Showing papers by "Michael Bachmann published in 1998"

The Multiligand-Binding Protein gC1qR, Putative C1q Receptor, Is a Mitochondrial Protein

Abstract: A protein of 33 kDa (p33) that tightly binds to the globular domains of the first complement component, C1q, is thought to serve as the major C1q receptor (gC1qR) on B cells, neutrophils, and mast cells. However, the cellular routing and the subcellular localization of p33/gC1qR are unknown. We have performed confocal laser-scanning microscopy and found that p33/gC1qR is present in intracellular compartments, where it colocalizes with the mitochondrial marker protein, pyruvate dehydrogenase. No surface staining for p33/gC1qR on endothelial EA.hy926 cells was observed. A fusion protein of the p33/gC1qR presequence with green fluorescent protein translocated to the mitochondria of transfected COS-7 cells. Concomitantly, a 6-kDa portion of the fusion protein was proteolytically removed. The 33 amino-terminal residues of the presequence proved sufficient to direct reporter constructs to mitochondria. Association of p33/gC1qR with mitoplasts indicated that the mature protein of 209 residues resides in the matrix and/or the inner membrane of mitochondria. Immunocytochemistry of fetal mice tissues revealed a ubiquitous expression of p33/gC1qR, most prominently in tissues that are rich in mitochondria. Thus, the candidate complement receptor p33/gC1qR of intact cells cannot interact with plasma C1q due to mutually exclusive localizations of the components. The functional role of p33/gC1qR needs to be reconsidered.

The value of synthetic linear epitope analogues of La/SSB for the detection of autoantibodies to La/SSB; specificity, sensitivity and comparison of methods.

Abstract: In a previous study it was shown that La/SSB contains four linear epitopes, p147–154, p291–302, p301–318 and p349–364. The aim of the present study was to investigate the value of the synthetic epitope analogues of the La/SSB autoantigen for the detection of antibodies to La/SSB, in comparison with recombinant La and fragments of this protein. A total of 122 sera with anti-La/SSB activity, from patients with primary Sjogren's syndrome (pSS) or systemic lupus erythematosus (SLE), were tested in various peptide-based assays. In addition, 62 sera from pSS or SLE patients with other autoantibody specificities and 95 sera from healthy individuals were used as controls. The autoantibody specificity was identified by counter immunoelectrophoresis and immunoblot. The peptide-based ELISA assays presented sensitivities ranging from 78% to 88.8% and specificities from 69% to 94.3%. Dot blot assays exhibited sensitivities ranging from 93.6% to 97%, but remarkably lower specificities from 56% to 88%. The most sensitive and specific peptide 349GSGKGKVQFQGKKTKF364 was synthesized and attached on a tetramer sequential oligopeptide carrier SOC4 and used for immunoassay development. Assays based on the recombinant native La protein, the La-C terminal (215 aa), and the N-terminal of La with a mutation at base pair 640 (nine adenines instead of eight) were also developed and compared with the SOC4 peptide-based assay. Of anti-La-positive sera, 88.1% were reactive with both the synthetic peptide SOC4-(349–364aa) and the recombinant La protein. Eighty-three percent of sera were reactive with the La N-terminus and 67.8% of sera were reactive with the La C-terminus. Using sera that were anti-Ro-positive but anti-La-negative, 37% were reactive with the recombinant protein, 26% with the La N-terminus, 33% with the La C-terminus and only 11% with the synthetic peptide. Our results suggest that the synthetic peptide epitopes exhibit high sensitivity and specificity for the detection of anti-La/SSB antibodies in ELISA and dot blot techniques. The peptide SOC4-(349–364aa) has the same sensitivity for the detection of anti-La/SSB antibodies as the recombinant protein.

Identification of human autoantigen La/SS-B as BC1/BC200 RNA-binding protein.

Abstract: Rodent BC1 RNA and primate BC200 RNA are small cytoplasmic non-messenger RNAs that are phylogenetically unrelated. Nevertheless, the two RNAs exhibit a large degree of parallelism. In addition to some sequence similarities in their 3′ domains, they are prevalently expressed in a similar subset of neurons and belong to a small group of transcripts with a somatodendritic location. Both RNAs are complexed with proteins as ribonucleoprotein particles (RNPs). Their similarities may even extend to analogous functional roles, for example, in the regulation of decentralized dendritic translation. To shed further light on the physiological role(s) of the BC1/BC200 RNPs, we began to analyze protein components that specifically bind to these RNAs. Ultraviolet-crosslinking experiments and affinity purification techniques revealed that the human autoantigen La/SS-B is associated with BC1/BC200 RNA in vitro and in vivo. As with other RNA polymerase III transcripts, La protein binds with high affinity to the 3′ end of B...

Analysis of epitope spreading over an eleven-year period in a patient with systemic lupus erythematosus.

Abstract: During a period of more than eleven years serum samples of a patient with Systemic Lupus Erythematosus were collected and analyzed for anti-nuclear autoantibodies. High titer of anti-La/SS-B were detectable in all serum samples. The La/SS-B epitopes remained constant. Besides anti-La/SS-B antibodies all serum samples contained traces of anti-Ro/SS-A including anti-Ro52 and anti-Ro60 antibodies. During disease flares anti-Ro/SS A antibodies were upregulated and anti-dsDNA antibodies appeared, thus supporting the concept of an antigen driven intermolecular epitope spreading to Ro/SS-A and dsDNA.

Sjögren's autoimmunity: how perturbation of recognition in endomembrane traffic may provoke pathological recognition at the cell surface.

Abstract: CD4 T cell antigen recognition requires presentation by major histocompatibility complex Class II molecules (MHC II). B cell surface immunoglobulins recognize antigens independently of MHC II, but activation typically requires CD4 cell cytokines as accessory signals. Plasma membrane-endomembrane traffic in lacrimal gland acinar cells, targets of autoimmune activity in Sjogren's syndrome, may satisfy both requirements. The Golgi protein galactosyltransferase and the lysosomal proteins cathepsin B and cathepsin D appear at the plasma membranes during sustained secretomotor stimulation. The RNA transcription termination factor La, a frequent target of Sjogren's autoantibodies, appears in the acinar cell cytoplasm and plasma membranes during viral infection and during in vitro exposure to cytokines. MHC II cycle through endomembrane compartments which contain La, galactosyltransferase, cathepsin B and cathepsin D and which are sites of proteolysis. This traffic may permit trilateral interactions in which B cells recognize autoantigens at the surface membranes, CD4 T cells recognize peptides presented by MHC II, B cells provide accessory signals to CD4 T cells, and CD4 T cells provide cytokines that activate B cells. Acinar cells stimulate lymphocyte proliferation in autologous mixed cell reactions, confirming that they are capable of provoking autoimmune responses.

The human autoantigen La/SS-B accelerates herpes simplex virus type 1 replication in transfected mouse 3T3 cells.

Abstract: Permanently transfected mouse cell lines which expressed different levels of the human autoantigen La/SS-B were infected with different strains of herpes simplex virus type 1, including the strains ANG, HSZP, 17syn+ and HFEM. During infection the localization of the human La protein was followed using an anti-La MoAb, which recognized only the human La protein but did not cross-react with either the endogenous mouse La protein or any viral encoded protein. After infection La protein was transported from the nucleus to the cytoplasm. The time course of translocation was dependent on the amount of human La protein expressed in the respective cell line. Moreover, acceleration of viral replication was dependent on the level of expression of human La protein, suggesting that La protein is a cellular factor that facilitates virus replication.