Recombinant bovine conglutinin, lacking the N-terminal and collagenous domains, has less conglutination activity but is able to inhibit haemagglutination by influenza A virus
Recombinant bovine conglutinin, lacking the N-terminal and collagenous domains, has less conglutination activity but is able to inhibit haemagglutination by influenza A virus
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DOI：
10.1042/bj3160043
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发表时间：
1996-05-15
影响因子：
4.1
通讯作者：
Wakamiya, N
中科院分区：
文献类型：
--
作者：
Eda, S;Suzuki, Y;Wakamiya, N
凝集素是一种牛血清蛋白，最初被描述为一种脊椎动物凝集素。这种蛋白属于c型凝集素家族。这些凝集素由四个特征结构域组成：(1)n端富含半胱氨酸结构域，(2)胶原样结构域，(3)颈部结构域和(4)碳水化合物识别结构域（CRD）。最近，由于补体介导的机制或调理，凝集素已被证明是免疫球蛋白不依赖的防御分子。我们先前的研究表明，牛凝集素可以抑制甲型流感病毒的血凝，并且由于其凝集素的特性直接中和它们。为了阐明胶原样结构域的生物学作用，在大肠杆菌系统中制备了缺乏该胶原样结构域的重组部分粘连蛋白，并对其生物活性进行了研究。通过逆转录酶PCR技术扩增出一个497 bp的序列，该序列包括一个短胶原区（两个重复的G-X-Y氨基酸序列）、颈部结构域和粘连素cDNA的CRD。将cDNA转移到细菌表达载体系统（pRSET-A）中，获得了高水平粘连素产量的稳定转染物。SDS/PAGE和Western blotting分析显示重组融合蛋白为27 kDa。交联研究和凝胶过滤实验结果表明，重组粘连蛋白可以形成三聚体结构，具有与天然粘连蛋白相似的糖结合活性和特异性。重组凝集素对甲型流感病毒引起的血液凝集有抑制作用，但凝集活性较低。这些结果表明，为了使粘连素抑制流感病毒引起的血凝，以及具有糖结合活性或形成三聚体，它不需要n端和胶原结构域；然而，它们对于充分的粘连活性是必不可少的。
Conglutinin is a bovine serum protein which was first described as a vertebrate lectin. This protein belongs to the family of C-type lectins. These lectins are composed of four characteristic domains: (1) an N-terminal cysteine-rich domain, (2) a collagen-like domain, (3) a neck domain and (4) a carbohydrate recognition domain (CRD). Recently lectins have been shown to function as immunoglobulin-independent defence molecules due to a complement-mediated mechanism or opsonization. Our previous study showed that bovine conglutinin can inhibit haemagglutination by influenza A viruses and act by directly neutralizing them due to its lectin properties. In order to elucidate the biological role of the collagen-like domain, a recombinant partial conglutinin lacking this collagen-like domain was produced in an Escherichia coli system and its biological activities were examined. A 497 bp sequence, consisting of a short collagen region (two repeats of G-X-Y amino acid sequences), the neck domain, and the CRD of conglutinin cDNA, was amplified by the reverse-transcriptase PCR technique. The cDNA was transferred to a bacterial expression vector system (pRSET-A) and stable transfectants with a high level of conglutinin production were obtained. SDS/PAGE and Western blotting analyses showed a recombinant fusion protein of 27 kDa. Results of a cross-linking study and gel-filtration assay indicated that the recombinant conglutinin can form a trimeric structure and that it has sugar binding activity and specificity similar to that of native conglutinin. The recombinant conglutinin was also found to inhibit haemagglutination caused by influenza A virus as well as to possess less conglutination activity. These results suggest that in order for conglutinin to inhibit haemagglutination caused by the influenza virus, as well as to have sugar binding activity or to form trimers, it does not require the N-terminal and collagenous domains; however, they are essential for full conglutination activity.