Hitoshi Shirakawa

Novel autoantigens of perinuclear anti‐neutrophil cytoplasmic antibodies (P‐ANCA) in ulcerative colitis: non‐histone chromosomal proteins, HMG1 and HMG2

Anti‐neutrophil cytoplasmic antibodies (ANCA) in sera from ulcerative colitis (UC) patients have been described as reacting with proteins in the granules of human neutrophils such as cathepsin G and lactoferrin and with yet unidentified antigens. Here we report the existence of a new member of perinuclear ANCA (P‐ANCA) in UC patients. In the previous study, we found that UC patients had a novel P‐ANCA against neutrophil 28‐kD protein. In this study, we purified the same antigens from HL‐60 lysates by using reversed phase high‐performance liquid chromatography, and revealed that the 28‐kD antigen consisted of two different proteins. The N‐terminus amino acids of these proteins are identical with those of high mobility group (HMG) non‐histone chromosomal proteins HMG1 and HMG2. Immunoblotting analysis of human neutrophil lysates using rabbit anti‐HMG1/2 antisera revealed a single band of 28 kD, and the 28‐kD band detected by immunoblotting analysis using patients serum IgG completely disappeared after preincubation with a mixture of HMG1 and HMG2. Furthermore, rabbit anti‐HMG1/2 antisera showed a perinuclear staining pattern in indirect immunofluorescence studies using ethanol‐fixed neutrophils. These data demonstrate that HMG1 and HMG2 are novel target antigens of P‐ANCA. HMG1 and HMG2 are distributed in the nuclei and cytoplasm of eukaryotic cells and act as transcription factors. Their intracellular localization and functions are distinct from those of the previously reported granular antigens of P‐ANCA.

Prevalence and characterization of perinuclear anti-neutrophil cytoplasmic antibodies (P-ANCA) directed against HMG1 and HMG2 in ulcerative colitis (UC)

In a previous study, we reported that the high mobility group (HMG) non‐histone chromosomal proteins HMG1 and HMG2 were novel target antigens of P‐ANCA. In this study, we determined the immunodiagnostic value of anti‐HMG1/HMG2 antibodies in patients with UC. Sixty sera from patients with UC were tested for reactivity with HMG1 and HMG2 by means of ELISA. Anti‐HMG1 antibody was detected in 32% of patients (40% of P‐ANCA+ patients). Anti‐HMG2 antibody was detected in 33% (40% of P‐ANCA+ patients). Thirty‐five percent of sera were positive for antibody to either HMG1 or HMG2 (43% of P‐ANCA+ patients). P‐ANCA+ patients expressed anti‐HMG1/HMG2 antibodies with significantly greater frequency compared with P‐ANCA− patients. Furthermore, the anti‐HMG1/HMG2 antibodies were significantly related to disease activity in UC. Sixteen of the 18 UC patients, who had high titres of anti‐HMG1 or ‐HMG2 antibody during the active phase, showed lower titres in the inactive phase. Anti‐HMG1/HMG2 antibodies appear to be useful as a marker for disease activity in UC.

Mitotic Phosphorylation Prevents the Binding of HMGN Proteins to Chromatin

ABSTRACT Condensation of the chromatin fiber and transcriptional inhibition during mitosis is associated with the redistribution of many DNA- and chromatin-binding proteins, including members of the high-mobility-group N (HMGN) family. Here we study the mechanism governing the organization of HMGN proteins in mitosis. Using site-specific antibodies and quantitative gel analysis with proteins extracted from synchronized HeLa cells, we demonstrate that, during mitosis, the conserved serine residues in the nucleosomal binding domain (NBD) of this protein family are highly and specifically phosphorylated. Nucleosome mobility shift assays with both in vitro-phosphorylated proteins and with point mutants bearing negative charges in the NBD demonstrate that the negative charge abolishes the ability of the proteins to bind to nucleosomes. Fluorescence loss of photobleaching demonstrates that, in living cells, the negative charge in the NBD increases the intranuclear mobility of the protein and significantly decreases the relative time that it is bound to chromatin. Expression of wild-type and mutant proteins inHmgN1−/− cells indicates that the negatively charged protein is not bound to chromosomes. We conclude that during mitosis the NBD of HMGN proteins is highly phosphorylated and that this modification regulates the interaction of the proteins with chromatin.

