Dam Tarun K, Gerken Thomas A, Cavada Benildo S, Nascimento Kyria S, Moura Tales R, Brewer C Fred
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
J Biol Chem. 2007 Sep 21;282(38):28256-63. doi: 10.1074/jbc.M704677200. Epub 2007 Jul 25.
Isothermal titration microcalorimetry (ITC) and hemagglutination inhibition measurements demonstrate that a chemically and enzymatically prepared form of porcine submaxillary mucin that possesses a molecular mass of approximately 10(6) daltons and approximately 2300 alpha-GalNAc residues (Tn-PSM) binds to the soybean agglutinin (SBA) with a K(d) of 0.2 nm, which is approximately 10(6)-fold enhanced affinity relative to GalNAcalpha1-O-Ser (Tn), the pancarcinoma carbohydrate antigen. The enzymatically derived 81 amino acid tandem repeat domain of Tn-PSM containing approximately 23 alpha-GalNAc residues binds with approximately 10(3)-fold enhanced affinity, while the enzymatically derived 38/40 amino acid cleavage product(s) of Tn-PSM containing approximately 11-12 alpha-GalNAc residues shows approximately 10(2)-fold enhanced affinity. A natural carbohydrate decorated form of PSM (Fd-PSM) containing 40% of the core 1 blood group type A tetrasaccharide, and 58% peptide-linked GalNAcalpha1-O-Ser/Thr residues, with 45% of the peptide-linked alpha-GalNAc residues linked alpha-(2,6) to N-glycolylneuraminic acid, shows approximately 10(4) enhanced affinity for SBA. Vatairea macrocarpa lectin (VML), which is also a GalNAc binding lectin, displays a similar pattern of binding to the four forms of PSM, although there are quantitative differences in its affinities as compared with SBA. The higher affinities of SBA and VML for Tn-PSM relative to Fd-PSM indicate the importance of carbohydrate composition and epitope density of mucins on their affinities for lectins. The higher affinities of SBA and VML for Tn-PSM relative to its two shorter chain analogs demonstrate that the length of a mucin polypeptide and hence total carbohydrate valence determines the affinities of the three Tn-PSM analogs. The results suggest a binding model in which lectin molecules "bind and jump" from alpha-GalNAc residue to alpha-GalNAc residue along the polypeptide chain of Tn-PSM before dissociating. The complete thermodynamic binding parameters for these mucins including their binding stoichiometries are presented. The results have important implications for the biological activities of mucins including those expressing the Tn cancer antigen.
等温滴定量热法（ITC）和血凝抑制测量表明，一种化学和酶法制备的猪下颌粘蛋白形式，其分子量约为10⁶道尔顿，含有约2300个α - GalNAc残基（Tn - PSM），与大豆凝集素（SBA）结合，解离常数（K(d)）为0.2纳米，相对于全癌碳水化合物抗原GalNAcalpha1 - O - Ser（Tn），其亲和力提高了约10⁶倍。Tn - PSM的酶促衍生的含约23个α - GalNAc残基的81个氨基酸串联重复结构域以约10³倍增强的亲和力结合，而Tn - PSM的酶促衍生的含约11 - 12个α - GalNAc残基的38/40个氨基酸裂解产物显示出约10²倍增强的亲和力。一种天然糖基化修饰的PSM形式（Fd - PSM），含有40%的核心1血型A四糖，58%的肽连接的GalNAcalpha1 - O - Ser/Thr残基，其中45%的肽连接的α - GalNAc残基以α - (2,6)连接到N - 羟乙酰神经氨酸，对SBA显示出约10⁴倍增强的亲和力。大果紫檀凝集素（VML）也是一种GalNAc结合凝集素，对四种PSM形式表现出类似的结合模式，尽管与SBA相比其亲和力存在定量差异。SBA和VML对Tn - PSM的亲和力高于Fd - PSM，这表明粘蛋白的碳水化合物组成和表位密度对其与凝集素的亲和力很重要。SBA和VML对Tn - PSM的亲和力高于其两种较短链类似物，这表明粘蛋白多肽的长度以及因此总的碳水化合物价决定了三种Tn - PSM类似物的亲和力。结果提示了一种结合模型，其中凝集素分子在解离前沿着Tn - PSM的多肽链从α - GalNAc残基“结合并跳跃”到α - GalNAc残基。给出了这些粘蛋白的完整热力学结合参数，包括它们的结合化学计量。这些结果对粘蛋白的生物活性具有重要意义，包括那些表达Tn癌抗原的粘蛋白。
