Recently, we published the existence of 2 populations of anti-␤ 2 -glycoprotein I (␤ 2 -GPI) IgG antibodies. Type A antibodies recognize epitope G40-R43 in domain I of ␤ 2 -GPI and are strongly associated with thrombosis. Type B antibodies recognize other parts of ␤ 2 -GPI and are not associated with thrombosis. In this study we demonstrate that type A antibodies only recognize plasma-purified ␤ 2 -GPI when coated onto a negatively charged surface and not when coated onto a neutrally charged surface. The affinity of type B antibodies toward plasma-purified ␤ 2 -GPI was independent of the charge of the surface to which ␤ 2 -GPI was coated. Type A antibodies did not recognize plasmapurified ␤ 2 -GPI in solution, whereas they did recognize recombinant ␤ 2 -GPI both in solution and coated onto a neutrally charged plate. When the carbohydrate chains were removed from plasma-purified ␤ 2 -GPI, we found that type A antibodies did recognize the protein in solution. This supports the hypothesis that the difference in recognition of plasma-purified and recombinant ␤ 2 -GPI is caused by the difference in glycosylation and that epitope G40-R43 of plasma-purified ␤ 2 -GPI is covered by a carbohydrate chain. Type A anti-␤ 2 -GPI antibodies can only recognize this epitope when this carbohydrate chain is displaced as a result of a conformational change. This finding has major implications both for the detection of pathogenic anti-␤ 2 -GPI antibodies and the comprehension of the pathophysiology of the antiphospholipid syndrome.

Identification of epitopes that are recognized by diabetogenic T cells and cause selective beta cell destruction in type 1 diabetes (T1D) has focused on peptides originating from native beta cell proteins. Translational errors represent a major potential source of antigenic peptides to which central immune tolerance is lacking. Here, we describe an alternative open reading frame within human insulin mRNA encoding a highly immunogenic polypeptide that is targeted by T cells in T1D patients. We show that cytotoxic T cells directed against the N-terminal peptide of this nonconventional product are present in the circulation of individuals diagnosed with T1D, and we provide direct evidence that such CD8 T cells are capable of killing human beta cells and thereby may be diabetogenic. This study reveals a new source of nonconventional polypeptides that act as self-epitopes in clinical autoimmune disease.

Posttranslational modification (PTM) of islet autoantigens can cause lack of central tolerance in type 1 diabetes (T1D). Tissue transglutaminase (tTG), involved in PTM of gluten antigens in celiac disease, creates negatively charged peptides favored by T1D-predisposing HLA-DQ molecules, offering an attractive candidate modifying islet autoantigens in T1D. The highly predisposing HLA-DQ8cis/trans molecules share preferences for negatively charged peptides, as well as distinct peptide-binding characteristics that distinguish their peptide-binding repertoire. We screened islet autoantigens with the tTG substrate motif for candidate-modified epitopes binding to HLA-DQ8cis/trans and identified 31 candidate islet epitopes. Deamidation was confirmed for 28 peptides (90%). Two of these epitopes preferentially bound to HLA-DQ8cis and six to HLA-DQ8trans upon deamidation, whereas all other peptides bound equally to HLA-DQ8cis/trans. HLA-DQ8cis-restricted T cells from a new-onset T1D patient could only be generated against a deamidated proinsulin peptide, but cross-reacted with native proinsulin peptide upon restimulation. The rate of T-cell autoreactivity in recent-onset T1D patients extended from 42% to native insulin to 68% adding responses to modified proinsulin, versus 20% and 37% respectively, in healthy donors. Most patients responded by interferon-g, whereas most healthy donors produced interleukin-10 only. Thus, T-cell autoreactivity exists to modified islet epitopes that differs in quality and quantity between patients and healthy donors.

