Chandrasekaran E V, Jain R K, Larsen R D, Wlasichuk K, Matta K L
Department of Gynecologic Oncology, Roswell Park Cancer Institute, Buffalo, New York 14263.
Biochemistry. 1995 Mar 7;34(9):2925-36. doi: 10.1021/bi00009a024.
The sequence in the assembly of the functional unit of selectin ligands containing sulfate, sialic acid, and fucose and also tumor-associated O-glycan structures was studied by examining the specificities of alpha 2,3-sialyltransferases (ST). The first enzyme, porcine liver ST, was 57, 37, and 79% active (Km: 0.105, 0.420, and 0.200 mM), respectively, toward 6-sulfo, 6-sialyl, or 6-O-methyl derivatives of the Gal beta 1,3GalNAc alpha- unit; C-3 or C-6 substitution on Gal abolished sialylation. An acrylamide copolymer (MW approximately 40,000) containing approximately 40 T-haptens and asialo Cowper's gland mucin (MW approximately 200,000) containing approximately 48 T-haptens was 5-fold more active as an acceptor as compared to Gal beta 1, 3GalNAc alpha-O-Al on a molecular weight basis. The second enzyme, a cloned alpha-2,3-ST specific for lactose-based structure, was 70, 102, and 108% active (Km: 0.500, 0.210, and 0.330 mM), respectively, toward 6-sialyl, 6-sulfo, or 6-O-methyl derivatives of the Gal beta 1,3GlcNAc beta- unit; C-3 and C-6 substitution on Gal abolished sialylation. Gal beta 1,4GlcNAc beta- and its 6-sulfo derivative were approximately 20% active; the Lewis a structure, Gal beta 1,3- (Fuc alpha 1,4)GlcNAc beta-, was not an acceptor. The acrylamide copolymers containing approximately 40 units of Gal beta 1,3GlcNAc beta-, Gal beta 1,3(6-sulfo)GlcNAc beta-, or fetuin triantennary asialo or bovine IgG diantennary glycopeptides were respectively 5.9-, 5.4-, 0.7-, and 0.1-fold as active. A transfer of 7-9 mol of NeuAc per mole of the above copolymers was catalyzed by this ST, the sialyl linkage being susceptible to alpha 2,3-specific sialidase. A partially purified Colo 205 Lewis type (alpha 1, 3/4) fucosyltransferase catalyzed the formation of 3'-sialyl-6-sulfo Lewis a from [9-3H]NeuAc alpha 2, 3Gal beta 1, 3(6-sulfo)GlcNAc beta-O-Allyl and copolymer containing [9-3H]NeuAc alpha 2, 3Gal beta 1, 3(6-sulfo)GlcNAc beta- units, using GDP[14C]Fuc as fucosyl donor. The third enzyme, HL-60 ST, was 103% active with Gal beta 3(6-sulfo)GalNAc alpha- but was only 8% active with 6-sialo compound; it showed 11.6-fold greater activity with the copolymer of T-hapten. Further, we observed the alpha 2,3 sialylation of Gal beta 1,4GlcNAc beta- but not Gal beta 1,3GlcNAc beta- by HL60-ST, consistent with the occurrence of 3'-sialyl LacNAc and 3'-sialyl Lewis x units in leukosialin of HL60.(ABSTRACT TRUNCATED AT 400 WORDS)
通过检测α2,3-唾液酸转移酶（ST）的特异性，研究了含硫酸酯、唾液酸和岩藻糖的选择素配体功能单元以及肿瘤相关O-聚糖结构的组装顺序。第一种酶是猪肝ST，对Galβ1,3GalNAcα-单元的6-磺酸基、6-唾液酸基或6-O-甲基衍生物的活性分别为57%、37%和79%（Km：0.105、0.420和0.200 mM）；Gal上的C-3或C-6取代会消除唾液酸化。与Galβ1,3GalNAcα-O-Al相比，含约40个T-抗原决定簇的丙烯酰胺共聚物（分子量约40,000）和含约48个T-抗原决定簇的去唾液酸考珀氏腺粘蛋白（分子量约200,000）作为受体时，按分子量计算活性高5倍。第二种酶是一种对基于乳糖结构具有特异性的克隆α-2,3-ST，对Galβ1,3GlcNAcβ-单元的6-唾液酸基、6-磺酸基或6-O-甲基衍生物的活性分别为70%、102%和108%（Km：0.500、0.210和0.330 mM）；Gal上的C-3和C-6取代会消除唾液酸化。Galβ1,4GlcNAcβ-及其6-磺酸基衍生物的活性约为20%；Lewis a结构Galβ1,3-(Fucα1,4)GlcNAcβ-不是受体。含约40个Galβ1,3GlcNAcβ-单元、Galβ1,3(6-磺酸基)GlcNAcβ-单元的丙烯酰胺共聚物，或去唾液酸胎球蛋白三分支或牛IgG双分支糖肽的活性分别为5.9倍、5.4倍、0.7倍和0.1倍。该ST催化每摩尔上述共聚物转移7 - 9摩尔NeuAc，唾液酸连接对α2,3特异性唾液酸酶敏感。一种部分纯化的Colo 205 Lewis型（α1,3/4）岩藻糖基转移酶，以GDP[14C]Fuc作为岩藻糖基供体，催化由[9-3H]NeuAcα2,3Galβ1,3(6-磺酸基)GlcNAcβ-O-烯丙基和含[9-3H]NeuAcα2,3Galβ1,3(6-磺酸基)GlcNAcβ-单元的共聚物形成3'-唾液酸基-6-磺酸基Lewis a。第三种酶是HL-60 ST，对Galβ3(6-磺酸基)GalNAcα-的活性为103%，但对6-唾液酸化合物的活性仅为8%；它对T-抗原决定簇共聚物的活性高11.6倍。此外，我们观察到HL60-ST可使Galβ1,4GlcNAcβ-发生α2,3唾液酸化，但不能使Galβ1,3GlcNAcβ-发生唾液酸化，这与HL60白细胞唾液酸蛋白中存在3'-唾液酸基乳糖胺和3'-唾液酸基Lewis x单元一致。（摘要截断于400字）
