Eda S, Suzuki Y, Kawai T, Ohtani K, Kase T, Fujinaga Y, Sakamoto T, Kurimura T, Wakamiya N
Department of Pathology, Osaka Prefectural Institute of Public Health, 1-3-69 Nakamichi, Higashinari, Osaka 537, Japan.
Biochem J. 1997 Apr 15;323 ( Pt 2)(Pt 2):393-9. doi: 10.1042/bj3230393.
Surfactant protein D (SP-D) is a lung-specific protein that is synthesized and secreted by lung epithelial cells and is believed to play an important role in lung host defence. This protein belongs to the C-type lectin family, which is characterized by an N-terminal cysteine-rich domain, a collagen-like domain, a neck domain and a carbohydrate recognition domain (CRD). To elucidate the biological actions of this animal lectin against such pathogens as micro-organisms, the biological activities of a recombinant partial SP-D lacking a collagen-like domain were examined. A recombinant human SP-D, consisting of a short collagen region (two repeats of Gly-Xaa-Yaa amino acid sequences), the neck domain and the CRD, was expressed in Escherichia coli. The recombinant SP-D was purified on a nickel column and then on a maltose-agarose column. This protein can form a trimeric structure owing to the neck domain and exhibits sugar-binding activity and specificity similar to those of native human SP-D. The recombinant SP-D caused dose-dependent and calcium-dependent agglutination of E. coli Y1088. The agglutination titre (the concentration required to achieve a 50% decrease in light transmission by agglutination) of recombinant SP-D was approx. 6-fold that of native SP-D. As for conglutination, the recombinant trimeric conglutinin required 8-16-fold higher concentrations than the native counterpart. In haemagglutination inhibition (HI) of influenza A virus, although native and recombinant conglutinin showed similar levels of HI activity, the recombinant SP-D was unable to inhibit haemagglutination, even at a concentration approx. 120-fold that of the native SP-D. The lectin precipitation and lectin blot assays showed that the truncated SP-D could bind to influenza A virus as well as native SP-D did. These results indicate that the agglutination activity of trimeric collectins can be largely retained, and furthermore that the oligomeric structure with several hands at opposite sites can enhance agglutination activity. The difference in HI activity against influenza A virus between native and recombinant SP-D suggests that SP-D uses a different mechanism from that of conglutinin to inhibit viral haemagglutination.
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，被认为在肺部宿主防御中发挥重要作用。该蛋白属于C型凝集素家族，其特征在于N端富含半胱氨酸结构域、胶原样结构域、颈部结构域和碳水化合物识别结构域（CRD）。为了阐明这种动物凝集素针对微生物等病原体的生物学作用，研究了缺乏胶原样结构域的重组部分SP-D的生物学活性。一种重组人SP-D，由短胶原区域（Gly-Xaa-Yaa氨基酸序列的两个重复）、颈部结构域和CRD组成，在大肠杆菌中表达。重组SP-D先在镍柱上纯化，然后在麦芽糖-琼脂糖柱上纯化。由于颈部结构域，该蛋白可形成三聚体结构，并表现出与天然人SP-D相似的糖结合活性和特异性。重组SP-D引起大肠杆菌Y1088的剂量依赖性和钙依赖性凝集。重组SP-D的凝集效价（通过凝集使透光率降低50%所需的浓度）约为天然SP-D的6倍。至于共凝集，重组三聚体共凝集素所需浓度比天然对应物高8至16倍。在甲型流感病毒的血凝抑制（HI）试验中，尽管天然和重组共凝集素表现出相似水平的HI活性，但重组SP-D即使在浓度约为天然SP-D的120倍时也无法抑制血凝。凝集素沉淀和凝集素印迹分析表明，截短的SP-D与天然SP-D一样能结合甲型流感病毒。这些结果表明三聚体凝集素的凝集活性可在很大程度上保留，此外，在相对位点具有多个臂的寡聚结构可增强凝集活性。天然和重组SP-D对甲型流感病毒HI活性的差异表明，SP-D使用与共凝集素不同的机制来抑制病毒血凝。
