Mo H, Van Damme E J, Peumans W J, Goldstein I J
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
Arch Biochem Biophys. 1993 Nov 1;306(2):431-8. doi: 10.1006/abbi.1993.1534.
A new mannose-binding lectin was isolated from shallot (Allium ascalonicum) bulbs by affinity chromatography on an immobilized D-mannose column. The lectin (A. ascalonicum agglutinin, AAA) appeared homogeneous by polyacrylamide gel electrophoresis at pH 4.3 and gave a single protein band with an apparent M(r) of 11 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a single symmetrical peak of 11 kDa by gel filtration on a Sephacryl S-200 HR column, indicating that AAA exists as a monomeric protein at neutral pH under the gel filtration condition employed. However, chemical cross-linking studies revealed that some degree of self-association of the lectin molecules occurs and that the lectin exists in solution as a mixture of monomers and oligomers. Scatchard analysis of equilibrium dialysis data showed the presence of one carbohydrate binding site for Man (alpha 1-3) Man-alpha-O-Me per monomer, with Ka = 1.62 x 10(4) M-1. The carbohydrate-binding properties of the purified AAA were investigated by quantitative precipitation and hapten inhibition assays. Purified AAA precipitated asialofetuin, asialotransferrin, asialothyroglobulin, asialoorosomucoid, as well as their agalacto derivatives, but did not precipitate either sialylated glycoproteins or mucins. AAA also reacted strongly with the highly branched yeast mannan obtained from Saccharomyces cerevisiae. Of the monosaccharides tested only D-mannose was a hapten inhibitor of the AAA-asialofetuin precipitation system, whereas D-glucose, D-altrose, D-talose, N-acetyl-D-mannosamine, and derivatives of D-mannose, including 2-deoxy-, 2-deoxy-2-fluoro-, 3-deoxy-, and 6-deoxy-D-mannose were noninhibitors. These results suggest that the presence of equatorial hydroxyl groups at the C-3 and C-4 positions, an axial hydroxyl group at the C-2 position, and a free hydroxyl group at the C-6 position of the pyranose ring are the most important loci for the binding of D-mannose to AAA. Of the oligosaccharides tested, the best inhibitors were oligosaccharides containing terminal Man(alpha 1-6) [Man(alpha 1-3)]Man groups. Oligosaccharides containing either Man(alpha 1-3)Man or Man(alpha 1-6)Man units were also moderately good inhibitors of the AAA-asialofetuin precipitation system. These results indicate that AAA has an extended carbohydrate-binding site, which is most complementary to a branched mannotriosyl residue, i.e., Man(alpha 1-6)[Man(alpha 1-3)]Man.(ABSTRACT TRUNCATED AT 400 WORDS)
通过在固定化D-甘露糖柱上进行亲和层析，从青葱（葱属）鳞茎中分离出一种新的甘露糖结合凝集素。在pH 4.3条件下，该凝集素（青葱凝集素，AAA）经聚丙烯酰胺凝胶电泳显示为均一性，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中呈现一条表观分子量为11 kDa的单一蛋白条带，在Sephacryl S-200 HR柱上进行凝胶过滤时呈现一个11 kDa的单一对称峰，这表明在所用的凝胶过滤条件下，中性pH时AAA以单体蛋白形式存在。然而，化学交联研究表明凝集素分子存在一定程度的自缔合，且该凝集素在溶液中以单体和寡聚体的混合物形式存在。对平衡透析数据的Scatchard分析表明，每个单体存在一个结合Man（α1-3）Man-α-O-Me的碳水化合物结合位点，解离常数Ka = 1.62×10⁴ M⁻¹。通过定量沉淀和半抗原抑制试验研究了纯化的AAA的碳水化合物结合特性。纯化的AAA能沉淀去唾液酸胎球蛋白、去唾液酸转铁蛋白、去唾液酸甲状腺球蛋白、去唾液酸血清类黏蛋白及其无糖基衍生物，但不能沉淀唾液酸化糖蛋白或黏蛋白。AAA还与从酿酒酵母获得的高度分支的酵母甘露聚糖强烈反应。在所测试的单糖中，只有D-甘露糖是AAA-去唾液酸胎球蛋白沉淀系统的半抗原抑制剂，而D-葡萄糖、D-阿洛糖、D-塔罗糖、N-乙酰-D-甘露糖胺以及D-甘露糖的衍生物，包括2-脱氧-、2-脱氧-2-氟-、3-脱氧-和6-脱氧-D-甘露糖均为非抑制剂。这些结果表明，吡喃糖环C-3和C-4位的赤道羟基、C-2位的轴向羟基以及C-6位的游离羟基是D-甘露糖与AAA结合的最重要位点。在所测试的寡糖中，最佳抑制剂是含有末端Man（α1-6）[Man（α1-叁）]Man基团寡糖。含有Man（α1-3）Man或Man（α1-6）Man单元的寡糖也是AAA-去唾液酸胎球蛋白沉淀系统的中度良好抑制剂。这些结果表明，AAA具有一个延伸的碳水化合物结合位点，该位点与分支的甘露三糖残基，即Man（α1-6）[Man（α1-3）]Man最为互补。（摘要截短于400字）
