Chandrasekaran E V, Chawda Ram, Locke Robert D, Piskorz Conrad F, Matta Khushi L
Molecular and Cellular Biophysics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Glycobiology. 2002 Mar;12(3):153-62. doi: 10.1093/glycob/12.3.153.
Prostate carcinoma LNCaP cells were unique among several human cancer cell lines which include two other prostate cancer cell lines, PC-3 and DU-145, in expressing alpha1,2-L-fucosyltransferase (FT) as an exclusive FT activity. Affinity gel-GDP and Sephacryl S100 HR columns were used for a partial purification of this enzyme from 3.9 x 10(9) LNCaP cells (approximately 200-fold; 40% yield). The K(m) value (2.7 mM) for the LacNAc type 2 acceptor was quite similar to the one reported for the cloned blood group H gene-specified alpha1,2-FT [Chandrasekaran et al. (1996) Biochemistry 35, 8914-8924]. N-Ethylmaleimide was a potent inhibitor (K(i ) 12.5 microM). The enzyme showed four-fold acceptor preference for the LacNAc type 2 unit in comparison to the T-hapten in mucin core 2 structure. Its main features were similar to those of the cloned enzyme: (1) C-6 sulfation of terminal Gal in the LacNAc unit increased the acceptor efficiency, whereas C-6 sialylation abolished acceptor ability; (2) C-6 sulfation of GlcNAc in LacNAc type 2 decreased by 80% the acceptor ability, whereas LacNAc type 1 was unaffected; (3) Lewis x did not serve as an acceptor; (4) the C-4 hydroxyl rather than the C-6 hydroxyl group of the GlcNAc moiety in LacNAc type1 was essential for activity; and (5) the acrylamide copolymer of Galbeta1,3GlcNAcbeta-O-Al was the best acceptor among the acrylamide copolymers. Additionally, highly significant biological features of alpha1,2FT were identified in the present study. The synthesis of Globo H and Lewis b determinants became evident from the fact that Galbeta1,3GalNAcbeta1,3Galalpha-O-Me and Galbeta1,3(Fucalpha1,4)Glc-NAcbeta1,3Galbeta-O-Me served as high-affinity acceptors for this enzyme. Further, D-Fucbeta1,3Gal-NAcbeta1,3Galalpha-O-Me was a very efficient acceptor, indicating that the C-6 hydroxyl group of the terminal Gal moiety in Globo H is not essential for the enzyme activity. Thus, the present study was able to demonstrate three different catalytic roles of LNCaP alpha1,2-FT, namely, the expressions of blood group H, Lewis b from Lewis a, and Globo H.
前列腺癌LNCaP细胞在几种人类癌细胞系中独具特色，这几种细胞系包括另外两种前列腺癌细胞系PC-3和DU-145，LNCaP细胞表达α1,2-L-岩藻糖基转移酶（FT），且仅有这种FT活性。亲和凝胶-GDP柱和Sephacryl S100 HR柱用于从3.9×10⁹个LNCaP细胞中对该酶进行部分纯化（约200倍；产率40%）。对于乳糖-N-新四糖2型受体，其米氏常数（K(m)）值（2.7 mM）与报道的克隆的血型H基因指定的α1,2-FT的K(m)值非常相似[钱德拉塞卡兰等人（1996年）《生物化学》35卷，8914 - 8924页]。N-乙基马来酰亚胺是一种强效抑制剂（抑制常数K(i)为12.5 μM）。与粘蛋白核心2结构中的T抗原决定簇相比，该酶对乳糖-N-新四糖2型单元的受体偏好性高四倍。其主要特征与克隆酶的特征相似：（1）乳糖-N-新四糖单元中末端半乳糖的C-6硫酸化增加了受体效率，而C-6唾液酸化则消除了受体能力；（2）乳糖-N-新四糖2型中N-乙酰葡糖胺的C-6硫酸化使受体能力降低80%，而乳糖-N-新四糖1型不受影响；（3）Lewis x不作为受体；（4）乳糖-N-新四糖1型中N-乙酰葡糖胺部分的C-4羟基而非C-6羟基对活性至关重要；（5）在丙烯酰胺共聚物中，Galβ1,3GlcNAcβ-O-Al的丙烯酰胺共聚物是最佳受体。此外，本研究还鉴定出了α1,2FT的高度显著的生物学特性。从Galβ1,3GalNAcβ1,3Galα-O-Me和Galβ1,3（Fucα1,4）GlcNAcβ- O-Me作为该酶的高亲和力受体这一事实可以明显看出，血型Glob H和Lewis b决定簇的合成。此外，D-Fucβ1,3GalNAcβ1,3Galα-O-Me是一种非常有效的受体，这表明血型Glob H中末端半乳糖部分的C-6羟基对于酶活性并非必不可少。因此，本研究能够证明LNCaP α1,2-FT的三种不同催化作用，即血型H、由Lewis a转变为Lewis b以及血型Glob H的表达。
