Michiteru Yoshida

Post-translational methylation of high mobility group box 1 (HMGB1) causes its cytoplasmic localization in neutrophils

High mobility group box 1 (HMGB1) protein plays multiple roles in transcription, replication, and cellular differentiation. HMGB1 is also secreted by activated monocytes and macrophages and passively released by necrotic or damaged cells, stimulating inflammation. HMGB1 is a novel antigen of anti-neutrophil cytoplasmic antibodies (ANCA) observed in the sera of patients with ulcerative colitis and autoimmune hepatitis, suggesting that HMGB1 is secreted from neutrophils to the extracellular milieu. However, the actual distribution of HMGB1 in the cytoplasm of neutrophils and the mechanisms responsible for it are obscure. Here we show that HMGB1 in neutrophils is post-translationally mono-methylated at Lys42. The methylation alters the conformation of HMGB1 and weakens its DNA binding activity, causing it to become largely distributed in the cytoplasm by passive diffusion out of the nucleus. Thus, post-translational methylation of HMGB1 causes its cytoplasmic localization in neutrophils. This novel pathway explains the distribution of nuclear HMGB1 to the cytoplasm and is important for understanding how neutrophils release HMGB1 to the extracellular milieu.

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.

HMGB proteins and transcriptional regulation.

In eukaryotic nuclei, transcription reactions on the chromatin are regulated by the binding of transcription regulators such as transcription factors and chromatin remodeling factors to chromatin. High-mobility-group-box (HMGB) protein family is a member of HMG super family proteins, the most abundant and ubiquitous non-histone chromatin binding proteins in eukaryotic cells. HMGB proteins bind to chromosomal DNA via their DNA binding motif, a HMG box, and induce structural changes of chromatin. Besides the chromatin binding property of HMGB proteins, HMGB proteins also interact with other proteins including transcription regulators and histones. In addition to those key transcriptional regulatory proteins, undoubtedly HMGB proteins bind dynamically to chromatin and interact with other proteins including transcription factors, thereby participating in transcription regulation in multiple processes. We will focus on the transcription regulation by HMGB proteins bound to chromatin, and possible roles of the unique structural and functional domain, the acidic C-tail region.

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.

The Plant Zinc Finger Protein ZPT2–2 Has a Unique Mode of DNA Interaction

ZPT2–2 is a DNA-binding protein of petunia that contains two canonical TFIIIA-type zinc finger motifs separated by a long linker. We previously reported that ZPT2–2 bound to two separate AGT core sites, with each zinc finger making contact with each core site. Here we present our further characterization of ZPT2–2 by using selected and amplified binding sequence imprinting and surface plasmon resonance analyses; together, these assays revealed some unusual features of the interaction between ZPT2–2 and DNA. These experiments allowed us to conclude that 1) the optimal binding sequence for the N-terminal zinc finger is AGC(T), and that of the C-terminal one is CAGT; 2) multiple arrangements of the two core sites accommodate binding; and 3) the spacing between the two core sites affects the binding affinity. In light of these observations, we propose a new model for the DNA-ZPT2–2 interaction. Further, consistent with this model, a high affinity binding site for ZPT2–2 was found in the promoter region of theZPT2–2 gene. This site may serve as acis-element for the autoregulation of ZPT2–2gene expression.

Lupus antibodies to the HMGB1 chromosomal protein: epitope mapping and association with disease activity

The high mobility group box 1 (HMGB1) protein is a non-histone chromosomal protein that acts as a potent proinflammatory cytokine when actively secreted from LPS- or TNF-activated macrophages, monocytes, and other cells. Anti-HMGB1/2 antibodies have been previously identified in sera from a high proportion of patients with autoimmune diseases. In this study, we examined anti-HMGB1 antibody titers in sera of patients with systemic rheumatic diseases and the correlations between the presence of anti-HMGB1 antibodies and disease activity in systemic lupus erythematosus (SLE) patients by enzyme-linked immunosorbent assay and western blotting. We detected increases in both the levels and the frequency of anti-HMGB1 antibodies in sera from SLE and polymyositis/dermatomyositis (PM/DM) patients, and observed that the presence of anti-HMGB1 antibodies positively correlates with SLE disease activity index. Through epitope mapping, we found that multiple HMGB1 epitopes were recognised in SLE sera, with the major epitope mapping to box A. Another epitope, the joiner region of HMGB1, was preferentially recognized by SLE sera, but not by PM/DM sera. Collectively, these observations suggest that the presence of anti-HMGB1 antibodies correlates with disease activity in SLE patients.

