Schuman J, Campbell A P, Koganty R R, Longenecker B M
Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle 98195, USA.
J Pept Res. 2003 Mar;61(3):91-108. doi: 10.1034/j.1399-3011.2003.00031.x.
MUC1 mucin is a large transmembrane glycoprotein, the extracellular domain of which is formed by a repeating 20 amino acid sequence, GVTSAPDTRPAPGSTAPPAH. In normal breast epithelial cells, the extracellular domain is densely covered with highly branched complex carbohydrate structures. However, in neoplastic breast tissue, the extracellular domain is under-glycosylated, resulting in the exposure of a highly immunogenic core peptide epitope (PDTRP in bold above), as well as in the exposure of normally cryptic core Tn (GalNAc), STn (sialyl alpha2-6 GalNAc) and TF (Gal beta1-3 GalNAc) carbohydrates. Here, we report the results of 1H NMR structural studies, natural abundance 13C NMR relaxation measurements and distance-restrained MD simulations designed to probe the structural and dynamical effects of Tn-glycosylation within the PDTRP core peptide epitope. Two synthetic peptides were studied: a nine-residue MUC1 peptide of the sequence, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6-Arg7-Pro8-Ala9, and a Tn-glycosylated version of this peptide, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6(alphaGalNAc)-Arg7-Pro8-Ala9. The results of these studies show that a type I beta-turn conformation is adopted by residues PDTR within the PDTRP region of the unglycosylated MUC1 sequence. The existence of a similar beta-turn within the PDTRP core peptide epitope of the under-glycosylated cancer-associated MUC1 mucin protein might explain the immunodominance of this region in vivo, as the presence of defined secondary structure within peptide epitope regions has been correlated with increased immunogenicity in other systems. Our results have also shown that Tn glycosylation at the central threonine within the PDTRP core epitope region shifts the conformational equilibrium away from the type I beta-turn conformation and toward a more rigid and extended state. The significance of these results are discussed in relation to the possible roles that peptide epitope secondary structure and glycosylation state may play in MUC1 tumor immunogenicity.
黏蛋白1（MUC1）是一种大型跨膜糖蛋白，其细胞外结构域由重复的20个氨基酸序列GVTSAPDTRPAPGSTAPPAH构成。在正常乳腺上皮细胞中，细胞外结构域被高度分支的复杂碳水化合物结构密集覆盖。然而，在乳腺肿瘤组织中，细胞外结构域糖基化不足，导致高免疫原性核心肽表位（上述加粗的PDTRP）暴露，同时也使正常情况下隐蔽的核心Tn（N-乙酰半乳糖胺）、STn（唾液酸α2-6 N-乙酰半乳糖胺）和TF（半乳糖β1-3 N-乙酰半乳糖胺）碳水化合物暴露。在此，我们报告了旨在探究PDTRP核心肽表位内Tn糖基化的结构和动力学效应的1H NMR结构研究、天然丰度13C NMR弛豫测量及距离约束MD模拟的结果。研究了两种合成肽：一种是序列为Thr1-Ser2-Ala3-Pro4-Asp5-Thr6-Arg7-Pro8-Ala9的九残基MUC1肽，以及该肽的Tn糖基化形式Thr1-Ser2-Ala3-Pro4-Asp5-Thr6(αGalNAc)-Arg7-Pro8-Ala9。这些研究结果表明，未糖基化MUC1序列的PDTRP区域内的PDTR残基采用I型β-转角构象。糖基化不足的癌症相关MUC1黏蛋白蛋白的PDTRP核心肽表位内存在类似的β-转角，这可能解释了该区域在体内的免疫优势，因为肽表位区域内确定的二级结构的存在与其他系统中免疫原性的增加相关。我们的结果还表明，PDTRP核心表位区域内中心苏氨酸处的Tn糖基化使构象平衡从I型β-转角构象转变为更刚性和伸展的状态。结合肽表位二级结构和糖基化状态可能在MUC1肿瘤免疫原性中发挥的作用，讨论了这些结果的意义。
