Bulgakov Aleksandr A, Eliseikina Marina G, Petrova Irina Yu, Nazarenko Evgeny L, Kovalchuk Svetlana N, Kozhemyako Valery B, Rasskazov Valery A
Pacific Institute of Bioorganic Chemistry Far Eastern Branch, Russian Academy of Science, Vladivostok, Russia.
Glycobiology. 2007 Dec;17(12):1284-98. doi: 10.1093/glycob/cwm093. Epub 2007 Sep 21.
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 Ca(2+)-, 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.
为阐明甘露聚糖结合凝集素（MBL）的起源与进化，从刺参体腔液中分离出一种对高甘露糖聚糖具有特异性的新型C型凝集素（CTL，MBL-AJ）。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上，MBL-AJ具有由相同17 kDa亚基组成的寡聚体形式。在天然配体中，从海洋嗜盐细菌中分离出的、由α-1,2和α-1,6连接的D-甘露糖残基组成的细胞外低分支而非高分支α-D-甘露聚糖可竞争性抑制凝集素的血凝活性。这表明该凝集素与主链或内侧链甘露糖残基相互作用，而非与末端残基相互作用。该凝集素的活性依赖于Ca(2+)、pH和温度。从刺参体腔细胞cDNA文库中克隆出MBL-AJ cDNA。成熟蛋白的亚基有159个氨基酸和一个CTL的单碳水化合物识别结构域（CRD）。CRD包含一个对所有已知MBL均保守的Glu-Pro-Asp氨基酸序列（EPN基序）。以34 kDa凝集素二聚体作为免疫原获得了一种针对MBL-AJ的单特异性多克隆抗体。MBL-AJ已证明与先前从另一种刺参日本瓜参中分离出的甘露聚糖结合CTL具有免疫化学同一性。但一个更有趣的发现是，针对MBL-AJ的抗体检测到MBL-AJ与人血清MBL具有交叉反应性。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有可能属于进化保守的甘露聚糖结合蛋白家族。
