Kubota Tomomi, Shiba Tomoo, Sugioka Shigemi, Furukawa Sanae, Sawaki Hiromichi, Kato Ryuich, Wakatsuki Soichi, Narimatsu Hisashi
Glycogene Function Team of Research Center for Glycoscience (RCG), National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
J Mol Biol. 2006 Jun 9;359(3):708-27. doi: 10.1016/j.jmb.2006.03.061. Epub 2006 Apr 19.
Mucin-type O-glycans are important carbohydrate chains involved in differentiation and malignant transformation. Biosynthesis of the O-glycan is initiated by the transfer of N-acetylgalactosamine (GalNAc) which is catalyzed by UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (pp-GalNAc-Ts). Here we present crystal structures of the pp-GalNAc-T10 isozyme, which has specificity for glycosylated peptides, in complex with the hydrolyzed donor substrate UDP-GalNAc and in complex with GalNAc-serine. A structural comparison with uncomplexed pp-GalNAc-T1 suggests that substantial conformational changes occur in two loops near the catalytic center upon donor substrate binding, and that a distinct interdomain arrangement between the catalytic and lectin domains forms a narrow cleft for acceptor substrates. The distance between the catalytic center and the carbohydrate-binding site on the lectin beta sub-domain influences the position of GalNAc glycosylation on GalNAc-glycosylated peptide substrates. A chimeric enzyme in which the two domains of pp-GalNAc-T10 are connected by a linker from pp-GalNAc-T1 acquires activity toward non-glycosylated acceptors, identifying a potential mechanism for generating the various acceptor specificities in different isozymes to produce a wide range of O-glycans.
粘蛋白型O-聚糖是参与分化和恶性转化的重要碳水化合物链。O-聚糖的生物合成由N-乙酰半乳糖胺（GalNAc）的转移启动，该转移由UDP-GalNAc：多肽α-N-乙酰半乳糖胺基转移酶（pp-GalNAc-Ts）催化。在此，我们展示了对糖基化肽具有特异性的pp-GalNAc-T10同工酶与水解的供体底物UDP-GalNAc以及与GalNAc-丝氨酸形成复合物的晶体结构。与未复合的pp-GalNAc-T1的结构比较表明，供体底物结合后，催化中心附近的两个环发生了显著的构象变化，并且催化结构域和凝集素结构域之间独特的结构域间排列形成了一个用于受体底物的狭窄裂隙。催化中心与凝集素β亚结构域上的碳水化合物结合位点之间的距离影响GalNAc在GalNAc糖基化肽底物上的糖基化位置。一种嵌合酶，其中pp-GalNAc-T10的两个结构域通过来自pp-GalNAc-T1的接头连接，获得了对非糖基化受体的活性，这确定了在不同同工酶中产生各种受体特异性以产生广泛O-聚糖的潜在机制。