Summary. Background: The major antigen implicated in the antiphospholipid syndrome is beta2-glycoprotein I (b 2 GPI). Dimerized b 2 GPI binds to apolipoprotein E receptor 2¢ (apoER2¢) on platelets and increases platelet adhesion to collagen under conditions of flow. Aim: To investigate whether the interaction between dimerized b 2 GPI and platelets is sufficiently strong to resist shear stresses. Methods: We studied the interaction of platelets with immobilized dimerized b 2 GPI under conditions of flow, and further analyzed the interaction using surface plasmon resonance and solid phase immunoassays. Results: We found that dimerized b 2 GPI supports platelet adhesion and aggregate formation under venous flow conditions. Adhesion of platelets to dimerized b 2 GPI was completely inhibited by the addition of soluble forms of both apoER2¢ and GPIba, and the addition of receptor-associated protein and the removal of GPIba from the platelet surface. GPIba co-precipitated with apoER2¢, suggesting the presence of complexes between GPIba and apoER2¢ on platelet membranes. The interaction between GPIba and dimeric b 2 GPI was of intermediate affinity (K d ¼ 180 nM) and Zn 2+, but not Ca 2+ -dependent. Deletion of domain V from dimeric b 2 GPI strongly reduced its binding to both GPIba and apoER2¢. Antibodies that inhibit the binding of thrombin to GPIba inhibited platelet adhesion to dimeric b 2 GPI completely, while antibodies blocking the binding of von Willebrand factor to GPIba had no effect. Dimeric b 2 GPI showed reduced binding to lowsulfated GPIba compared to the fully sulfated form. Conclusion: We show that platelets adhere to dimeric b 2 GPI under both arterial and venous shear stresses. Platelets adhere via two receptors: GPIba and apoER2¢. These receptors are present in a complex on the platelet surface.

The paradoxical correlation between thrombosis and the lupus anticoagulant (LAC) effect is an enigmatic feature of the antiphospholipid (aPL) syndrome. The Dutch authors previously reported that thrombosis-related anti-␤2-glycoprotein I (␤2GPI) antibodies recognize domain I and cause LAC. The American authors reported that aPLs disrupt an anticoagulant annexin A5 (AnxA5) crystal shield. We investigated whether antidomain I antibodies correlate with disruption of AnxA5-anticoagulant activity. We studied a well-characterized group of 33 patients including subgroups with ␤2GPI-dependent LAC that recognize domain I (n ‫؍‬ 11), with ␤2GPI-independent LAC (n ‫؍‬ 12), and lacking LAC (n ‫؍‬ 10). The effects on AnxA5-anticoagulant activity were determined with an AnxA5 resistance assay that measures coagulation times with and without AnxA5. Patients with ␤2GPI-dependent LAC (group A, all with thrombosis) had significantly lower AnxA5-anticoagulant ratios than those with ␤2GPI-independent LAC (group B, thrombosis n ‫؍‬ 4; 157.8% versus 235.6%, P < .001) and those without LAC (group C, thrombosis n ‫؍‬ 2; 157.8% versus 232.5%, P < .001). There was no difference in the ratios between groups B and C (P ‫؍‬ .92). Plasmas with ␤2GPI-dependent LAC that recognize domain I displayed significantly increased AnxA5 resistance, suggesting that specifically anti-␤2GPI antibodies compete with AnxA5 for anionic phospholipids. These results are consistent with a model in which aPL antibodies may promote thrombosis by interfering with the anticoagulant activity of AnxA5.

See also Pierangeli SS. In search for a receptor for antiphospholipid antibodies on target cells. This issue, pp 1678-9.Summary. The antiphospholipid syndrome (APS) is a noninflammatory autoimmune disease characterized by arterial and/or venous thrombosis and/or pregnancy morbidity in the presence of autoantibodies that recognize beta2-glycoprotein I (b 2 GPI) bound to phospholipids. We have previously demonstrated that dimerization of b 2 GPI by autoantibodies induces platelet activation, involving the platelet receptor apolipoprotein E receptor 2Õ (apoER2¢) a receptor belonging to the lowdensity lipoprotein receptor (LDL-R) family. Here, we show that dimeric b 2 GPI, but not monomeric b 2 GPI, interacts with four other LDL-R family members: the LDL-R related protein (LRP), megalin, the LDL-R and the very-low density lipoprotein receptor (VLDL-R). Interaction between dimeric b 2 GPI and LDL-R, apoER2¢ and VLDL-R was best described with a one-site binding model (half-maximal binding; $20 nM for apoER2¢ and VLDL-R and $300 nM for LDL-R), whereas the interaction between dimeric b 2 GPI and LRP or megalin was best described with a two-site binding model, representing a high-($3 nM) and a low-affinity site ($0.2 lM). Binding to all receptors tested was unaffected by a tryptophane to serine (W316S) substitution in domain V of b 2 GPI, which is known to disrupt the phospholipid binding site of b 2 GPI. Also deletion of domain I or II left the interaction with the receptors unaffected. Deletion of domain V, however, significantly decreased the affinity for the receptors. In conclusion, our data show that dimeric b 2 GPI can interact with different LDL-R family members. This interaction is dependent on a binding site within domain V of b 2 GPI, which does not overlap with the phospholipid-binding site within domain V.