High mobility group (HMG) non-histone chromosomal proteins HMG1 and HMG2 are significant target antigens of perinuclear anti-neutrophil cytoplasmic antibodies in autoimmune hepatitis

BACKGROUND High mobility group (HMG) non-histone chromosomal proteins HMG1 and HMG2 have been identified as novel antigens of perinuclear anti-neutrophil cytoplasmic antibodies (p-ANCAs), and the existence of anti-HMG1 and anti-HMG2 antibodies in a population of patients with ulcerative colitis has been reported. AIMS To investigate whether HMG1 and HMG2 are target antigens for p-ANCAs in autoimmune hepatitis (AIH). PATIENTS Serum samples from 28 patients with AIH, 44 patients with primary biliary cirrhosis (PBC), 27 patients with chronic hepatitis C, and 23 patients with chronic hepatitis B were tested. METHODS ANCAs were detected by routine indirect immunofluorescence (IIF). Anti-HMG1 and anti-HMG2 antibodies were assayed by enzyme linked immunosorbent assay. RESULTS p-ANCAs were detected in 89% (25/28) of patients with AIH, 36% (16/44) of patients with PBC, 11% (3/27) of patients with chronic hepatitis C, and 13% (3/23) of patients with chronic hepatitis B. Anti-HMG1 and/or anti-HMG2 antibodies were detected in 89% (25/28) of patients with AIH, 70% (31/44) with PBC, 26% (7/27) with chronic hepatitis C, and 9% (2/23) with chronic hepatitis B. In AIH, anti-HMG1 and/or anti-HMG2 antibodies were detected in 96% (24/25) of p-ANCA positive patients. The p-ANCA staining pattern detected by IIF using sera from patients with AIH disappeared or decreased in titre after preincubation with a mixture of HMG1/HMG2. The presence and titres of those antibodies in AIH correlated significantly with those of p-ANCA, but not with those of anti-nuclear antibody or anti-smooth muscle antibody. CONCLUSIONS HMG1 and HMG2 are significant target antigens of p-ANCA in AIH.

Cloning and sequencing of the gene encoding the plasma membrane H+-ATPase from an acidophilic red alga, Cyanidium caldarium

A cDNA containing an open reading frame encoding the putative plasma membrane H(+)-ATPase in an acidophilic red alga, Cyanidium caldarium, was cloned and sequenced by means of PCR and Southern hybridization based on homologous sequences of P-type ATPases found in other organisms. The cloned cDNA is 3300 bp in length, containing a 2865 bp open reading frame encoding a polypeptide of 955 amino acids which has a predicted molecular mass of 105,371. The deduced amino acid sequence was found to be more homologous to those of P-type H(+)-ATPases from higher plants than that from the green alga Dunaliella bioculata.

Detection of Anti-Neutrophil Cytoplasmic Antibodies in MRL/Mp-lpr/lpr Mice and Analysis of their Target Antigens

Anti-neutrophil cytoplasmic antibodies (ANCA) have been widely studied and recognized to be clinically very important for some human diseases including systemic rheumatic diseases. We analyzed ANCA response and their target antigens in MRL/Mp-lpr/lpr (MRL-lpr) mice, an animal model of systemic rheumatic disease. P-ANCA was detected in 57% of the mice. Antibodies to the known P-ANCA target antigens at the same age were examined. Among these, antibodies to high mobility group (HMG) proteins HMG1 and HMG2 were detected in 57% of the mice, 75% of which were also positive for P-ANCA. These anti-HMGl/HMG2 activities were absorbed by preincubation with a mixture of HMG1 and HMG2. In contrast, antibodies to myeloperoxidase and cathepsin G were detected in 14% and 7%, respectively, but these activities were not inhibited by preincubation with corresponding antigens. In addition, the titers of P-ANCA and anti-HMGl/HMG2 antibodies in MRL-lpr mice were significantly correlated with each other. Thus, HMG1 and HMG2 were considered to be significant target antigens of P-ANCA in MRL-lpr mice

Involvement of HMGB1 and HMGB2 proteins in exogenous DNA integration reaction into the genome of HeLa S3 cells

High mobility group 1 and 2 proteins (HMGB1 and HMGB2) are abundant chromosomal proteins in eukaryotic cells. We examined the involvement of HMGB1 and HMGB2 in nonhomologous illegitimate recombination. The HMGB1 or HMGB2 expression plasmid, carrying the neo(r) gene as a selection marker, was introduced into HeLa S3 cells to obtain stably-transfected cells. The number of G418-resistant colonies was about 10 times the number of colonies of control cells transfected with plasmids not carrying the HMGB genes. The copy number of the stably-integrated neo(r) gene was higher in the cells transfected with the HMGB expression plasmids than in control cells. The exogenous DNA integration was suggested to have occurred by nonhomologous illegitimate recombination. On the contrary, the introduction of the HMGB antisense RNA expression plasmid with a reporter plasmid carrying the neo(r) gene into HeLa S3 cells decreased the number of G418-resistant colonies. These results indicate that HMGB1 and HMGB2 each have a novel function as stimulators of stable integration of plasmid DNA into the host genome and that they may be important for the process of spontaneous DNA integration in living cells.