Ulrich Krawinkel

MATRIX METALLOPROTEINASE MMP-19 (RASI-1) IS EXPRESSED ON THE SURFACE OF ACTIVATED PERIPHERAL BLOOD MONONUCLEAR CELLS AND IS DETECTED AS AN AUTOANTIGEN IN RHEUMATOID ARTHRITIS

In order to characterize the autoimmune response participating in the pathogenesis of rheumatoid arthritis (RA) a cDNA expression library constructed from mRNAs which had been isolated from the inflamed synovium of an RA patients was screened with autologous IgG autoantibodies. This led to the identification of gene rasi-1 which encodes a protein showing sequence identity with the zinc-binding matrix metalloproteinase MMP-19. MMP-19 is detected on the surface of activated PBMCs, TH1 lymphocytes, and Jurkat T lymphoma cells. It exhibits gelatinolytic activity and is recognized by autoantibodies in 26% and, respectively, 33% of sera collected from RA patients and systemic lupus erythematosus (SLE) patients. The novel autoantigen MMP-19 thus could play a role in the pathological processes participating in RA-associated joint tissue destruction.

The matrix metalloproteinase RASI-1 is expressed in synovial blood vessels of a rheumatoid arthritis patient

RASI-1 is a novel matrix metalloproteinase which we isolated from an expression cDNA library representing the mRNA of an inflamed synovium obtained from a patient with rheumatoid arthritis (RA). To investigate the involvement of RASI-1 in the pathology of RA, we examined synovial specimens from RA patients with antibodies directed against an unique RASI-1-derived peptide. In comparison to interstitial collagenase, gelatinase A and B, and stromelysin 1, the RASI-1 expression in the RA-synovium is located mainly in the tunica media of blood vessel walls and its synovial localization is not as ubiquitous as that of other MMPs. The tissue inhibitor of metalloproteinases (TIMP-1), although also widely expressed in the synovium, exhibits strong colocalization with RASI-1 in blood vessel walls. While RASI-1 is expressed in blood vessels of the inflamed synovium of an RA patient, its expression was not found in control synovial specimens from patients with luxation and arthrosis. However, RASI-1 expression can also be found in non-inflamed blood vessels of uterine ligaments and skin. RASI-1, although its function and substrates are unknown, could be involved in processes such as neovascularization and angiogenesis or lymphocyte extravasation and thus may participate in joint tissue destruction during RA.

Mass Spectrometric Identification of Leucine Zipper-like Homodimer Complexes of the Autoantigen L7

The eucaryotic protein L7 has been shown to associate in the cytoplasm with the large subunit of ribosomes and to interact specifically with as yet unknown cognate sites of mRNA, thereby inhibiting cell-free translation (Neumann, F., Hemmerich, P., von Mikecz, A., Peter, H. H., and Krawinkel, U. (1995) Nucleic Acids Res. 23, 195-202). The N-terminal region of protein L7 contains a sequence motif similar to the leucine zipper domain of eucaryotic transcription factors, which promotes dimerization through α-helical coiled coil formation. Using electrospray-ionization mass spectrometry as a method of molecular specificity, we have directly identified the dimeric complexes comprising the leucine zipper-like region of protein L7 and have determined the dissociation constant of L7 homodimers in an affinity binding assay. We also demonstrate the high content of α-helicity of the dimer by circular dichroism spectra and computer-based structure simulation and show that the leucine zipper region of protein L7 is fully sufficient to mediate the inhibition of cell-free mRNA translation. A structural basis for the function of L7 to regulate translation is discussed. From the present results we conclude that L7 interacts with double stranded mRNA in a similar fashion as leucine zipper proteins with specific cognate sites on double stranded DNA.

Autoantigenic epitopes on eukaryotic L7.

Ribosomal protein L7 has been established recently as a novel autoantigen representing a frequent target for autoantibodies from patients with systemic autoimmune diseases. Up to 75% of systemic lupus erythematosus (SLE) patients and 50% of mixed connective tissue disease (MCTD) and progressive systemic sclerosis (PSS) patients produce antibodies against in vitro translated L7 and form immunoprecipitable complexes. In this study the B cell response to protein L7 was investigated with respect to the immunogenic determinants recognized by autoantibodies. Eighteen truncated fragments of protein L7 were generated as recombinant fusions with glutathione‐S‐transferase and examined by immunoblotting for their reactivity with sera from patients suffering from systemic rheumatic diseases. Anti‐L7 antibodies target three major non‐overlapping autoepitopes. Two epitopes reside in the highly conserved C‐terminal part of the protein, whereas the N‐terminal autoepitope is not conserved during evolution. The N‐terminal epitope comprises 24 amino acid residues. Ten amino acid resides of this epitope are shared with the BZIP‐like RNA binding domain of protein L7. Autoantibodies recognizing this epitope cross‐react with the corresponding region of a L7 homologue, namely ribosomal protein L7 (RPL7) from Dictyostelium discoideum. This indicates that amino acid residues 14VPE…KKR22, which are conserved between humans and fungi, contribute essentially to the formation of autoantibody‐autoantigen complexes.

