Ramsland P A, Movafagh B F, Reichlin M, Edmundson A B
Crystallography Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.
J Mol Recognit. 1999 Jul-Aug;12(4):249-57. doi: 10.1002/(SICI)1099-1352(199907/08)12:4<249::AID-JMR463>3.0.CO;2-B.
Circulating autoimmune complexes of IgM rheumatoid factors (RF) bound to the Fc portions of normal, polyclonal IgG antibodies are frequently present in humans with rheumatoid arthritis (RA). The sweet tasting methyl ester of L-Asp-L-Phe (aspartame or APM) was found to relieve pain and improve joint mobility in subjects with osteo- and mixed osteo/rheumatoid arthritis [Edmundson, A. B. and Manion, C. V. (1998). Clin. Pharmac. Ther. 63, 580-593]. These clinical observations prompted the testing of the inhibition by APM of the binding interactions of human IgM RFs with IgG Fc regions. The propensity of APM to inhibit IgM RF binding was assessed by competitive enzyme immunoassays with solid-phase human IgG. Ten RA serum samples and three purified monoclonal cryoglobulins, all of which had RF activity, were tested in this system. We found that the presence of APM significantly reduced the binding of IgM RFs. The inhibitory propensity of APM with monoclonal RF cryoglobulins was increased by the addition of CaCl(2) to the binding buffer. Similar inhibition of the binding of RA derived RFs to IgG was observed for Asp-Phe and its amidated derivative, indicating that the methyl ester is not required for APM's interaction with IgM antibodies. A human (Mez) IgM known to bind octameric peptides derived from the Fc portion of a human IgG(1) antibody was tested for binding of dipeptides by the Pepscan method of combinatorial chemistry. The relative binding constants of Asp-Phe and Phe-Asp were ranked among the highest values for 400 possible combinations of the 20 most common amino acids. Possible blocking interactions of APM were explored by computer-assisted docking studies with the model of a complex of an RF Fab with the Fc of a human IgG(4) antibody. Modeling of ternary immune complexes revealed a few key residues, which could act as molecular recognition sites for APM. A structural hypothesis is presented to explain the observed interference with RF reactivity by APM. Extrapolations of the current results suggest that APM may inhibit the binding of IgG in a substantial proportion of IgM RFs. Interference of RF reactivity, especially in RA patients, may alleviate the pain and immobility resulting from chronic inflammation of the joints.
与正常多克隆IgG抗体Fc部分结合的IgM类风湿因子（RF）循环自身免疫复合物在类风湿关节炎（RA）患者中经常出现。L-天冬氨酰-L-苯丙氨酸甲酯（阿斯巴甜或APM）有甜味，被发现可缓解骨关节炎和混合性骨关节炎/类风湿关节炎患者的疼痛并改善关节活动度[埃德蒙森，A.B.和马尼恩，C.V.（1998年）。临床药理学与治疗学63，580 - 593]。这些临床观察促使人们测试APM对人IgM RF与IgG Fc区域结合相互作用的抑制作用。通过与固相人IgG的竞争性酶免疫测定评估APM抑制IgM RF结合的倾向。在该系统中测试了10份RA血清样本和3种纯化的单克隆冷球蛋白，它们都具有RF活性。我们发现APM的存在显著降低了IgM RF的结合。向结合缓冲液中添加CaCl₂可增强APM对单克隆RF冷球蛋白的抑制倾向。观察到Asp-Phe及其酰胺化衍生物对源自RA的RF与IgG结合有类似的抑制作用，这表明甲酯并非APM与IgM抗体相互作用所必需。使用组合化学的Pepscan方法测试了一种已知能与人IgG(1)抗体Fc部分衍生的八聚体肽结合的人（Mez）IgM与二肽的结合情况。Asp-Phe和Phe-Asp的相对结合常数在20种最常见氨基酸的400种可能组合中排名最高。通过计算机辅助对接研究，利用RF Fab与人IgG(4)抗体Fc复合物的模型探索了APM可能的阻断相互作用。三元免疫复合物的建模揭示了一些关键残基，它们可能作为APM的分子识别位点。提出了一个结构假说来解释观察到的APM对RF反应性的干扰。当前结果的推断表明，APM可能在很大比例的IgM RF中抑制IgG的结合。RF反应性的干扰，尤其是在RA患者中，可能减轻关节慢性炎症引起的疼痛和活动受限。