The antiphospholipid syndrome is caused by autoantibodies directed against ␤ 2 -glycoprotein I (␤ 2 GPI). Dimerization of ␤ 2 GPI results in an increased platelet deposition to collagen. We found that apolipoprotein E receptor 2 (apoER2 ), a member of the low density lipoprotein receptor family, is involved in activation of platelets by dimeric ␤ 2 GPI. To identify which domain of dimeric ␤ 2 GPI interacts with apoER2 , we have constructed domain deletion mutants of dimeric ␤ 2 GPI, lacking domain I (⌬I), II (⌬II), or V (⌬V), and a mutant with a W316S substitution in the phospholipid (PL)-insertion loop of domain V. ⌬I and ⌬II prolonged the clotting time, as did full-length dimeric ␤ 2 GPI; ⌬V had no effect on the clotting time. Second, ⌬I and ⌬II bound to anionic PL, comparable with full-length dimeric ␤ 2 GPI. ⌬V and the W316S mutant bound with decreased affinity to anionic PL. Platelet adhesion to collagen increased significantly when full-length dimeric ␤ 2 GPI, ⌬I, or ⌬II (mean increase 150%) were added to whole blood. No increase was found with plasma ␤ 2 GPI, ⌬V, or the W316S mutant. Immunoprecipitation indicated that full-length dimeric ␤ 2 GPI, ⌬I, ⌬II, and the W316S mutant can interact with apoER2 on platelets. ⌬V did not associate with apoER2 . We conclude that domain V is involved in both binding ␤ 2 GPI to anionic PL and in interaction with apoER2 and subsequent activation of platelets. The binding site in ␤ 2 GPI for interaction with apoER2 does not overlap with the hydrophobic insertion loop in domain V.The antiphospholipid syndrome is a non-inflammatory autoimmune disease associated with a wide variety of clinical symptoms. The main clinical features are arterial, venous, or small vessel thrombosis, both early and late pregnancy losses, and pre-eclampsia (1-4). The syndrome is diagnosed when one of the above clinical criteria is accompanied by the persistent presence of antiphospholipid antibodies (aPL) 3 (lupus anticoagulants and anticardiolipin antibodies) in the plasma of patients. These aPL are a heterogeneous group of antibodies directed to plasma proteins with affinity for anionic phospholipids (PL). We now know that the most important plasma protein, to which the aPL are directed, is ␤ 2 -glycoprotein I (␤ 2 GPI or apolipoprotein H) (5, 6).␤ 2 -Glycoprotein I is abundantly present in plasma (ϳ200 g/ml) and is mainly synthesized in the liver, although mRNA coding for ␤ 2 GPI has been found in a variety of cells such as trophoblasts, placental cells, endothelial cells, and neurons (7-9). The mature sequence of human ␤ 2 GPI consists of 326 (44 kDa) amino acids (aa) with four N-linked glycosylation sites. It is composed of five repeating units that belong to the complement control protein family. The first four domains have ϳ60 aa residues and 4 cysteines each, with potential disulfide bridges joining the first to third and the second to fourth cysteines to contribute to a "looped-back " structure, called Sushi domains. The fifth domain is aberrant, having 82 aa and three disulfide bridges. A...

Background: HLA-DQ2/8 heterozygous individuals have the highest risk for development of type 1 diabetes. Results: The disease-associated HLA-DQ2/8 transdimer exhibits unique peptide binding features compared with other HLA-DQ2/8 dimers. Conclusion: This newly identified binding motif predicts islet autoantigen-derived peptides as candidate T cell epitopes. Significance: Predicting new HLA-DQ2/8 transdimer-specific candidate T cell epitopes sets the stage for testing candidate diabetogenic epitopes.