The acceptor specificities of rat liver Gal(@ -4)GlcNAc a-2,6-sialyltransferase, recombinant fulllength human liver Gal(j31-4)GlcNAc cx-2,6-sialyltransferase, and a soluble form of recombinant rat liver Gal(/j1-3/4)GlcNAc a-2,3-sialyltransferase were studied with a panel of analogues of the trisaccharide Gal(j31-4)GlcNAc(pl-2)Man(al -O)(CH,),CH,. These analogues contain structural variants of D-galactose, modified at either C3, C4 or C5 by deoxygenation, fluorination, 0-methylation, epimerization, or by the introduction of an amino group. In addition, the enantiomer of D-galactose is included. The a-2,6-sialyltransferases tolerated most of the modifications at the galactose residue to some extent, whereas the a-2,3-sialyltransferase displayed a narrower specificity. Molecular dynamics simulations were performed in order to correlate enzymatic activity to three-dimensional structure. Ineffective acceptors for rat liver n-2,6-sialyltransferase were shown to be inhibitory towards the enzyme; likewise, the a-2,3-sialyltransferase was found to be inhibited by all non-substrates. Modified sialyloligosaccharides were obtained on a milligram scale by incubation of effective acceptors with one of each of the three enzymes, and characterized by 500-MHz 'H-NMR spectroscopy.Keywords: sialyltransferase ; substrate specificity ; enzymatic synthesis ; glycoprotein.Sialic acids that are located at the periphery of glycoconjugate glycans can be involved in a variety of biological phenomena, such as cell-cell and receptor-ligand interactions, cell differentiation, or tumor progression and metastasis [l -51. The various types of sialylation patterns found in either glycolipid or glycoprotein glycans are the result of the action of at least 12 sialyltransferases, that can be readily distinguished by their strict in vivo specificity for acceptor substrates, including the type of linkage formed in the product [l]. However, in patients suffering from a-mannosidosis, we found the unusual trisaccharide Neu5-Ac(a2-6)Man@1-4)GlcNAc [6], and it has been suggested that this compound is formed by transfer of Neu5Ac from CMPNeuSAc to H06' in the accumulated disaccharide Manwl-4)-GlcNAc. Additional studies by us 17, 81 and by others 191 have shown that the purified rat liver Gal(/j-4)GlcNAc a-2,6-sialyltransferase, though specific for the B-1,4-linkage in the Nacetyllactosamine epitope in N-glycans, tolerated modifications in the accepting terminal monosaccharide in vitro, producing varying yields of sialyloligosaccharides. These results indicated that some of the hydroxyl groups of the terminal monosaccha- Enzymes. CMP-NeuSAc :Gal@ -4)GlcNAc-R a-2,6-sialyltransferase (EC 2.4.99.1 ): CMP-Neu5Ac:Gal(/~l-3/4)GlcNAc-R rr-2,3-sialyltransferase (EC 2.4.99.6); orthophosphoric monoester phosphohydrolase, alkaline phosphatase (EC 3.1 3.1).ride are of minor importance for effective sialylation, at least by the applied a-2,6-sialyltransferase, and prompted us to a program aimed at the exploration of the specific topology required by sialyltransferases....

A protease-defective strain of Succharomyces cerevisiae (BT 150) was used to express fulllength cDNA of HeLa cell P-D-N-acetylglucosarninide-P-1,4-galactosyltransferase (gal-T). To ascertain import of the recombinant gal-T into the secretory pathway of yeast cells, metabolically labeled enzyme was immunoprecipitated from extracts of yeast transformants, analysed by SDS/PAGE/ fluorography and tested for sensitivity to treatment with endoglycosidase-H. Untreated recombinant gal-T had an apparent molecular mass of 48 kDa, which was reduced to 47 kDa after treatment, indicating that the recombinant enzyme was N-glycosylated and, therefore, competent for translocation across the membranes of the endoplasmic reticulum.Using specific gal-T assays employing N-acetylglucosamine or glucose in combination with alactalbumin as exogenous acceptor substrates, recombinant gal-T enzyme activity could readily be detected in crude homogenates. Analysis of the disaccharide products by 'H-NMR spectroscopy demonstrated that only 8-1-4 linkages were formed by the recombinant gal-T. The recombinant gal-T was detergent solubilized and subsequently purified by affinity chromatography on N-acetylglucosamine-derivatized Sepharose followed by a-lactalbumin-Sepharose. The purified enzyme preparation had a specific activity comparable to that of the soluble gal-T isolated from human milk. Furthermore, kinetic parameters determined for both acceptor and donor substrates of both enzymes differed only slightly.This work shows that yeast provides an appropriate host system for the heterologous expression of mammalian glycosyltransferases. 8-D-N-Acetylglucosaminide-P-1,4-galactosyltransferase(gal-T) is a glycosyltransferase which catalyzes the transfer of galactose from UDP-galactose to GlcNAc or GlcNAc-R, forming a fi-1 + 4 linkage according to the following reaction :UDP-galactose + acceptor + galactose 8-1,4-acceptor + UDP .

Biosynthesis and intracellular transport of recombinant human full-length Pl,4 galactosyltransferase (GT) and full-length a2,6 sialyltransferase (ST) were investigated in Saccharomyces cerevisiae. Recently, enzymic activity of recombinant GT (rGT) in crude homogenates of S. cerevisiae could successfully be demonstrated [Krezdorn, C . , Watzele, G., Kleene, R. B., Ivanov, S. X. & Berger, E. G. (1993) Eur: J. Biochem. 212, 113-1201. In the present work, we show that, in yeast strains transformed with plasmid pDPSIA containing the cDNA coding for human ST, rST enzymic activity using asialo-fetuin or N-acetyllactosamine as acceptor substrates could readily be detected. Analysis by 'H-NMR spectroscopy of the disaccharide product of rGT, as recently reported, and the trisaccharide product of rST demonstrated that only the expected glycosidic linkages were formed. Following mechanical disruption of yeast cells, both enzymes sedimented with a fraction enriched in membranes of the endoplasmic reticulum (ER) and were activated by Triton X-100 3-5-fold. rGT and rST could be immunoprecipitated from their [35S]Met-labelled transformed yeast extracts using polyclonal antibodies raised against fusion proteins consisting of P-galactosidase-GT or P-galactosidase-ST, respectively, expressed in Escherichia coli. For rGT a single glycosylated form of apparent molecular mass 48 kDa was reported, but for rST two main bands corresponding to apparent molecular masses of 48 kDa and 44 kDa, respectively, were detected. Immunoprecipitation from either tunicamycin-treated [35S]Met-labelled transformed yeast cells or labelling with radioactive sugars both indicated that the 44-kDa form of rST was non-glycosylated and that the 48-kDa form of rST was core N-glycosylated. In addition, core glycosylation of both recombinant enzymes demonstrated that they were competent for translocation across the ER membranes. However, the 44-kDa form of rST was converted to the 48-kDa glycosylated form only slowly, suggesting a mechanism of posttranslational translocation. Absence of hyperglycosylation of rST and rGT in wild type and lack of the Golgi-specific man-al,6-man epitope suggest that the recombinant enzymes did not enter the yeast Golgi apparatus.These results indicated that both rGT and rST are retained as enzymically active enzymes in the ER of yeast and suggest a ribonucleoprotein-independent import of rST into the ER.

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