Timofeeva Anna M, Buneva Valentina N, Nevinsky Georgy A
SB RAS, Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Ave., 630090, Novosibirsk, Russia.
Novosibirsk State University, 2 Pirogova St., 630090, Novosibirsk, Russia.
J Mol Recognit. 2015 Oct;28(10):614-27. doi: 10.1002/jmr.2476. Epub 2015 Apr 13.
Antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. Small pools of phage particles displaying light chains with different affinities for MBP were isolated by affinity chromatography on MBP-Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27 kDa). Seventy-two of 440 individual colonies were randomly chosen, expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography. Twenty-two of 72 MLChs have high affinity and efficiently hydrolyze only MBP (not other control proteins) demonstrating various pH optima in a 5.7-9.0 range and different substrate specificity in the hydrolysis of four different MBP oligopeptides. Four MLChs demonstrated serine protease-like and three thiol protease-like activities, while 11 MLChs were metalloproteases. The activity of three MLChs was inhibited by both phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA), two other by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. The ratio of relative activity in the presence of Ca(2+), Mg(2+), Mn(2+), Ni(2+), Zn(2+), Cu(2+), and Co(2+) was individual for each of 22 MLCh preparations. It is the first examples of human MLChs, which probably can possess two or even three different proteolytic activities. These observations suggest an extreme diversity of anti-MBP abzymes in SLE patients. The immune systems of individual SLE patients can generate a variety of anti-MBP abzymes, which can attack MBP of myelin-proteolipid sheath of axons and play an important role in MS and SLE pathogenesis.
水解髓鞘碱性蛋白（MBP）的抗体在多发性硬化症（MS）和系统性红斑狼疮（SLE）的发病机制中可能起重要作用。使用了一个源自SLE患者外周血淋巴细胞的免疫球蛋白轻链噬菌粒文库。通过在MBP-琼脂糖凝胶上进行亲和层析，分离出展示对MBP具有不同亲和力的轻链的小噬菌体颗粒池，并用0.5 M NaCl洗脱的部分用于制备单个单克隆轻链（MLChs，26 - 27 kDa）。从440个单个菌落中随机选择72个，在大肠杆菌中以可溶形式表达，并通过金属螯合层析纯化MLChs。72个MLChs中有22个具有高亲和力，仅高效水解MBP（而非其他对照蛋白），在5.7 - 9.0范围内表现出不同的最适pH值，并且在水解四种不同的MBP寡肽时具有不同的底物特异性。4个MLChs表现出丝氨酸蛋白酶样活性，3个表现出硫醇蛋白酶样活性，而11个MLChs是金属蛋白酶。3个MLChs的活性被苯甲基磺酰氟（PMSF）和乙二胺四乙酸（EDTA）同时抑制，另外2个被EDTA和碘乙酰胺抑制，1个被PMSF、EDTA和碘乙酰胺抑制。在存在Ca(2+)、Mg(2+)、Mn(2+)、Ni(2+)、Zn(2+)、Cu(2+)和Co(2+)的情况下，22种MLCh制剂中每种的相对活性比例都是独特的。这是人类MLChs的首个例子，其可能具有两种甚至三种不同的蛋白水解活性。这些观察结果表明SLE患者中抗MBP抗体酶具有极端多样性。个体SLE患者的免疫系统可产生多种抗MBP抗体酶，这些抗体酶可攻击轴突髓鞘-蛋白脂质鞘的MBP，并在MS和SLE发病机制中起重要作用。
