Eda S, Suzuki Y, Kase T, Kawai T, Ohtani K, Sakamoto T, Kurimura T, Wakamiya N
Osaka Prefectural Institute of Public Health, Department of Pathology, Japan.
Biochem J. 1996 May 15;316 ( Pt 1)(Pt 1):43-8. doi: 10.1042/bj3160043.
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.
胶固素是一种牛血清蛋白，最初被描述为一种脊椎动物凝集素。这种蛋白质属于C型凝集素家族。这些凝集素由四个特征结构域组成：（1）N端富含半胱氨酸的结构域，（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）。最近研究表明，由于补体介导机制或调理作用，凝集素可作为不依赖免疫球蛋白的防御分子发挥作用。我们之前的研究表明，牛胶固素可抑制甲型流感病毒的血凝作用，并因其凝集素特性直接中和病毒。为了阐明胶原样结构域的生物学作用，我们在大肠杆菌系统中制备了一种缺乏该胶原样结构域的重组部分胶固素，并检测了其生物学活性。通过逆转录酶PCR技术扩增了一段497 bp的序列，该序列由一个短胶原区域（G-X-Y氨基酸序列的两个重复）、颈部结构域和胶固素cDNA的CRD组成。将该cDNA转移到细菌表达载体系统（pRSET-A）中，获得了高水平表达胶固素的稳定转染子。SDS/PAGE和蛋白质印迹分析显示有一个27 kDa的重组融合蛋白。交联研究和凝胶过滤分析结果表明，重组胶固素可形成三聚体结构，并且具有与天然胶固素相似的糖结合活性和特异性。还发现重组胶固素可抑制甲型流感病毒引起的血凝作用，且具有较低的胶固活性。这些结果表明，胶固素为了抑制流感病毒引起的血凝作用以及具有糖结合活性或形成三聚体，并不需要N端和胶原结构域；然而，它们对于充分的胶固活性是必不可少的。
