THERMODYNAMICS OF LECTIN-CARBOHYDRATE INTERACTIONS - TITRATION MICROCALORIMETRY MEASUREMENTS OF THE BINDING OF N-LINKED CARBOHYDRATES AND OVALBUMIN TO CONCANAVALIN-A
THERMODYNAMICS OF LECTIN-CARBOHYDRATE INTERACTIONS - TITRATION MICROCALORIMETRY MEASUREMENTS OF THE BINDING OF N-LINKED CARBOHYDRATES AND OVALBUMIN TO CONCANAVALIN-A
复制标题
DOI：
10.1021/bi00171a014
复制
发表时间：
1994-02-08
期刊：
影响因子：
2.9
通讯作者：
BREWER, CF
中科院分区：
文献类型：
--
作者：
MANDAL, DK;KISHORE, N;BREWER, CF
本文用微量滴定量热法测定了伴刀豆球蛋白A（Con;A）与一系列直链和支链寡糖（包括某些N-连接复合型和寡甘露糖型糖类）以及一部分含有Man 7和Man 8寡甘露糖链的鹌鹑卵清蛋白的结合热力学。甲基3，6-二-O-（α-D-吡喃甘露糖基）-α-D-吡喃甘露糖苷是在所有N-连接碳水化合物中发现的一种分支链三糖部分，其亲和力比甲基α-D-吡喃甘露糖苷高约60倍，与单糖的结合焓变化（Δ H）为-8.2 kcal mol（-1）相比，其结合焓变化（Δ H）为-14.4 kcal mol（-1）。这表明Con A具有三甘露糖苷的延伸结合位点。然而，具有末端β（1，2）-GlcNAc残基的双触角复合型碳水化合物的Δ H仅为-10.6 kcal mol（-1），其结合亲和力比三甘露糖苷高3倍。本研究中碳水化合物的-Δ H与-T Δ S的曲线图显示，复合型碳水化合物以及作为寡甘露糖型碳水化合物结构的一部分的α（1，2）-二甘露糖基和三甘露糖基寡糖的-T Δ S存在正偏差。这些化合物的结合熵的相对有利的变化归因于每个分子中存在多个内部和末端残基，其可以独立地结合到凝集素的单糖结合位点。复合型碳水化合物和α（1，2）甘露寡糖的Δ H值也比甲基α-D-吡喃甘露糖苷的Δ H值大约-2.5 kcal mol（-1），表明存在一些扩展的结合位点相互作用。N-连接的寡聚甘露糖型碳水化合物的二聚体Con A及其琥珀酰基和乙酰基衍生物的结合的热力学进行了测定，因为这些碳水化合物是二价的，沉淀与天然的四聚体凝集素，但不与二聚体蛋白质及其两个衍生物。用Man 5寡甘露糖型寡糖滴定琥珀酰-Con A得到的Δ H为-14.5 kcal mol（-1），与分支链三甘露糖苷相似。这表明含有三甘露糖基部分的Man 5的α（1，6）核心臂是Con A的主要结合表位。在单个糖基化位点含有Man 7和Man 8链混合物的鹌鹑卵清蛋白部分显示与琥珀酰-Con A的单价结合和-13.6 kcal mol（-1）的Δ H。这些结果表明碳水化合物链的α（1，6）臂上的三甘露糖苷部分是主要结合表位，并且其与凝集素的相互作用相对不受卵清蛋白的蛋白质基质的影响。
The thermodynamics of binding of concanavalin A (Con;A) with a series of linear and branched chain oligosaccharides including certain N-linked complex type and oligomannose type carbohydrates and a fraction of quail ovalbumin containing Man7 and Man8 oligomannose chains have been determined using titration microcalorimetry. Methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, a branch chain trisaccharide moiety found in all N-linked carbohydrates which possesses approximately 60-fold higher affinity than methyl alpha-D-mannopyranoside, exhibited a change in enthalpy of binding (Delta H) of -14.4 kcal mol(-1) as compared to -8.2 kcal mol(-1) for the monosaccharide. This demonstrates that Con A possesses an extended binding site for the trimannoside. However, a biantennary complex type carbohydrate with terminal beta(1,2)-GlcNAc residues which binds with 3-fold higher affinity than the trimannoside possesses a Delta H of only -10.6 kcal mol(-1). A plot of -Delta H versus -T Delta S for the carbohydrates in the present study showed positive deviations in -T Delta S for the complex type carbohydrate, as well as alpha(1,2)-di- and trimannosyl oligosaccharides which are part of the structures of oligomannose type carbohydrates. The relative favorable changes in binding entropies of these compounds are attributed to the presence of multiple internal and terminal residues in each molecule which can independently bind to the monosaccharide binding site of the lectin. The Delta H values for the complex type carbohydrate and the alpha(1,2) mannose oligosaccharides were also approximately -2.5 kcal mol(-1) greater than that of methyl alpha-D-mannopyranoside, indicating some extended binding site interactions. The thermodynamics of binding of N-linked oligomannose type carbohydrates to dimeric Con A and its succinyl and acetyl derivatives were determined since these carbohydrates are bivalent and precipitate with the native tetrameric lectin but not with the dimeric protein and its two derivatives. Titration of succinyl-Con A with a Man5 oligomannose type oligosaccharide gave a Delta H of -14.5 kcal mol(-1), which is similar to that of the branch chain trimannoside. This indicates that the alpha(1,6) core arm of Man5 which contains the trimannosyl moiety is the primary binding epitope for Con A. A fraction of quail ovalbumin containing a mixture of Man7 and Man8 chains at a single glycosylation site showed univalent binding to succinyl-Con A and a Delta H of -13.6 kcal mol(-1). These results indicate that the trimannoside moiety on the alpha(1,6) arm(s) of the carbohydrate chains is the primary binding epitope and that its interactions with the lectin are relatively unaffected by the protein matrix of ovalbumin.