The ␣2,6-sialyltransferase (ST) is a Golgi glycosyltransferase that adds sialic acid residues to glycoprotein N-linked oligosaccharides. Here we show that two forms of ␣2,6-sialyltransferase are expressed by the liver and are encoded by two different RNAs that differ by a single nucleotide. The ST tyr possesses a Tyr at amino acid 123, whereas the ST cys possesses a Cys at this position. The ST tyr is more catalytically active than the ST cys; however, both are functional when introduced into tissue culture cells. The proteolytic processing and turnover of the ST tyr and ST cys proteins differ dramatically. The ST cys is retained intact in COS-1 cells, whereas the ST tyr is rapidly cleaved and secreted. Analysis of the N-linked oligosaccharides of these proteins demonstrates that both proteins enter the late Golgi. However, differences in ST tyr and ST cys proteolytic processing may be related to differences in their localization, because ST tyr but not ST cys is expressed at low levels on the cell surface. The possibility that the ST tyr is cleaved in a post-Golgi compartment is supported by the observation that a 20°C temperature block, which stops protein transport in the trans Golgi network, blocks both cleavage and secretion of the ST tyr.

ST6Gal-I (␣2,6-sialyltransferase) is expressed as two isoforms, ST Tyr and ST Cys , which exhibit differences in catalytic activity, trafficking through the secretory pathway, and proteolytic processing and secretion. We have found that the ST6Gal-I isoforms are phosphorylated on luminal Ser and Thr residues. Immunoprecipitation of 35 S-and 32 P-labeled proteins expressed in COS-1 cells suggests that the ST Tyr isoform is phosphorylated to a greater extent than the ST Cys isoform. Analysis of domain deletion mutants revealed that ST Tyr is phosphorylated on stem and catalytic domain amino acids, whereas ST Cys is phosphorylated on catalytic domain amino acids. An endoplasmic reticulum retained/ retrieved chimeric Iip33-ST protein demonstrates drastically lower phosphorylation than does the wild type ST Tyr isoform. This suggests that the bulk of the ST6Gal-I phosphorylation is occurring in the Golgi. Treatment of cells with the ionophore monensin does not significantly block phosphorylation of the ST Tyr isoform, suggesting that phosphorylation is occurring in the cis-medial Golgi prior to the monensin block. This study demonstrates the presence of kinase activities in the cis-medial Golgi and the substantial phosphorylation of the luminal sequences of a glycosyltransferase.The sialyltransferases are a large family of glycosyltransferases that act to modify N-linked and O-linked oligosaccharides and glycolipids as these molecules traverse the Golgi apparatus of the cell. Their activity is required for the synthesis of important sialylated oligosaccharide structures that modulate or mediate a variety of interactions. These include selectin-leukocyte interactions in inflammation and lymphocyte homing; virus, parasite, and toxin binding to host cells; maintenance of glycoproteins in the circulation; cell interactions in B cell maturation and activation; and antiadhesive effects during metastasis and development (for review, see Ref. 1).Many of the glycosyltransferases have been precisely localized in the cisternae of the Golgi of various cell types (for review, see Ref. 2). From these localization studies, it appears that the glycosyltransferases are organized throughout the Golgi cisternae in roughly the same order in which they act to add sugar residues to the growing oligosaccharide chains. It has been presumed that this relatively strict localization pattern allows for efficient glycosylation by ensuring that enzymes are compartmentalized with their glycoconjugate substrates and sugar nucleotide donors. In support of this idea, recent studies have shown that differential compartmentalization of enzymes that compete for the same substrates does alter the types of oligosaccharide structures made by the cell (3, 4).In addition to compartmentalization, other post-transcriptional or post-translational events may control glycosyltransferase activity. Glycosyltransferases have been found as soluble forms in a variety of body fluids (5-11). Not surprisingly, many of these enzymes are cleaved and secreted after expressi...

Many Golgi glycosyltransferases are type II membrane proteins which are cleaved to produce soluble forms that are released from cells. Cho and Cummings recently reported that a soluble form of alpha1, 3-galactosyltransferase was comparable to its membrane bound counterpart in its ability to galactosylate newly synthesized glycoproteins (Cho,S.K. and Cummings,R.D. (1997) J. Biol. Chem., 272, 13622-13628). To test the generality of their findings, we compared the activities of the full length and soluble forms of two such glycosyltransferases, ss1,4 N-Acetylgalactosaminyltransferase (GM2/GD2/ GA2 synthase; GalNAcT) and beta galactoside alpha2,6 sialyltransferase (alpha2,6-ST; ST6Gal I), for production of their glycoconjugate products in vivo . Unlike the full length form of GalNAcT which produced ganglioside GM2 in transfected cells, soluble GalNAcT did not produce detectable GM2 in vivo even though it possessed in vitro GalNAcT activity comparable to that of full length GalNAcT. When compared with cells expressing full length alpha2,6-ST, cells expressing a soluble form of alpha2,6-ST contained 3-fold higher alpha2,6-ST mRNA levels and secreted 7-fold greater alpha2,6-ST activity as measured in vitro , but in striking contrast contained 2- to 4-fold less of the alpha2,6-linked sialic acid moiety in cellular glycoproteins in vivo . In summary these results suggest that unlike alpha1,3-galactosyltransferase the soluble forms of these two glycosyltransferases are less efficient at glycosylation of membrane proteins and lipids in vivo than their membrane bound counterparts.

A significant proportion of the ␣2,6-sialyltransferase of protein Asn-linked glycosylation (ST6Gal I) forms disulfide-bonded dimers that exhibit decreased activity, but retain the ability to bind asialoglycoprotein substrates. Here, we have investigated the subcellular location and mechanism of ST6Gal I dimer formation, as well as the role of Cys residues in the enzyme's trafficking, localization, and catalytic activity. Pulse-chase analysis demonstrated that the ST6Gal I disulfidebonded dimer forms in the endoplasmic reticulum. Mutagenesis experiments showed that Cys-24 in the transmembrane region is required for dimerization, while catalytic domain Cys residues are required for trafficking and catalytic activity. Replacement of Cys-181 and Cys-332 generated proteins that are largely retained in the endoplasmic reticulum and minimally active or inactive, respectively. Replacement of Cys-350 or Cys-361 inactivated the enzyme without compromising its localization or processing, suggesting that these amino acids are part of the enzyme's active site. Replacement of Cys-139 or Cys-403 generated proteins that are catalytically active and appear to be more stably localized in the Golgi, since they exhibited decreased cleavage and secretion. The Cys-139 mutant also exhibited increased dimer formation suggesting that ST6Gal I dimers may be critical in the oligomerization process involved in stable ST6Gal I Golgi localization.The sialyltransferases are a large family of glycosyltransferases that function in the Golgi apparatus to transfer NeuAc from the sugar nucleotide donor, CMP-NeuAc, to terminal positions of N-linked and O-linked oligosaccharides of glycoproteins and the oligosaccharides of glycolipids. The sialylated oligosaccharides that are products of these enzymes' action have a variety of important roles in the normal cell and during development and disease (reviewed in Ref. 1). For example, sialylated oligosaccharides function as selectin ligands during inflammation and the homing of lymphocytes; act as receptors for viruses, toxins, and parasites; function in B cell maturation and activation; maintain glycoproteins in the circulation; and play roles in negatively modulating cell adhesion during development and oncogenesis (1).