Miyamura K, Leigh L E, Lu J, Hopkin J, López Bernal A, Reid K B
Department of Biochemistry, University of Oxford, U.K.
Biochem J. 1994 May 15;300 ( Pt 1)(Pt 1):237-42. doi: 10.1042/bj3000237.
Surfactant protein D (SP-D) is a lung-specific protein, synthesized and secreted by lung epithelial cells. It belongs to group III of the family of C-type lectins; each member of this group has an unusual overall structure consisting of multiple globular 'head' regions (which contain the C-type lectin domains) linked by triple-helical, collagen-like, strands. This group includes the surfactant protein A (SP-A) and the serum proteins mannan-binding protein, conglutinin and collectin-43, all of which have been shown to bind to the C1q receptor found on a wide variety of cells, including macrophages. Both SP-D and SP-A have been shown to enhance oxygen radical production by alveolar macrophages. Although this strongly suggests a direct interaction between SP-D and a specific receptor on alveolar macrophages, it is still unclear whether SP-D binds to the same receptor used by SP-A and/or C1q. Human SP-D was isolated from amniotic fluid and was radiolabelled using 125I. Alveolar macrophages were isolated from human bronchioalveolar lavage fluid, and also from bovine lung washings, by differential adhesion to 24-well tissue-culture plates. The study was carried out using EDTA-containing buffers, to eliminate Ca(2+)-dependent C-type lectin binding, and was also carried out at 4 degrees C to eliminate possible internalization by the cells. 125I-SP-D showed specific binding to alveolar macrophages in both a time- and concentration-saturable manner. The binding was inhibited, by approx. 90%, on addition of a 200-fold excess of unlabelled SP-D. The apparent dissociation constant (Kd) was (3.6 +/- 1.3) x 10(-11) M, based on the assumption that native SP-D is assembled as a dodecamer of 12 identical polypeptides of 43 kDa to yield a protein of 516 kDa. C1q was also shown to bind alveolar macrophages (Kd 3 x 10(-6) M), but addition of C1q did not show inhibition of the binding of 125I-SP-D to the macrophages. We conclude that SP-D binds specifically to alveolar macrophages and the receptor involved is different from that utilized by C1q.
表面活性蛋白D（SP-D）是一种肺特异性蛋白，由肺上皮细胞合成并分泌。它属于C型凝集素家族的III组；该组的每个成员都有一个不同寻常的整体结构，由多个球状“头部”区域（包含C型凝集素结构域）通过三螺旋、胶原样链连接而成。该组包括表面活性蛋白A（SP-A）以及血清蛋白甘露聚糖结合蛋白、胶固素和collectin-43，所有这些蛋白都已被证明能与多种细胞（包括巨噬细胞）上发现的C1q受体结合。SP-D和SP-A都已被证明能增强肺泡巨噬细胞的氧自由基产生。尽管这强烈表明SP-D与肺泡巨噬细胞上的特定受体之间存在直接相互作用，但仍不清楚SP-D是否与SP-A和/或C1q使用的相同受体结合。人SP-D从羊水分离出来，并用125I进行放射性标记。肺泡巨噬细胞从人支气管肺泡灌洗液以及牛肺灌洗液中通过对24孔组织培养板的差异黏附分离出来。该研究使用含EDTA的缓冲液进行，以消除Ca(2+)依赖性C型凝集素结合，并且也在4℃下进行以消除细胞可能的内化作用。125I-SP-D以时间和浓度饱和的方式显示出与肺泡巨噬细胞的特异性结合。加入200倍过量的未标记SP-D后，结合被抑制了约90%。基于天然SP-D组装成由12个相同的43 kDa多肽组成的十二聚体以产生516 kDa蛋白质的假设，表观解离常数（Kd）为(3.6 +/- 1.3) x 10(-11) M。C1q也被证明能结合肺泡巨噬细胞（Kd 3 x 10(-6) M），但加入C1q并未显示出对125I-SP-D与巨噬细胞结合的抑制作用。我们得出结论，SP-D特异性结合肺泡巨噬细胞，且所涉及的受体与C1q使用的受体不同。
