Holmskov U, Laursen S B, Malhotra R, Wiedemann H, Timpl R, Stuart G R, Tornøe I, Madsen P S, Reid K B, Jensenius J C
Department of Medical Microbiology, University of Odense, Denmark.
Biochem J. 1995 Feb 1;305 ( Pt 3)(Pt 3):889-96. doi: 10.1042/bj3050889.
Collectin-43 (CL-43) is a recently described bovine plasma protein containing both collagenous regions and C-type-lectin domains [Holmskov, Teisner, Willis, Reid and Jensenius (1993) J. Biol. Chem. 268, 10120-10125; Lim, Willis, Reid, Lu, Laursen, Jensenius and Holmskov (1994) J. Biol. Chem. 269, 11820-11824]. CL-43 was purified by affinity chromatography on mannan-Sepharose. On SDS/PAGE under reducing conditions the purified lectin showed a double band at about 43 kDa, with the upper band representing the intact molecule and the lower band a truncated form that lacked the N-terminal nine amino acid residues. Under non-reducing conditions, only one band was seen at 120 kDa. Analytical gel chromatography and sucrose-density-gradient centrifugation of the purified molecule, showed a Stokes radius of 9.1 +/- 0.3 nm (91 +/- 3 A) and a sedimentation coefficient (s20,w) of 3.6 +/- 0.1 S. These values correspond to a molecular mass of 119-138 kDa under non-denaturing condition in solution. The frictional coefficient (f/f0) was 2.7, indicating extreme elongation due to the collagenous segment. Only monomer subunits, with 37.4 +/- 1.7-nm-long rods, were seen by electron microscopy. These findings indicate that CL-43, in contrast with the other circulating collectins, is found only as a single subunit composed of three polypeptide chains. Two-dimensional gel electrophoresis showed that CL-43 has two isoforms, with pI values of 4.9 and 5.3, corresponding to the native form and the truncated form of the molecule respectively. CL-43, like conglutinin, lung surfactant protein A and mannan-binding protein (MBP), was shown to bind to the collectin receptor. Bovine MBP caused the activation of the complement system as revealed by the deposition of complement component C4 upon incubation of diluted serum in wells containing MBP bound to solid-phase mannan. CL-43, lung surfactant protein D (SP-D) and conglutinin showed no complement-activating properties under the same conditions. Conglutinin binds fluid- and solid-phase iC3b, while CL-43 and MBP do not show such reactivity.
收集素-43（CL-43）是最近描述的一种牛血浆蛋白，含有胶原区域和C型凝集素结构域[霍尔姆斯科夫、泰斯纳、威利斯、里德和延森纽斯（1993年）《生物化学杂志》268卷，10120 - 10125页；林、威利斯、里德、卢、劳尔森、延森纽斯和霍尔姆斯科夫（1994年）《生物化学杂志》269卷，11820 - 11824页]。CL-43通过甘露聚糖-琼脂糖亲和层析进行纯化。在还原条件下的SDS/PAGE上，纯化的凝集素在约43 kDa处显示出一条双链带，上带代表完整分子，下带是一种截短形式，缺少N端的九个氨基酸残基。在非还原条件下，在120 kDa处仅可见一条带。对纯化分子进行分析凝胶过滤和蔗糖密度梯度离心，结果显示斯托克斯半径为9.1±0.3 nm（91±3 Å），沉降系数（s20,w）为3.6±0.1 S。这些值对应于在溶液中非变性条件下119 - 138 kDa的分子量。摩擦系数（f/f0）为2.7，表明由于胶原片段导致极度伸长。通过电子显微镜仅观察到单体亚基，其杆状长度为37.4±1.7 nm。这些发现表明，与其他循环收集素不同，CL-43仅以由三条多肽链组成的单个亚基形式存在。二维凝胶电泳显示CL-43有两种同工型，pI值分别为4.9和5.3，分别对应于分子的天然形式和截短形式。CL-43与胶固素、肺表面活性蛋白A和甘露聚糖结合蛋白（MBP）一样，被证明可与收集素受体结合。牛MBP可激活补体系统，如在含有与固相甘露聚糖结合的MBP的孔中孵育稀释血清后，补体成分C4的沉积所揭示。在相同条件下，CL-43、肺表面活性蛋白D（SP-D）和胶固素未显示出补体激活特性。胶固素结合液相和固相结合的iC3b，而CL-43和MBP不显示这种反应性。
