Biosynthesis of the carbohydrate antigenic determinants, Globo H, blood group H, and Lewis b: a role for prostate cancer cell alpha1,2-L-fucosyltransferase.
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.
Chandrasekaran
E V
EV
Molecular and Cellular Biophysics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Chawda
Ram
R
Locke
Robert D
RD
Piskorz
Conrad F
CF
Matta
Khushi L
KL
D000017:ABO Blood-Group System / Q000276:immunology*; D002240:Carbohydrate Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D000939:Epitopes / Q000096:biosynthesis* / Q000737:chemistry; D005033:Ethylmaleimide / Q000494:pharmacology; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007983:Lewis Blood Group Antigens / Q000096:biosynthesis* / Q000737:chemistry; D008297:Male; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000737:chemistry; D011471:Prostatic Neoplasms; D014407:Tumor Cells, Cultured; D000097631:Galactoside 2-alpha-L-fucosyltransferase
D000017:ABO Blood-Group System; D002413:Cations, Divalent; D000939:Epitopes; D005033:Ethylmaleimide; D005647:Fucosyltransferases; D007983:Lewis Blood Group Antigens; D009844:Oligosaccharides; D000097631:Galactoside 2-alpha-L-fucosyltransferase