Topological Requirements and Signaling Properties of T Cell–activating, Anti-CD28 Antibody Superagonists

Full activation of naive T cells requires both engagement of the T cell antigen receptor (TCR; signal 1) and costimulatory signaling by CD28 (signal 2). We previously identified two types of rat CD28-specific monoclonal antibodies (mAbs): “conventional,” TCR signaling–dependent costimulatory mAbs and “superagonistic” mAbs capable of inducing the full activation of primary resting T cells in the absence of TCR ligation both in vitro and in vivo. Using chimeric rat/mouse CD28 molecules, we show that the superagonists bind exclusively to the laterally exposed C′′D loop of the immunoglobulin-like domain of CD28 whereas conventional, costimulatory mAbs recognize an epitope close to the binding site for the natural CD80/CD86 ligands. Unexpectedly, the C′′D loop reactivity of a panel of new antibodies raised against human CD28 could be predicted solely on the basis of their superagonistic properties. Moreover, mouse CD28 molecules engineered to express the rat or human C′′D loop sequences activated T cell hybridomas without TCR ligation when cross-linked by superagonistic mAbs. Finally, biochemical analysis revealed that superagonistic CD28 signaling activates the nuclear factor κB pathway without inducing phosphorylation of either TCRζ or ZAP70. Our findings indicate that the topologically constrained interactions of anti-CD28 superagonists bypass the requirement for signal 1 in T cell activation. Antibodies with this property may prove useful for the development of T cell stimulatory drugs

Differences in CD80 and CD86 transendocytosis reveal CD86 as a key target for CTLA-4 immune regulation

CD28 and CTLA-4 (CD152) play essential roles in regulating T cell immunity, balancing the activation and inhibition of T cell responses, respectively. Although both receptors share the same ligands, CD80 and CD86, the specific requirement for two distinct ligands remains obscure. In the present study, we demonstrate that, although CTLA-4 targets both CD80 and CD86 for destruction via transendocytosis, this process results in separate fates for CTLA-4 itself. In the presence of CD80, CTLA-4 remained ligand bound, and was ubiquitylated and trafficked via late endosomes and lysosomes. In contrast, in the presence of CD86, CTLA-4 detached in a pH-dependent manner and recycled back to the cell surface to permit further transendocytosis. Furthermore, we identified clinically relevant mutations that cause autoimmune disease, which selectively disrupted CD86 transendocytosis, by affecting either CTLA-4 recycling or CD86 binding. These observations provide a rationale for two distinct ligands and show that defects in CTLA-4-mediated transendocytosis of CD86 are associated with autoimmunity

Ｘ線結晶構造解析によるω-アミノ酸 : ピルビン酸アミノ基転移酵素の反応機構の研究

　アミノ基転移酵素はピリドキザール5\u27-リン酸(PLP)を補酵素として持つPLP依存酵素で基質のアミノ基をケト酸に転移する反応を触媒する｡アミノ基転移酵素は共通の性質として活性部位にあるリジン残基がPLPとの間でシッフ塩基を形成しており､また反応は共通の中間体即ちPLP-基質アミノ酸のシッフ塩基の形成を経て進行すると考えられてきた｡天然には多くのアミノ基転移酵素が存在し､既に40種以上の酵素のアミノ酸配列が決定されており､酵素化学的な方法で反応の研究もなされている｡しかしながら､3次元構造が得られているものは､アスパラギン酸アミノ基転移酵素(AspAT),ホスホセリンアミノ基転移酵素,最近構造決定されたD-アミノ酸アミノ基転移酵素及びω-アミノ酸:ピルビン酸アミノ基転移酵素(ω-APT)の4種である｡そのうち結晶学的な方法で反応機構の研究かなされているものはAspATのみである｡　Psedomonas sp.F-128の生産するω-APTはPLP依存酵素で､β-alanincからピルビン酸へのアミノ基の転移を触媒し､アミノドナーに対しては特異性が低く､アミノアクセプターに対しては極めて高い特異性を示す｡この分子は449個のアミノ酸残基からからなる結晶学的に同一なザブユニット4個からなり､アミノ酸配列は与那覇らにより決定された｡結晶構造解析は渡辺らによりまず異常分散を考慮した多重同型置換法により分解能が2.0人のデータを用いて行われ､得られた構造は更に分解能が1.6人のデータでプログラムPROLSQにより精密化された｡本論文ではω-APTと種々のアミノドナー(β-Alaninc,γ-Amino-n-butyratc,6-Aminohexonatc,L-Alaninc,Isoamylamine)及びアミノアクセプターであるピルビン酸との複合体結晶の構造解析を行い､それらの構造に基づいて提唱された反応機構について報告する｡　複合体結晶は透析法による共結晶法によってpH7.0の38%硫酸アンモニウム水溶液から調整された｡強度データの収集は高エネルギー物理学研究所･放射光実験施設･BL6A2に設置された巨大分子用ワイセンベルグカメラにより200nm×400mmのイメージングプレートを記録媒体として行われた｡L-Alaninc複合体結晶の場合はまず3軸のまわりで1.5Å分解能まで､次いで露光時間を約3倍にして1.4Åまでのデータ収集を行った｡Isoamylaminc複合体結晶については1.5Å分解能､その他については1.8Å分解能までのデータ収集を行った｡複合体結晶はいずれもNative結晶(空間群1222､a=124.7,b=137.9,c=61.5Å)と同型であった｡強度データの処理にはプログラムWEISを用いた｡精密化の初期構造としてはNative結晶の構造パラメータを使用し､プログラムはPROLSQとXPLORを利用した｡基質複合体モデルの構築をプログラムFRODOを使用して3次元グラフィックスにより行った｡精密化の結果､L-Alaninc複合体結晶では分解能10.0?1.6ÅでR値が0.159､β-Alaninc,γ-Amino-n-butyrate,6-Aminohexonate,Isoamylaminc及びピルビン酸複合体結晶ではR値それぞれ0.220,0.165,0.196,0.152,0.208の構造を得ることが出来た｡図にω-APT及び種々の複合体の結晶構造に基づく反応機構を示す｡図中Aの構造については既に渡辺らの解析で得られいたが､AspATのNativeの構造と異なり活性部位にあるLys288とPLPの間にはシッフ塩基の形成はみられない｡このことはPLP酵素に対して50年間共通の性質と考えられてきた定説を覆すことになる｡そこで､結晶化条件を変えたり､精密化の方法を変えたりして検討を続けたが結果は同じであった｡最近杉尾らにより構造解析されたD-アミノ酸アミノ基転移酵素は､ポリエチレングリコールを沈殿剤に用いて結晶化されているにも拘わらず活性部位にシッフ塩基が形成されていない結果が得られた｡さらにこの経路中､D中間体はβ-Alaninc複合体の構造に相当し､I中間体の構造はL-Alaninc複合体の構造に相当する｡またPLPとPMPにより周りの環境に大きな変化がないので､Fの構造はピルビン酸とPLPの複合体結晶の構造と同様な配置をとると考えられる｡これらの中間体の構造を基に図の反応機構は矛盾なく説明できる｡なお酵素反応機構はAspATとは異なっている