Specific Interactions of the Autoantigen L7 with Multi-zinc Finger Protein ZNF7 and Ribosomal Protein S7

The eucaryotic protein L7, which associates with the large subunit of ribosomes, has been shown to be a major autoantigen in systemic autoimmune arthritis. The N terminus carries a sequence motif that is similar to the leucine zipper domain of eucaryotic transcription factors. This domain promotes the homodimerization of protein L7 through α-helical coiled-coil formation and binds to distinct mRNAs, thereby inhibiting their cell-free translation. Using a yeast two-hybrid selection, we have identified from a Jurkat T lymphoma cDNA library ribosomal protein S7 and the multi-zinc finger protein ZNF7 as proteins that interact with protein L7. A fragment of L7 carrying the leucine zipper-like domain is fully sufficient to mediate these interactions. Their potential biological significance is indicated by low apparent dissociation constants of S7-L7 (15 × 10−9 m) and, respectively, ZNF7-L7 (2 × 10−9 m) complexes and co-immunoprecipitation of proteins S7, ZNF7, and L7 from a cell lysate with an anti-L7 antibody. We also show that ZNF7-like L7 and S7 can exist in a ribosome-bound form. This study provides further evidence suggesting that L7 is involved in translational regulation through interactions with components of the translational apparatus.

Mass Spectrometric Analysis of Noncovalent Complexes Between Synthetic Peptides from Human Ribosomal Protein L7 and Protein G

In order to gain a comprehensive insight into the complexes of human ribosomal protein L7 with protein G in a certain degree, an investigation on the complexes of five synthetic L7 peptides, containing the basic-region-leucine-zipper (BZIP)-like domain (aa 15–49), with protein G was performed using nanoelectrospray ionization mass spectrometry (nanoESI-MS). Circular dichroism (CD) was used to characterize the secondary structures of L7 peptides. The characteristics of the complexes between L7 peptides and protein G were studied under various conditions, such as molar ratio of ligands, solvent condition, declustering potential, and peptide sequence. The stability of the complexes is found to decrease with increased declustering potential (>20 V), decreased pH (<5), increased pH (>5), while L7 peptide sequence had no obvious effect on the complex formation. Taken together, the complexes of L7 peptides with protein G are specific noncovalent binding with 1:1 stoichiometry. Because of the availability of synthetic L7 peptides, they might be used as baits to discover the binding partners of protein L7. Furthermore, the elaboration of the binding mechanisms of L7 peptides with protein G could benefit further application of protein G.

Specific interactions of the autoantigen L7 with multi zinc finger protein ZNF7 and ribosomal protein S7

The eucaryotic protein L7, which associates with the large subunit of ribosomes, has been shown to be a major autoantigen in systemic autoimmune arthritis. The N terminus carries a sequence motif that is similar to the leucine zipper domain of eucaryotic transcription factors. This domain promotes the homodimerization of protein L7 through alpha-helical coiled-coil formation and binds to distinct mRNAs, thereby inhibiting their cell-free translation. Using a yeast two-hybrid selection, we have identified from a Jurkat T lymphoma cDNA library ribosomal protein S7 and the multi-zinc finger protein ZNF7 as proteins that interact with protein L7. A fragment of L7 carrying the leucine zipper-like domain is fully sufficient to mediate these interactions. Their potential biological significance is indicated by low apparent dissociation constants of S7-L7 (15 x 10(-9) M) and, respectively, ZNF7-L7 (2 x 10(-9) M) complexes and co-immunoprecipitation of proteins S7, ZNF7, and L7 from a cell lysate with an anti-L7 antibody. We also show that ZNF7-like L7 and S7 can exist in a ribosome-bound form. This study provides further evidence suggesting that L7 is involved in translational regulation through interactions with components of the translational apparatus.