and blotting of proteins on PVDF membrane showed that MAb 3G2 detected a single polypeptide of apparent molecular mass of 48 kDa and an isoelectric point (pI) of 5.2, designated EP48. A transient expression during somatic embryogenesis was observed for EP48. Indirect immunofluorescence showed that this protein highly accumulated in the cell walls of some single cells, microclusters and partly in proembryogenic masses (PEMs), but not in globular embryos of the embryogenic cell line and microclusters from the non-embryogenic cell line. Signal intensity varied between individual cells of the same population and in successive stages of somatic embryo development. Screening of several D. glomerata L. embryogenic and non-embryogenic cell lines with MAb 3G2 indicated the presence of ECP48 in only embryogenic suspension cultures at early stages of embryo development long before morphological changes have taken place and thus it could serve as an early marker for embryogenic potential in D. glomerata L. suspension cultures.

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 Arg(114) and Arg(129) of the ghB module, in the C1q-IgG interaction.