News and Publications

Autoantibodies in Neonatal Lupus Erythematosus Syndrome

D. Wang, J.P. Buyon,* E.K.L. Chan

* Hospital for Joint Diseases, New York, NY

Most mothers of children with neonatal lupus erythematosus syndrome have detectable antibodies to the antigen SS-A/Ro in their sera. In addition, these autoantibodies are also found in patients who have Sjögren's syndrome or systemic lupus erythematosus. Antibodies to SS-A/Ro recognize a 52-kD protein and a family of ribonucleoprotein particles present in small quantities that contain a 60-kD protein and small cytoplasmic RNAs known as hY1-5.

We used yeast 2-hybrid cloning to screen for proteins associated with the 60-kD SS-A/Ro protein. We found that a novel 75-kD protein, pp75, interacts with the carboxyl 70% of the 60-kD protein. The association between these 2 proteins and the specificity of interaction was confirmed by using a mammalian 2-hybrid assay. The identified full-length cDNA was 3.3 kb. The open reading frame consisting of 541 amino acids had a relatively high percentage of serine residues (15%). This result is consistent with the finding that pp75 is a phosphorylated polypeptide, as shown by immunoprecipitation assays with extracts from radiolabeled HeLa cells. In Northern blot analysis, pp75 was expressed in all tissues analyzed, including human heart. Rabbit antibodies to recombinant pp75 gave predominantly cytoplasmic staining and recognized the 75-kD protein in Western blot assays with a HeLa cell extract. The complete pp75 cDNA was subcloned into a eukaryotic expression vector with a N-terminal epitope tag T7. Transient transfection in HeLa cells showed that pp75 was also localized predominantly in the cytoplasm.

Western blot and enzyme-linked immunosorbent assays indicated that sera from 15 (18%) of 82 mothers of babies with neonatal lupus erythematosus recognized the recombinant protein. Eleven of these 15 mothers had babies with cardiac and cutaneous manifestations, 3 had babies with skin involvement only, and 1 had a baby with congenital heart block without antibodies to the antigen Ro/La. Five of the 15 seropositive mothers were asymptomatic, 3 had systemic lupus erythematosus, 2 had Sjögren's syndrome, 2 had an undifferentiated autoimmune syndrome, and 1 had an unknown disorder. Our data suggest that pp75 is a novel autoantigen that may interact with the 60-kD SS-A/Ro protein transiently in vivo. Further work is needed to determine if pp75 is directly involved in the pathogenesis of neonatal lupus erythematosus.

Golgins: Coiled Coil--Rich Proteins Associated With the Golgi Complex

M. Lu, J.C. Hamel, M.J. Fritzler,* E.K.L. Chan

* University of Calgary, Calgary, Alberta

Autoantibodies directed against self-antigens are characteristic features of a number of human diseases. In systemic rheumatic diseases, these autoantibodies include ones that react with extracellular molecules, the cell membrane, and nuclear and cytoplasmic components. We are interested in understanding autoimmune responses in systemic lupus erythematosus and related disease states. We use human autoantibodies as molecular probes to study the cell biology of target antigens.

Since 1993, we have cloned 4 of 5 known autoantigens of the Golgi complex: golgin-95/GM130, golgin-97, golgin-160/GCP170, and golgin-245. The fifth human autoantigen is giantin, also known as macrogolgin or GCP372. Interestingly, the amino acid sequences deduced for all 5 autoantigens indicate that the antigens are proteins with predominantly -helical coiled-coil domains and nonhelical domains at the N- and C-termini.

The current postulate is that these coiled coil--rich proteins such as golgin-95/GM130 form intermolecular complexes with a docking protein, p115, that is important for Golgi vesicular traffic. An alternative nonexclusive hypothesis is that these coiled coil--rich proteins are the components of the "strings" that are important in linking and guiding vesicles in intercisternal traffic. How this family of coiled-coil proteins and their complexes become autoimmune targets remains to be determined.

PUBLICATIONS

Chan, E.K.L., Fritzler, M.J. Golgins: Coiled-coil proteins associated with the Golgi complex. Electron. J. Biotechnol. [on-line serial]. 1(2), May 1998. Available at http://www.ejb.org/content/vol1/issue2/full/1

Furuta, K., Matsuoka, S., Hildebrandt, B., Kiyosawa, K., Reimer, G., Luderschmidt, C., Chan, E.K.L., Tan, E.M. Immunologic characterization of heterochromatin protein p25ß autoantibodies and relationship with centromere autoantibodies and pulmonary fibrosis in systemic scleroderma. J. Mol. Med. 76:54, 1998.

Hassfeld, W.G., Chan, E.K.L., Mathison, D.A., Portman, D., Dreyfuss, G., Steiner, G., Tan, E.M. Molecular definition of heterogeneous nuclear ribonucleoprotein R (hnRNP-R) using autoimmune autoantibody: Immunological relationship with hnRNP-P. Nucleic Acids Res. 26:439, 1998.

Miranda-Carus, M.E., Boutjdir, M., Tseng, C.-E., DiDonato, F., Chan, E.K.L., Buyon, J.P. Induction of antibodies reactive with SSA/Ro-SSB/La and development of congenital heart block in a murine model. J. Immunol., in press.

Miranda-Carus, M.E., Tseng, C.-E., Rashbaum, W.M., Ochs, R.L., Casiano, C.A., DiDonato, F., Chan, E.K.L., Buyon, J.P. Accessibility of SSA/Ro and SSB/La antigens to maternal autoantibodies in apoptotic human fetal cardiac myocytes. J. Immunol., in press.

Seo, B.B., Kitajima-Ihara, T., Chan, E.K.L., Scheffler, I.E., Matsuno-Yagi, A., Yagi, T. Molecular remedy of complex I defects: Rotenone-insensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae mitochondria restores the NADH oxidase activity of complex I-deficient mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 95:9167, 1998..

Tseng, C., Miranda, E., DiDonato, F., Boutjdir, M., Rashbaum, W., Chan, E.K.L., Buyon, J.P. mRNA and protein expression of SS-A/Ro and SS-B/La in human fetal cardiac myocytes cultured using a novel application of the Langendorf procedure. Pediatr. Res., in press.

von Mühlen, C.A., Chan, E.K.L., Angles-Cano, E., Mamula, M.J., Garcia-De La Torre, I., Fritzler, M.J. Advances in autoantibodies in SLE. Lupus. in press.