Human lung adenocarcinoma alpha1,3/4-L-fucosyltransferase displays two molecular forms, high substrate affinity for clustered sialyl LacNAc type 1 units as well as mucin core 2 sialyl LacNAc type 2 unit and novel alpha1,2-L-fucosylating activity.
Human lung tumor alpha1,3/4-L-fucosyltransferase (FT) was purified (2000-fold, 29% recovery) from 290 g of tissue by including a chromatography step on Affinity Gel-GDP. Two molecular forms (FTA, larger size carrying 15% alpha1,4-FT activity; FTB, the major form with 85% activity) were separated by further fractionation on a Sephacryl S-100 HR column. A difference in the electrophoretic mobilities of these two activities was also found on native polyacrylamide gel electrophoresis (PAGE). Both forms were devoid of typical alpha1,2-fucosylating activity but were associated with the novel alpha1,2-fucosylating ability of converting the Lewis a determinant to Lewis b. Based on percentage activity toward 2-O-MeGalbeta1,3GlcNAcbeta-O-Bn, both forms exhibited the same extent of activity toward various acceptors, which included sulfated, sialylated, or methylated LacNAc type 1 or type 2 as well as mucin core 2 acceptors. However, FTA and FTB exhibited a difference in their ability to act on mucin core 2 3'-sialyl LacNAc (activities 24.2% and 40.8%, respectively, as compared to 2-O-MeGalbeta1,3GlcNAcbeta-O-Bn). The unsubstituted LacNAc type 1 acceptors were 15-20 times as active as the corresponding LacNAc type 2 acceptors. The 3-O-substitution on the beta1,4-linked Gal (methyl, sulfate, or sialyl) in mucin core 2 acceptors increased the efficiency of these acceptors five- to eightfold. The most efficient acceptor for FTA and FTB was 3-O-sulfoGalbeta1,3GlcNAcbeta-O-Al (K(m) 100 and 47 microM, respectively). The K(m) (mM) values for 2-O-methyl Galbeta1,3GlcNAcbeta-O-Bn and 3-O-sialyl Galbeta1,3GlcNAcbeta-O-Bn were 0.40 and 2.5 (FTA) and 0.16 and 0.67 (FTB), respectively. The 35-kDa glycoprotein ancrod (from Malayan pit viper venom) containing 36% complex N-glycans with the antennae NeuAcalpha2,3Galbeta1,3GlcNAcbeta- acted as the best macromolecular acceptor substrate (K(m): 45 microM), as examined with FTB. On desialylation the acceptor efficiency dropped to approximately 50% (K(m) for asialo ancrod: 167 microM). Sialylglycoproteins, such as carcinoembryonic antigen, fetuin, and bovine alpha(1)-acid glycoprotein, were better acceptors than asialo fetuin. On the contrary, fetuin triantennary glycopeptide containing predominantly NeuAcalpha2,3Galbeta1,4GlcNAcbeta- was only 55% active as compared to the asialo glycopeptide (K(m): 1.43 and 0.63 mM, respectively). Thus, the human lung tumor alpha1,3/4-L-FT has the potential to generate clustered sialyl Lewis a and Lewis b determinants in N-glycans and sialyl Lewis x determinant in mucin core 2 structures.
Chandrasekaran
E V
EV
Molecular and Cellular Biophysics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.
Chawda
R
R
Rhodes
J M
JM
Xia
J
J
Piskorz
C
C
Matta
K L
KL
D002288:Adenocarcinoma, Mucinous / Q000201:enzymology*; D000951:Antigens, Neoplasm / Q000737:chemistry; D002240:Carbohydrate Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005647:Fucosyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006020:Glycopeptides; D006023:Glycoproteins; D006801:Humans; D066298:In Vitro Techniques; D008175:Lung Neoplasms / Q000201:enzymology*; D008875:Middle Aged; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry; D000080506:Sialyl Lewis X Antigen; D013379:Substrate Specificity
D000951:Antigens, Neoplasm; C042317:DU-PAN-2 antigen, human; D006020:Glycopeptides; D006023:Glycoproteins; D009077:Mucins; D009844:Oligosaccharides; D000080506:Sialyl Lewis X Antigen; D005647:Fucosyltransferases; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase