Holmskov U, Teisner B, Willis A C, Reid K B, Jensenius J C
Department of Medical Microbiology, University of Odense, Denmark.
J Biol Chem. 1993 May 15;268(14):10120-5.
A previously undescribed bovine serum lectin (designated CL-43) was identified by its Ca(2+)-dependent binding to mannan and by its molecular mass of 43 kDa under reducing conditions on SDS-PAGE. The lectin was isolated by polyethylene glycol precipitation, affinity chromatography on mannan-Sepharose (followed by elution with EDTA), and absorption on Sepharose-4B-coupled rabbit anti-bovine Ig (to remove anti-mannan antibodies). Fractions containing the lectin were reapplied to mannan-Sepharose. Bound conglutinin was eluted with GlcNAc, and then the 43-kDa lectin, together with mannan-binding protein (MBP), was eluted with mannose. The 43-kDa lectin was separated from MBP by ion exchange chromatography on Mono-Q. On SDS-PAGE under nonreducing conditions the lectin showed a molecular mass of 120 kDa. On gel chromatography under nondissociating conditions the protein was eluted at a volume corresponding to a molecular mass of approximately 750 kDa. Amino acid analysis showed the presence of hydroxyproline and hydroxylysine and a high content of glycine (24.3%) indicating the presence of a collagen-like structure. This was supported by the susceptibility of the protein to collagenase digestion. The designation CL-43 was chosen since this molecule appears to belong to the collectins, i.e. proteins with collagen structure and lectin activity. The N-terminal sequence (27 amino acids) showed 56% identity with bovine SP-D and 44% identity to bovine conglutinin. An inhibition assay with biotinylated CL-43, using solid-phase mannan as ligand, revealed the following carbohydrate inhibition pattern: mannose and ManNAc > fucose > GlcNAc > glucose and maltose > galactose > lactose >> GalNAc. We conclude that CL-43 is a circulating lectin, with structural similarities to bovine conglutinin and SP-D, and a ligand binding profile resembling that of MBP.
一种先前未被描述的牛血清凝集素（命名为CL - 43）通过其对甘露聚糖的Ca(2+)依赖性结合以及在SDS - PAGE还原条件下43 kDa的分子量被鉴定出来。该凝集素通过聚乙二醇沉淀、甘露聚糖 - 琼脂糖亲和层析（随后用EDTA洗脱）以及琼脂糖 - 4B偶联的兔抗牛Ig吸附（以去除抗甘露聚糖抗体）进行分离。含有凝集素的组分重新应用于甘露聚糖 - 琼脂糖。结合的胶固素用N - 乙酰葡糖胺洗脱，然后43 kDa的凝集素与甘露糖结合蛋白（MBP）一起用甘露糖洗脱。通过Mono - Q离子交换层析将43 kDa的凝集素与MBP分离。在非还原条件下的SDS - PAGE上，该凝集素显示分子量为120 kDa。在非解离条件下的凝胶层析中，该蛋白在对应于约750 kDa分子量的体积处被洗脱。氨基酸分析表明存在羟脯氨酸和羟赖氨酸以及高含量的甘氨酸（24.3%），表明存在类似胶原蛋白的结构。这通过该蛋白对胶原酶消化的敏感性得到支持。选择CL - 43这个名称是因为该分子似乎属于凝集素家族，即具有胶原蛋白结构和凝集素活性的蛋白质。N端序列（27个氨基酸）与牛SP - D有56%的同一性，与牛胶固素有44%的同一性。使用固相甘露聚糖作为配体的生物素化CL - 43抑制试验揭示了以下碳水化合物抑制模式：甘露糖和N - 乙酰甘露糖胺＞岩藻糖＞N - 乙酰葡糖胺＞葡萄糖和麦芽糖＞半乳糖＞乳糖＞＞N - 乙酰半乳糖胺。我们得出结论，CL - 43是一种循环凝集素，与牛胶固素和SP - D在结构上有相似性，并且配体结合谱类似于MBP。
