Objective. To investigate protein citrullination by the periodontal pathogen Porphyromonas gingivalis as a potential mechanism for breaking tolerance to citrullinated proteins in rheumatoid arthritis (RA).Methods. The expression of endogenous citrullinated proteins was analyzed by immunoblotting of cell extracts from P gingivalis and 10 other oral bacteria. P gingivalis-knockout strains lacking the bacterial peptidylarginine deiminases (PADs) or gingipains were created to assess the role of these enzymes in citrullination. Citrullination of human fibrinogen and ␣-enolase by P gingivalis was studied by incubating live wild-type and knockout strains with the proteins and analyzing the products by immunoblotting and mass spectrometry.Results. Endogenous protein citrullination was abundant in P gingivalis but lacking in the other oral bacteria. Deletion of the bacterial PAD gene resulted in complete abrogation of protein citrullination. Inactivation of arginine gingipains, but not lysine gingipains, led to decreased citrullination. Incubation of wild-type P gingivalis with fibrinogen or ␣-enolase caused degradation of the proteins and citrullination of the resulting peptides at carboxy-terminal arginine residues, which were identified by mass spectrometry.Conclusion. Our findings demonstrate that among the oral bacterial pathogens tested, P gingivalis is unique in its ability to citrullinate proteins. We further show that P gingivalis rapidly generates citrullinated host peptides by proteolytic cleavage at Arg-X peptide bonds by arginine gingipains, followed by citrullination of carboxy-terminal arginines by bacterial PAD. Our results suggest a novel model where P gingivalismediated citrullination of bacterial and host proteins provides a molecular mechanism for generating antigens that drive the autoimmune response in RA.Rheumatoid arthritis (RA) is characterized by disease-specific autoimmunity to citrullinated proteins. Citrullination is a posttranslational modification of arginine residues that is mediated by the family of peptidylarginine deiminases (PADs). Citrullinated fibrin(ogen) and ␣-enolase are 2 of the physiologic proteins that are targeted by anti-citrullinated protein antibodies in RA (1-5). Fibrinogen is the precursor of fibrin, and autoanMs. Wegner and Drs.

Protein phosphorylation in amyloplasts and chloroplasts of Triticum aestivum (wheat) was investigated after the incubation of intact plastids with g-32 P-ATP. Among the soluble phosphoproteins detected in plastids, three forms of starch branching enzyme (SBE) were phosphorylated in amyloplasts (SBEI, SBEIIa, and SBEIIb), and both forms of SBE in chloroplasts (SBEI and SBEIIa) were shown to be phosphorylated after sequencing of the immunoprecipitated 32 P-labeled phosphoproteins using quadrupole-orthogonal acceleration time of flight mass spectrometry. Phosphoamino acid analysis of the phosphorylated SBE forms indicated that the proteins are all phosphorylated on Ser residues. Analysis of starch granuleassociated phosphoproteins after incubation of intact amyloplasts with g-32 P-ATP indicated that the granule-associated forms of SBEII and two granule-associated forms of starch synthase (SS) are phosphorylated, including SSIIa. Measurement of SBE activity in amyloplasts and chloroplasts showed that phosphorylation activated SBEIIa (and SBEIIb in amyloplasts), whereas dephosphorylation using alkaline phosphatase reduced the catalytic activity of both enzymes. Phosphorylation and dephosphorylation had no effect on the measurable activity of SBEI in amyloplasts and chloroplasts, and the activities of both granule-bound forms of SBEII in amyloplasts were unaffected by dephosphorylation. Immunoprecipitation experiments using peptide-specific anti-SBE antibodies showed that SBEIIb and starch phosphorylase each coimmunoprecipitated with SBEI in a phosphorylation-dependent manner, suggesting that these enzymes may form protein complexes within the amyloplast in vivo. Conversely, dephosphorylation of immunoprecipitated protein complex led to its disassembly. This article reports direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation and protein-protein interactions in the control of starch anabolism and catabolism.

