Cocuzzi E, Szczotka L B, Brodbeck W G, Bardenstein D S, Wei T, Medof M E
Department of Pathology and Center For Vision Research, Case Western Reserve University, Cleveland, OH 44106-2622, USA.
Clin Exp Immunol. 2001 Feb;123(2):188-95. doi: 10.1046/j.1365-2249.2001.01408.x.
Previous studies have shown that DAF (or CD55), a cell surface inhibitor of autologous C3 activation, is present in tears and that > 90% of the C3 convertase regulatory activity in tear fluid resides in this protein (Lass JH et al., Invest Ophth Vis Sci 1990; 31:1136-48). This study investigated whether (i) the membrane cofactor protein (MCP or CD46), an additional factor that regulates C3 activation, and (ii) the membrane inhibitor of reactive lysis (MIRL or CD59), a cell surface regulator that acts to prevent formation of the membrane attack complex, are also present in tears, and if so, are functional. Two-site immunoradiometric assays showed that MCP is present in tears at low levels (42 + 8 ng/ml, n = 8) while CD59 is present at levels (222 + 78 ng/ml, n = 14) comparable to those of DAF (325 + 289 ng/ml, n = 12). The concentrations of CD59 (i) were increased two-fold or more in closed eye tears, and (ii) were decreased in reflex tears. Western blotting showed that CD59 protein in tears migrates with an apparent mol. wt similar to membrane CD59 protein. Phenyl-Sepharose adsorption and Triton X-114 partitioning of tear CD59 as well as of tear DAF however, showed that both proteins are devoid of GPI anchors. Assays using cobra venom factor-activated human serum and guinea pig erythrocytes showed that CD59 is functionally active in inhibiting autologous C5b-9-mediated lysis and, under constitutive conditions, accounts for > 85% of the C9 inhibitory activity in tear fluid.
以往的研究表明,衰变加速因子(DAF,即CD55)是一种细胞表面自体C3激活抑制剂,存在于泪液中,且泪液中>90%的C3转化酶调节活性存在于该蛋白中(Lass JH等人,《Invest Ophth Vis Sci》1990年;31:1136 - 48)。本研究调查了:(i)膜辅因子蛋白(MCP,即CD46),一种调节C3激活的额外因子;以及(ii)反应性溶解膜抑制剂(MIRL,即CD59),一种防止膜攻击复合物形成的细胞表面调节剂,是否也存在于泪液中,若存在,是否具有功能。双位点免疫放射分析表明,MCP以低水平存在于泪液中(42 + 8 ng/ml,n = 8),而CD59的存在水平(222 + 78 ng/ml,n = 14)与DAF(325 + 289 ng/ml,n = 12)相当。CD59的浓度:(i)在闭眼泪液中增加了两倍或更多;(ii)在反射性泪液中降低。蛋白质印迹法显示,泪液中的CD59蛋白迁移时的表观分子量与膜CD59蛋白相似。然而,泪液CD59以及泪液DAF的苯基 - 琼脂糖吸附和Triton X - 114分配表明,这两种蛋白都没有糖基磷脂酰肌醇(GPI)锚。使用眼镜蛇毒因子激活的人血清和豚鼠红细胞进行的分析表明,CD59在抑制自体C5b - 9介导的溶解方面具有功能活性,并且在组成性条件下,占泪液中C9抑制活性的>85%。