Intramolecular chaperone and inhibitor activities of a propeptide from a bacterial zinc aminopeptidase.

An aminopeptidase from Aeromonas caviae T-64 was translated as a preproprotein consisting of three domains; a signal peptide (19 amino acid residues), an N-terminal propeptide (101 residues) and a mature region (273 residues). We demonstrated that a proteinase, which was isolated from the culture filtrate of A. caviae T-64, activated the recombinant pro-aminopeptidase by removal of the majority of the propeptide. Using L-Leu-p-nitroanilide as a substrate, the processed aminopeptidase showed a large increase in kcat when compared with the unprocessed enzyme, whereas the Km value remained relatively unchanged. The similar Km values for the pro-aminopeptidase and the mature aminopeptidase indicated that the N-terminal propeptide of the pro-aminopeptidase did not influence the formation of the enzyme-substrate complex, suggesting the absence of marked conformational changes in the active domain. In contrast, the marked difference in kcat suggests a significant decrease in the energy of one or more of the transition states of the enzyme-substrate reaction coordinate. Moreover, we showed that the activity of the urea-denatured pro-aminopeptidase could be recovered by dialysis, whereas the activity of the urea-denatured mature aminopeptidase, which lacked the propeptide, could not. Further to this, the propeptide-deleted aminopeptidase formed an inclusion body in the cytoplasmic space in Escherichia coli and was not secreted at all. These results suggested that the propeptide of the pro-aminopeptidase acted as an intramolecular chaperone that was involved with the correct folding of the enzyme in vitro and was required for extracellular secretion in E. coli.

Efficient large-scale purification of non-histone chromosomal proteins HMG1 and HMG2 by using Polybuffer-exchanger PBE94.

A method for the efficient and practical large-scale purification of high-mobility group (HMG) non-histone chromosomal proteins, HMG1 and HMG2, from porcine thymus applying Polybuffer-exchanger PBE94 gel as anion-exchanger has been developed. This method affords higher resolution, purity and yield, than the conventional procedure of CM-Sephadex C-25 ion-exchange column chromatography. Furthermore, use of Polybuffer-exchanger PBE94 column chromatography led to direct preparation of HMG1 and HMG2 from loosely bound non-histone chromosomal protein fraction of chromatin without prefractional precipitation with trichloroacetic acid or prior extraction with perchloric acid. Thus, the application of PBE94 gel as an anion-exchanger to the subfractionation of other kinds of homologous protein is possible.

Distinct domains in HMGB1 are involved in specific intramolecular and nucleosomal interactions.

HMGB1 is composed of two DNA-binding domains and a long acidic tail at the C-terminus. The acidic tail interacts with the DNA-binding domains of HMGB1 and with core histone H3 in the nucleosome. These interactions are important for modulation of the DNA and chromatin binding activities of HMGB1, as well as biological functions of HMGB1. However, the interactions are not fully characterized, because the tertiary structure of full-length HMGB1 is still unknown. Here we use chemical cross-linking, mass spectrometry, and epitope masking analysis to perform a detailed characterization of the inter- and intramolecular protein interactions of the acidic tail of HMGB1. We show that specific regions of the acidic tail participate in intramolecular interactions with Lys2 of HMGB1 and in intermolecular interactions with Lys36 and Lys37 of histone H3. The acidic tail is oriented by its location adjacent to the C-terminus of helix III of DNA-binding HMG box A in the HMGB1 molecule. These results suggest that the acidic tail modulates the biological functions of HMGB1 through these specific interactions.