The naturally occurring population of dedicated regulatory T cells that coexpress CD4 and CD25 is known to play a key role in the maintenance of peripheral T-cell tolerance; however, their mechanism of action has remained obscure. Here we report that a member of the family of ␤-galactoside-binding proteins, galectin-1, is overexpressed in regulatory T cells, and that expression is increased after activation. Most importantly, blockade of galectin-1 binding significantly reduced the inhibitory effects of human and mouse CD4 ؉ CD25 ؉ T cells. Reduced regulatory activity was observed in CD4 ؉ CD25 ؉ T cells obtained from galectin-1-homozygous null mutant mice. These results suggest that galectin-1 is a key effector of the regulation mediated by these cells. IntroductionMore than 10 years ago, T lymphocytes, which constitutively coexpress CD4 and CD25 and represent approximately 5% of murine peripheral T cells, were shown to play a key role in the prevention of autoimmunity. 1,2 Similar cells have since been characterized in human peripheral blood. 3,4 These cells differ from recently activated CD4 ϩ T cells expressing CD25 because of their low-level expression of surface markers of activation such as CD69. In contrast to CD4 ϩ CD25 Ϫ T cells, CD4 ϩ CD25 ϩ T cells are hyporesponsive in vitro to polyclonal T-cell-receptor (TCR) stimuli. After activation they do not produce interleukin-2 (IL-2), IL-4, interferon (IFN)-␥, or IL-10, and their anergic state can be partially restored by IL-2. The mechanism of regulation appears to involve the inhibition of transcription of cytokine genes, most notably IL-2 and IFN-␥, leading to reduced proliferation. 5,6 In mouse models, Papiernik et al have demonstrated that CD4 ϩ CD25 ϩ T cells originate in the thymus, where they are induced to express CD25 at the CD4 single-positive stage, before migration to the periphery. 7 Although the generation of CD4 ϩ CD25 ϩ T cells during T-cell differentiation has not yet been fully characterized, it appears that the relatively high affinity of these cells for self-antigens falls between that required for positive and negative selection. 8 The importance of naturally occurring CD4 ϩ CD25 ϩ regulatory T (Treg) cells for maintenance of peripheral T-cell tolerance is well established, but their mechanisms of action remain elusive. Treg cells express high levels of cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), which, it has been suggested, may act by sequestering B7 family molecules and inhibiting the costimulation of neighboring T cells. 9 However, since CD4 ϩ CD25 ϩ Treg cells effect suppression in the absence of B7-expressing antigenpresenting cells, this cannot be a universal explanation. 3 Cellsurface-transforming growth factor ␤-1 (TGF-␤1) has also been implicated, although this has not been reproduced. 10,11 It has recently been reported that Lag-3 contributes to the function of naturally occurring regulatory cells, but the reversal of suppression achieved by Lag-3 blockade was only modest. 5,6,12 Although cytokines such as IL-10 a...

Objective. To map the antibody response to human citrullinated ␣-enolase, a candidate autoantigen in rheumatoid arthritis (RA), and to examine crossreactivity with bacterial enolase.Methods. Serum samples obtained from patients with RA, disease control subjects, and healthy control subjects were tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with citrullinated ␣-enolase peptides. Antibodies specific for the immunodominant epitope were raised in rabbits or were purified from RA sera. Cross-reactivity with other citrullinated epitopes was investigated by inhibition ELISAs, and cross-reactivity with bacterial enolase was investigated by immunoblotting.Results. An immunodominant peptide, citrullinated ␣-enolase peptide 1, was identified. Antibodies to this epitope were observed in 37-62% of sera obtained from patients with RA, 3% of sera obtained from disease control subjects, and 2% of sera obtained from healthy control subjects. Binding was inhibited with homologous peptide but not with the arginine-containing control peptide or with 4 citrullinated peptides from elsewhere on the molecule, indicating that antibody binding was dependent on both citrulline and flanking amino acids. The immunodominant peptide showed 82% homology with enolase from Porphyromonas gingivalis, and the levels of antibodies to citrullinated ␣-enolase peptide 1 correlated with the levels of antibodies to the bacterial peptide (r 2 ‫؍‬ 0.803, P < 0.0001). Affinitypurified antibodies to the human peptide cross-reacted with citrullinated recombinant P gingivalis enolase.Conclusion. We have identified an immunodominant epitope in citrullinated ␣-enolase, to which antibodies are specific for RA. Our data on sequence similarity and cross-reactivity with bacterial enolase may indicate a role for bacterial infection, particularly with P gingivalis, in priming autoimmunity in a subset of patients with RA.

The growing availability of genomic sequence information, together with improvements in analytical methodology, have enabled high throughput, high sensitivity protein identification. Silver staining remains the most sensitive method for visualization of proteins separated by two-dimensional gel electrophoresis (2-D PAGE). Several silver staining protocols have been developed which offer improved compatibility with subsequent mass spectrometric analysis. We describe a modified silver staining method that is available as a commercial kit (Silver Stain PlusOne; Amersham Pharmacia Biotech, Amersham, UK). The 2-D patterns abtained with this modified protocol are comparable to those from other silver staining methods. Omitting the sensitizing reagent allows higher loading without saturation, which facilitates protein identification and quantitation. We show that tryptic digests of proteins visualized by the modified stain afford excellent mass spectra by both matrix-assisted laser desorption/ionization and tandem electrospray ionization. We conclude that the modified silver staining protocol is highly compatible with subsequent mass spectrometric analysis.