Crystallization and preliminary X-ray analysis of human MTH1 complexed with two oxidized nucleotides, 8-oxo-dGMP and 2-oxo-dATP

The complexes of human MTH1 with two oxidized nucleotides, 8-oxo-dGMP and 2-oxo-dATP, were crystallized. The crystals diffracted to 1.95 and 2.22 Å resolution, respectively

doi:10.1093/nar/gkr575 Diverse substrate recognition and hydrolysis mechanisms of human NUDT5

Human NUDT5 (hNUDT5) hydrolyzes various modified nucleoside diphosphates including 8-oxo-dGDP, 8-oxo-dADP and ADP-ribose (ADPR). However, the structural basis of the broad substrate specificity remains unknown. Here, we report the crystal structures of hNUDT5 complexed with 8-oxo-dGDP and 8-oxo-dADP. These structures reveal an unusually different substrate-binding mode. In particular, the positions of two phosphates (a and b phosphates) of substrate in the 8-oxo-dGDP and 8-oxo-dADP complexes are completely inverted compared with those in the previously reported hNUDT5–ADPR complex structure. This result suggests that the nucleophilic substitution sites of the substrates involved in hydrolysis reactions differ despite the similarities in the chemical structures of the substrates and products. To clarify this hypothesis, we employed the isotope-labeling method and revealed that 8-oxo-dGDP is attacked by nucleophilic water at Pb, whereas ADPR is attacked at Pa. This observation reveals that the broad substrate specificity of hNUDT5 is achieved by a diversity of not only substrate recognition, but also hydrolysis mechanisms and leads to a novel aspect that enzymes do not always catalyze the reaction of substrates with similar chemical structures by using the chemically equivalent reaction site

Crystal structure of the CD2-binding domain of CD58 (lymphocyte function-associated antigen 3) at 1.8-Å resolution

The binding of the cell surface molecule CD58 (formerly lymphocyte function-associated antigen 3) to its ligand, CD2, significantly increases the sensitivity of antigen recognition by T cells. This was the first heterophilic cell adhesion interaction to be discovered and is now an important paradigm for analyzing the structural basis of cell–cell recognition. The crystal structure of a CD2-binding chimeric form of CD58, solved to 1.8-Å resolution, reveals that the ligand binding domain of CD58 has the expected Ig superfamily V-set topology and shares several of the hitherto unique structural features of CD2, consistent with previous speculation that the genes encoding these molecules arose via duplication of a common precursor. Nevertheless, evidence for considerable divergence of CD2 and CD58 is also implicit in the structures. Mutations that disrupt CD2 binding map to the highly acidic surface of the AGFCC′C′′ β-sheet of CD58, which, unexpectedly, lacks marked shape complementarity to the equivalent, rather more basic CD58-binding face of human CD2. The specificity of the very weak interactions of proteins mediating cell–cell recognition may often derive largely from electrostatic complementarity, with shape matching at the protein–protein interface being less exact than for interactions that combine specificity with high affinity, such as those involving antibodies