Since the FG-repeat motifs of p62 are flexible and mobile in contrast to nucleoporin p58 and nucleoporin p54, which are very similar proteins, this flexible motif might be the reason of strong immunogenicity of p62 [8]

Since the FG-repeat motifs of p62 are flexible and mobile in contrast to nucleoporin p58 and nucleoporin p54, which are very similar proteins, this flexible motif might be the reason of strong immunogenicity of p62 [8]. lamin B receptor Bergamottin and lamin-associated polypeptides as well as autoantibodies against nuclear pore proteins (gp210, Nup153, Tpr, p62) have been defined in different autoimmune diseases [2-4]. The nuclear pore complex (NPC) serves as the sole gate between the nucleoplasm and the cytoplasm and contains several different nuclear pore proteins such as nucleoporin p62. Nucleoporin p62 was cloned and sequenced in 1991 [5] and found not to be identical with the cytoplasmic RNA-binding protein p62 which is an autoantigen in human hepatocellular carcinoma [6]. The amino acid sequence of nucleoporin p62 contains phenylalanine- and glycine- rich (FG) repeat motifs, which are characteristic for nuclear pore proteins [5]. Nucleoporin p62 is usually a glycoprotein and probably therefore highly immunogenic [7]. Since the FG-repeat motifs of p62 are flexible and mobile in contrast to nucleoporin p58 and nucleoporin p54, which are very similar proteins, this flexible motif might be the reason of strong immunogenicity of p62 [8]. One trial explained p62 Bergamottin as an oxalate-binding protein with increased expression in different kidney disorders [9]. Nucleoporins are well explained autoantigens in main biliary cirrhosis where their presence might indicate a more severe course of the disease [10-15]. Anti-nucleoporin antibodies can be also found in systemic connective tissue diseases. For example, in four cases of Sj?grens syndrome [16], four cases of systemic lupus Rabbit polyclonal to HSP90B.Molecular chaperone.Has ATPase activity. erythematosus [9, 10] and one case of Sharp syndrome [17] p62 autoantibodies were identified. In a previous Bergamottin study, we expressed p62 in three recombinant fusion proteins. Autoantibodies in main biliary cirrhosis are realizing the amino terminal fragment of p62 [17]. Since the aminoterminal fragment therefore seems to be the antigen, we used this fragment for further investigations in Lupus erythematosus, to evaluate, if p62 serves as an autoantigen in this disease too. We investigated twenty five patients suffering from the disease. Six of the twenty five patients showed anti-p62 autoantibodies. MATERIALS AND METHODS Collection of Blood Samples Serum blood samples of 25 patients suffering from systemic lupus erythematosus were collected. Prior to the trial the project has been approved by the ethical committee in Wrzburg/Germany. For each patient, the trial was discussed in detail and the approved consent form was signed prior to participation. Systemic lupus erythematosus was diagnosed according the revised criteria of the American College of Rheumatology for the classification of SLE [1, 18]. All patients were characterized by a prolonged time since diagnosis (13.1 years in average) and had received a variety of immunosuppressive drug therapies. 22 patients were female, 3 patients were male. Patient age ranged from 24 years to 64 years. Median age was 41.72. To quantify the disease we used the SLICC/SDI damage score [19]. As controls we used a 24 12 months aged and a 41 12 months old healthy Bergamottin woman. Both controls experienced no antinuclear antibodies or anti-native DNA antibodies. Expression and Purification of p62 Fusion Protein The amino terminal domain name of p62 which contains the phenylalanine- and glycine-repeat (FG-repeat) domain name (amino acid residue 1 to 329) was expressed using a set of non degenerate oligonucleotide primers (5-ATGAGCGGC TTTAATTTTGGAGG-3 and 5-GGTCATGGCGGAGCT GGCAG-3). Primers contained additional restriction sites for HindIII and XHOI. The corresponding PCR product was inserted between the HindIII- and XHOI restriction sites in pET21-b vector (Novagen, Madison, Wisconsin) made up of a six-histidine tag. The construct was transformed into BL21 (DE3). P62-His6 fusion protein was purified under denaturing conditions as explained previously [17]. Gel Electrophoresis and Immunoblotting P62 fusion protein was analyzed by 15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE; 10g of protein/lane) and transferred to Hybond ECL membranes (Amersham, Braunschweig, Germany) at 50 V for 3 h. After a blocking step with 5% skimmed milk in phosphate-buffered saline (PBS; pH 7.4) for 1h at room heat (RT), the Hybond linens were incubated overnight at 40C with the control- and patient-sera (diluted 1:500 in 5% milk). Bound antibodies were visualized with horseradish peroxidase-labelled goat anti-human IgG (Jackson Immunoresearch Laboratories, West Grove, Pa.) diluted 1:4,000 in PBS, including 5% milk by using enhanced chemiluminescence (Amersham, Braunschweig, Germany). RESULTS To investigate, if SLE is usually characterized by p62-autoantibodies, we expressed the amino terminal fragment of nucleoporin p62 in This fragment contains the FG-repeat domains, which are not only the adhesion site for nuclear transport factors, but also serve as the antigenic epitope for p62 autoantibodies [17]. We collected sera of 25 patients experiencing SLE. Data evaluation of the individuals demonstrated, that seven individuals had nephritis throughout their health background, six individuals had been suffering.