UDPgalactose : N-acetyl-D-glucosamine 4-P-~-galactosyltransferase (EC 2.4.1.38) (GalT) is a Golgi-membranebound enzyme that participates in the biosynthesis of the oligosaccharide structures of glycoproteins and gl ycolipids.Synthetic DNA oligomers representing segments of the published partial cDNA sequence for bovine GalT were used as molecular probes to isolate from bovine-liver cDNA libraries overlapping cDNA clones that span 1728 nucleotides and potentially code for the entire polypeptide chain of bovine galactosyltransferase. The cDNA sequence for bovine GalT reveals a 1206-base-pair open reading frame that codes for 402 amino acids, including a presumptive N-terminal membrane anchoring domain of 20 hydrophobic amino acids. The colinearity between the cDNA sequence and 29 non-overlapping amino acid residues which were positively identified by N-terminal sequencing of two polypeptides isolated form the soluble form of the enzyme was consistent with the translation frame and confirmed the authenticity of the cDNA clones.The finding of an N-terminal hydrophobic segment which serves as the membrane anchor and signal sequence suggests that the C-terminal region of the GalT polypeptide is oriented within the lumen of the Golgi membranes. This conclusion is in agreement with previous biochemical studies which indicated that the 51-kDa and 42-kDa soluble forms of the enzyme which encompass the C-terminal324 and 297 amino acid residues of the entire GalT polypeptide, respectively, include the catalytic site.Current evidence supports the model that cell-surface carbohydrates may act as carriers of specific information in cell interaction. Stage-specific changes of cell-surface carbohydrates have been observed during embryogenesis and differentiation [l, 21. Qualitative and quantitative alterations in cell-surface carbohydrates have been associated with both neoplastic transformation and metastatic progression [I, 31. The carbohydrate moieties of glycoproteins and glycolipids are synthesized through a series of elongation reactions which are catalyzed by specific glycosyltransferases [4]. The coordinate interplay of these enzymes determines the classes of oligosaccharide structures displayed at the cell surface, and ultimately the ability of a cell to interact with its fluid and cellular environment. The molecular cloning of the glycosyltransferase genes will provide a powerful tool for elucidating the genetic control of cell-surface carbohydrate biosynthesis and its role in development and neoplastic transformation.UDPgalactose : N-acetyl-D-glucosamine 4-j?-~-galactosyltransferase (GalT) is a glycosyltransferase that participates in the biosynthesis of the oligosaccharide structures of glycoproteins and glycolipids [ 5 ] . The enzyme catalyzes the specific Correspondence to

UDP-N-acetyl-D-glucosamine:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II (EC 2.4.1.143) (GnT II) is a Golgi resident enzyme that catalyzes an essential step in the biosynthetic pathway leading from high mannose to complex N-linked oligosaccharides. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the enzyme purified from rat liver revealed a polypeptide of 42 kDa. Amino acid sequences were obtained from the N terminus and a tryptic peptide. Overlapping cDNA clones coding for the full-length rat GnT II were obtained. The complete nucleotide sequence revealed a 1326-base pair open reading frame that codes for a polypeptide of 442 amino acids, including a presumptive N-terminal membrane-anchoring domain. The region of cDNA coding for the C-terminal 389 amino acids of rat GnT II was linked in frame to a cDNA segment encoding the cleavable signal sequence of the human interleukin-2 receptor and transiently expressed in COS-7 cells. A 77-fold enhancement of GnT II activity over a control carrying the GnT II cDNA out-of-frame was detected in the culture medium at 72 h after transfection. 1H-NMR spectroscopy confirmed that the oligosaccharide synthesized in vitro by the recombinant enzyme was the product of GnT II activity. These data verify the identity of the cloned GnT II cDNA and demonstrate that the C-terminal region of the protein includes the catalytic domain.

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