Integral membrane proteins associated with the nuclear lamina are novel autoimmune antigens of the nuclear envelope.

We have analyzed sera from 55 patients, most with rheumatic diseases, that all react with the nuclear envelope of human cells in immunofluorescence microscopy. The molecular targets of these autoantibodies were characterized by immunoblot analysis of fractions derived from rat liver nuclear envelopes. While numerous sera were found to react with previously characterized autoimmune antigens of the nuclear envelope including nuclear lamins and the pore complex glycoprotein gp210, a substantial number of the sera were found to recognize relatively minor integral membrane proteins of the nuclear envelope associated with the nuclear lamina (LAP 1A and LAP 2), which have not been previously identified as autoantigens. Autoantibodies to LAP 1A and LAP 2 are present in 9 and 29% of the patient sera, respectively. Only autoantibodies to lamins A/C were encountered more frequently (in 31% of the sera) than autoantibodies to LAP 2, suggesting that LAP 2 may be among one of the most prominent autoantigens of the nuclear envelope in rheumatic disease patients. Since recent studies have suggested that LAP 1A and LAP 2 may be involved in attaching lamins and chromosomes to the nuclear envelope, these findings could promoted understanding of nuclear envelope functions as well as autoimmunity.

Synthetic compound peptide simulating antigenicity of conformation-dependent autoepitope.

Proliferating cell nuclear antigen (PCNA), also known as auxiliary protein of DNA polymerase delta, is involved in DNA replication and repair. Certain patients with systemic lupus erythematosus (lupus) produce autoantibodies to PCNA and the autoantibody-defined epitopes of PCNA have been inferred to be conformation-dependent. Based on antigenic properties of continuous primary structure peptides, compound peptides composed of covalently linked discontinuous sequences were synthesized and used as immunogens in rabbits. One compound peptide induced an antibody response to a nuclear antigen which demonstrated a cell cycle-related pattern of nuclear immunofluorescence with maximum expression in the DNA synthesis phase of the cell cycle associated with other features which simulated the conformation-dependent properties of lupus defined auto-epitopes. Other rabbit antibodies raised against different compound peptides did not show such properties and recognized epitopes which were continuous primary sequence regions. These results show that from analysis of antigenic sites on a protein, it might be possible to construct synthetic compound peptides which can mimic conformation-dependent epitopes recognized by spontaneously occurring autoantibodies.

Activin A-induced differentiation in K562 cells is associated with a transient hypophosphorylation of RB protein and the concomitant block of cell cycle at G1 phase.

The human erythroleukemic cell line, K562, can be induced to differentiate by the addition of activin A, a newly purified protein belonging to the TGF-beta 1 family. The present studies used flow cytometric cell cycle analysis, indirect immunofluorescence staining of the proliferating cell nuclear antigen (PCNA), and thymidine incorporation assay of cell proliferation to study the effects of activin A on the cell cycle during differentiation in K562 cells. Activin A-treated K562 cells were found to undergo a transient block in cell cycle, temporarily halting progression from G1 to S phase. The latter can be observed after approximately 24 hr of incubation with activin A and then disappears after this early stage of induction of differentiation. Cell cycle kinetics analysis using synchronized K562 cells also confirms that in the presence of activin A, K562 cells progress normally through various phases of cell cycle, except that there is prolongation of the G1 phase between 10 to 24 hr of culture. Furthermore, this transient arrest in G1 is correlated with dephosphorylation of a nucleoprotein, the RB gene product, which occurs within 9-24 hr of incubation with activin A; and phosphorylation of RB protein then develops afterward. In addition, these cell cycle-related events are observed to occur earlier than the accumulation of hemoglobins in K562 cells. It is concluded that transient dephosphorylation of RB protein and prolongation of G1 phase of cell cycle precede and accompany erythroid differentiation caused by activin A and chemical inducers, thus constituting part of the mechanism for induction of differentiation in the erythroleukemia cells.

Influence of free thiol group(s) on autoantibody-defined epitope of proliferating cell nuclear antigen.

Proliferating cell nuclear Ag (PCNA) is an intranuclear protein involved in DNA replication directed by DNA polymerase delta and is the target Ag of autoantibodies in some patients with SLE. There is evidence that the epitope on PCNA recognized by human autoantibodies is conformation-dependent and is not a continuous peptide sequence. Thimerosal, a mercury-containing sulfhydryl blocking compound, markedly reduced or abolished the reactivity of this autoantibody-defined PCNA epitope. The thimerosal effect was observed in various Ag-detecting systems including indirect immunofluorescence, immunodiffusion, immunoprecipitation, and flow cytometry. The mechanism of the thimerosal effect appeared to be mediated through free but not readily accessible sulfhydryl group or groups. The sulfhydryl-modification by thimerosal could be reversed by competition with thiol-containing compounds. Experimentally induced mAb to PCNA generated by immunization with purified PCNA have been shown to recognize epitopes that are continuous peptide sequences and these epitopes were not affected by thimerosal. It has been shown that the human autoantibody-defined epitope is related to the function of PCNA because autoantibodies are able to inhibit DNA polymerase delta directed DNA replication, whereas experimentally induced antibodies are not. These studies show that certain sulfhydryl groups in PCNA have a role in determining the antigenicity of the epitope recognized by autoantibody and raise the possibility that certain sulfhydryl groups might also be associated with its function.

Cloning and DNA sequence of the omc gene encoding the outer membrane protein-macromolecular complex from Neisseria gonorrhoeae.

The omc gene, encoding the outer membrane protein-macromolecular complex (OMP-MC), was cloned in two pieces from Neisseria gonorrhoeae 2686. The 5' fragment of the omc gene included a promoter sequence, as indicated by its unregulated expression in Escherichia coli. Attempts to reconstruct an intact omc gene were unsuccessful, suggesting that expression of the complete OMP-MC protein was toxic to E. coli. Complete sequence determination revealed a coding sequence of 2,133 nucleotides; the deduced amino acid sequence indicated a mature protein of 687 amino acids with an NH2-terminal signal peptide of 24 amino acids. Analysis of the deduced amino acid sequence revealed that the NH2-terminal half of OMP-MC is generally hydrophilic, while the COOH-terminal portion contains alternating hydrophobic and hydrophilic regions. Serological analyses demonstrated that the NH2-terminal portion of OMP-MC is exposed on the gonococcal surface and the COOH-terminal portion is membrane associated.