C-reactive protein binds to Fc gamma RI in transfected COS cells.

C-Reactive protein (CRP) is an acute phase serum protein in man that binds to certain bacterial polysaccharides and to components exposed on damaged cells. CRP is bound by receptors on phagocytic cells and functions as an opsonin for its ligands. Interactions of CRP with a specific CRP receptor (CRP-R) and with the high affinity receptor for IgG, Fc gamma RI, on monocytic cells have previously been demonstrated. It was not possible to fully characterize CRP binding to Fc gamma RI in these studies, since cells and cell lines expressing Fc gamma RI also have the CRP-R. In the present study we examined the interaction of CRP with Fc gamma RI in COS-7 cells transfected with a cDNA encoding this receptor. Expression of Fc gamma RI and specific CRP binding to transfected cells were demonstrated by flow cytometry. By two-color analysis, the cell population binding CRP was the same as the population that bound the Fc gamma RI-specific mAb 10.1 and 32.2 CRP inhibited the binding of radiolabeled IgG1 and IgG4 by up to 60%. A CRP molecule that was mutated in the amino acid sequence homologous to the IgG sequence proposed to interact with Fc gamma RI failed to bind to transfected cells, but retained the ability to bind to the CRP-R on monocytic cells. These studies confirm the binding of CRP to Fc gamma RI and identify a site on CRP that is essential for this binding.

C-reactive protein (CRP) binding to the Sm-D protein of snRNPS. Identification of a short polypeptide binding region.

C-reactive protein (CRP) binds to chromatin, histones, and small nuclear ribonucleoproteins (snRNPs) through a phosphocholine (PC)-inhibitable, calcium-dependent binding site. snRNPs process pre-mRNA to mature mRNA and are composed of small uridine-rich RNAs (designated U1, U2, U5 and U4/U6) and associated proteins. We have shown that CRP binds to snRNPs in intact cells and to the U1 snRNP-specific 70 K protein in cell extracts. To determine whether CRP bound to other snRNP proteins, snRNPs were purified from rabbit thymus extract and CRP binding was assessed by blotting. CRP bound to a protein with the same mobility as Sm-D as well as to the 70 K protein. CRP specifically bound to and precipitated a fusion protein containing full-length Sm-D, confirming the binding of CRP to Sm-D. Binding was inhibited by PC and by EDTA. Binding studies using deletion mutants of the Sm-D fusion protein revealed that CRP binding was mediated by the C-terminal region of Sm-D, a region which binds autoantibodies and is proposed to bind to RNA. A comparison of the peptide regions on different autoantigens suggests that there is a shared motif to which CRP binds.

Anti-class II beta-chain antibodies in the serum and synovial fluid of rheumatoid arthritis patients.

Sera and synovial fluid (SF) from rheumatoid arthritis (RA) patients were evaluated for anti-HLA class II beta-chain antibodies using single and two-dimensional immunoblots. The antibodies from RA sera and SFs which reacted with class II beta-chain determinants were predominantly IgM and IgA with minimal IgG. This reactivity was also present in SFs from other rheumatic diseases. Anti-class II beta-chain antibodies were also shown to be present simultaneously in RA sera and SF.

anti-DR antibodies inhibit in vitro production of human rheumatoid factor.

Anti-MHC class II antibodies have been shown to have a profound effect on the immune system and have been used successfully in the therapy of human and animal autoimmune diseases. In addition, naturally-occurring anti-DR antibodies have been observed in the sera of rheumatoid arthritis (RA) and systemic lupus erythematosus patients. Now, we demonstrate that several monoclonal anti-DR, but not anti-DQ antibodies specifically inhibit the production of rheumatoid factor (RF) in pokeweed-stimulated cultures of peripheral blood mononuclear cells from RA patients. Moreover, partially-purified anti-DR antibodies from RA sera have similar inhibitory effects on in vitro RF synthesis. These results indicate the inhibition of autoantibody production as a possible mechanism operative in immunotherapy using anti-class II antibodies. Furthermore, this data also suggests a protective, beneficial role for the endogenous anti-DR autoantibodies in RA.

Network regulation in rheumatoid arthritis. Studies of DR+ T cells, anti-DR, antiidiotypic antibodies, and clinical disease activity.

We prospectively studied rheumatoid arthritis patients with various degrees of clinical disease activity, for the presence of DR+ T cells by flow cytometry, for anti-DR using immunoblot analysis, and for antiidiotypic (anti-id) antibodies by enzyme-linked immunosorbent assay using F(ab')2 monoclonal antibody anti-DR L243 as idiotype. DR+ T cells correlated positively with anti-DR, and anti-id correlated negatively with both DR+ T cells and anti-DR. Active clinical disease correlated positively with both DR+ T cells and anti-DR, and correlated negatively with anti-id. This DR antigen/anti-DR/anti-id network may control disease activity in rheumatoid arthritis patients.

Characterization of the phosphorylated small enzyme subunit, NS, of the vesicular stomatitis virus RNA polymerase.

NS protein of vesicular stomatitis virus was shown to migrate with a mobility consistent with the molecular weight predicted from the published cDNA sequence on polyacrylamide gels containing the detergent cetyltrimethylammonium bromide at low pH. Cyanogen bromide cleavage of NS protein produced a large acidic amino-terminal peptide, as predicted by the sequence, which contained the majority of the phosphate residues. However, analysis of tryptic peptides by high-performance liquid chromatography suggested that there may be inaccuracies in the sequence of the carboxyl terminus of the sequence.

The major ribonucleoprotein-associated protein kinase of vesicular stomatitis virus is a host cell protein.

Ribonucleoprotein particles (RNPs) of vesicular stomatitis virus (VSV) were fractionated by column chromatography through Fractogel TSK HW-55F and by centrifugation through KCl sucrose. Analyses of fractions for protein content and for protein kinase activity indicated that the major peak of kinase activity did not correspond exactly with any of the VSV-specific proteins. Neither anti-NS nor anti-M IgG preparations inhibited protein kinase activity, and IgG did not act as an exogenous phosphate acceptor. Reconstitution of an RNP-enzyme complex did not result in a restoration of protein kinase activity. In vitro translation of VSV-specific poly(A)-containing RNA did not result in any detectable production of kinase activity. Thus, the major RNP-associated kinase is a host cell protein which is tightly bound to the RNP particle.

Effect of a mutation in the VSV G protein on the synthesis and maturation of nucleocapsids.

The tsO45 (V) mutant of vesicular stomatitis virus (VSV) has been used to demonstrate that discrimination between positive (+) and negative (-) strand nucleocapsids in the process of budding mature virions is maintained despite the lack of detectable G protein in either the plasma membrane or the virus membrane. These data indicate that the VSV G protein plays no active role in the discrimination between negative- and positive-strand nucleocapsids which will mature to virions. The synthesis of intracellular nucleocapsids in tsO45 (V)-infected cells at permissive and non-permissive conditions was also examined in comparison with that of the wild-type virus. No differences were observed in intracellular nucleocapsid quantity or polarity in any of these cases despite a 97% inhibition of virus yield in the case of tsO45 (V) at non-permissive temperature.