Molecular and biological characterization of a mannan-binding lectin from the holothurian Apostichopus japonicus
Molecular and biological characterization of a mannan-binding lectin from the holothurian Apostichopus japonicus
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DOI：
10.1093/glycob/cwm093
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发表时间：
2007-12-01
期刊：
影响因子：
4.3
通讯作者：
Rasskazov, Valery A.
中科院分区：
文献类型：
--
作者：
Bulgakov, Aleksandr A.;Eliseikina, Marina G.;Rasskazov, Valery A.
为了阐明甘露聚糖结合凝集素（MBL）的起源和进化，从海参Apostichopus sp.的体腔血浆中分离到一种新的高甘露聚糖特异性C型凝集素（MBL-AJ）。MBL-AJ在SDS-PAGE上具有相同的17-kDa亚基的寡聚体形式。在天然配体中，凝集素血凝活性被从海洋嗜盐菌中分离的细胞外低分支但不高分支的α-D-甘露聚糖竞争性抑制，所述α-D-甘露聚糖由α-1，2和α-1，6连接的D-甘露糖残基组成。这表明凝集素与主链或内侧链甘露糖残基相互作用，但不与末端甘露糖残基相互作用。该凝集素的活性具有Ca ~（2+）、pH和温度依赖性。从海参体腔细胞cDNA文库中克隆了MBL-AJ cDNA。成熟蛋白的亚基由159个氨基酸组成，含有一个CTL的糖识别结构域（CRD）。CRD含有所有已知MBL保守的Glu-Pro-Asp氨基酸序列（EPN基序）。用34-kDa凝集素二聚体作为免疫原，获得了抗MBL-AJ的单特异性多克隆抗体。MBL-AJ已被证明与早期从另一种海参（日本黄瓜）中分离的甘露聚糖结合CTL具有免疫化学同一性。但一个更有趣的发现是MBL-AJ与人血清MBL的交叉反应性由抗MBL-AJ的抗体检测。考虑到MBL-AJ的碳水化合物特异性、形成甘露糖结合位点的保守区域的存在、与人MBL的共同抗原决定簇以及参与防御反应等特性，MBL-AJ可能属于进化上保守的甘露聚糖结合蛋白家族。
To elucidate the origin and evolution of mannan-binding lectins (MBL), a new C-type lectin (CTL) specific for high-mannose glycans (MBL-AJ) was isolated from the coelomic plasma of the holothurian Apostichopus japonicus. MBL-AJ has oligomeric forms with identical 17-kDa subunits on SDS-PAGE. Among natural ligands, lectin hemagglutination activity was competitively inhibited by extracellular low-branched, but not high-branched, alpha-D-mannans isolated from marine halophilic bacteria and composed of alpha-1,2 and alpha-1,6 linked D-mannose residues. This suggests that the lectin interacts with backbone or inner side chain mannose residues, but not with terminal ones. The activity of the lectin was Ca2+-, pH-, and temperature-dependent. MBL-AJ cDNA was cloned from a holothurian coelomocyte cDNA library. The subunit of the mature protein has 159 amino acids and a single carbohydrate-recognition domain (CRD) of CTL. CRD contains a Glu-Pro-Asp amino acid sequence (EPN-motif) conserved for all known MBLs. A monospecific polyclonal antibody against MBL-AJ was obtained using the 34-kDa lectin dimer as an immunogen. The MBL-AJ has demonstrated immunochemical identity to the earlier isolated mannan-binding CTL from another holothurian, Cucumaria japonica. But a more interesting finding was cross-reactivity of MBL-AJ and human serum MBL detected by the antibody against MBL-AJ. Taking into consideration such MBL-AJ peculiarities as its carbohydrate specificity, the presence of a conserved region forming the mannose-binding site, common antigenic determinants with human MBL, and participation in defense reactions, it is possible that MBL-AJ belongs to the family of evolutionary conserved mannan-binding proteins.