Abstract
The human H(O) blood group is specified by the structure Fucalpha1-2Galbeta1-R, but the factors regulating expression of this determinant on cell surface glycoconjugates are not well understood. To learn more about the regulation of H blood group expression, cDNA encoding the human H-type GDPFuc:beta-D-galactoside alpha1, 2-fucosyltransferase (alpha1,2FT) was stably transfected into Chinese hamster ovary (CHO) cells. The new cell line, designated CHO(alpha1,2)FT, expressed surface neoglycans containing the H antigen. The structures of the fucosylated neoglycans in CHO(alpha1, 2)FT cells and the distribution of these glycans on glycoproteins were characterized. Seventeen percent of the [3H]Gal-labeled glycopeptides from CHO(alpha1,2)FT cells bound to the immobilized H blood group-specific lectin Ulex europaeus agglutinin-I (UEA-I), whereas none from parental CHO cells bound to the lectin. The glycopeptides from CHO(alpha1,2)FT cells binding to UEA-I contained polylactosamine [3Galbeta1-4GlcNAcbeta1-]n with the terminal sequence Fucalpha1-2Galbeta1- 4GlcNAc-R. Fucosylation of the polylactosamine sequences on complex-type N-glycans in CHO(alpha1, 2)FT cells caused a decrease in both sialylation and length of polylactosamine. Unexpectedly, only small amounts of terminal fucosylation was found in diantennary complex-type N-glycans. The O-glycans and glycolipids were not fucosylated by the H-type alpha1, 2FT. Two major high molecular weight glycoproteins, one of which was shown to be the lysosome-associated membrane glycoprotein LAMP-1, preferentially contained the H-type structure and were bound by immobilized UEA-I. These results demonstrate that in CHO cells the expressed H-type alpha1,2FT does not indiscriminately fucosylate terminal galactosyl residues in complex-type N-glycans, but it favors glycans containing polylactosamine and dramatically alters their length and sialylation.
Highlights
The human H(O) blood group is specified by the structure Fuc␣1–2Gal1-R, but the factors regulating expression of this determinant on cell surface glycoconjugates are not well understood
The oligosaccharide precursors synthesized by Chinese hamster ovary (CHO) cells have been extensively characterized previously (14 –17), and represent a prototypical cell line with which to explore the in vivo acceptor substrate utilization properties of the human H blood group ␣1,2FT
We have previously shown that in CHO cells polylactosamine sequences are enriched on two major glycoproteins, one identified as lysosome-associated membrane glycoprotein 1 (LAMP-1), and the other, which has the properties of LAMP-2 [33]
Summary
Materials—Galactose, lactose, fucose, raffinose, ␣-methylmannoside, ␣-methylglucoside, Triton X-100, Nonidet P-40, dimethyl sulfoxide, iodomethane, 2-acetamido-2-deoxy-D-glucose, anhydrous sodium acetate, phenyl--D-galactoside, ␣-methylgalactoside, and 1-methylimidazole, Sephadex G-25, Arthrobacter urefaciens neuraminidase, pepstatin, leupeptin, trypsin inhibitor, phenylmethylsulfonyl fluoride, Tetragonolobus purpureas agglutinin, Ulex europaeus agglutinin I (UEA-I), Anguilla anguilla agglutinin, Griffonia simplicifolia I-B4 isolectin, and Euonymus europaeus agglutinin were obtained from Sigma. Assay for ␣1,2FT—Extracts were prepared from CHO cell lines by homogenizing washed cell pellets in 1% Triton X-100, as described previously [24]. Labeled glycopeptides and glycolipids from the same cell pellets were separately obtained essentially according to the method of Finne and Krusius [29], modified as described previously [15]. The pelleted residues obtained after extraction of radiolabeled glycolipids were washed with ethanol to remove excess organic solvents and resuspended in 0.1 M Tris, 1 mM CaCl2, pH 8.0, containing Pronase (10 mg/ml). After the chromatograms were dried, 1-cm segments were transferred to 12 ϫ 75-mm test tubes containing 1 ml of 0.1 M pyridine acetate buffer, pH 5.4, to elute radiolabeled oligosaccharides, and aliquots were assayed for radioactivity by liquid scintillation counting. The retention times for each of the partially O-methylated, alditol acetates were consistent with previously reported values [36]
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