Human β1,4 galactosyltransferase and α2,6 sialyltransferase expressed in Saccharomyces cerevisiae are retained as active enzymes in the endoplasmic reticulum

Abstract

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.

Glycosylation Defect in Lec1 Chinese Hamster Ovary Mutant Is Due to a Point Mutation in N-Acetylglucosaminyltransferase I Gene

“…Bound antibody was detected by peroxidase-conjugated anti-sheep immunoglobulin and enhanced chemiluminescence. Previous studies have demonstrated that ␤1,4-galactosyltransferase and ␣2,6-sialyltransferase can be expressed in S. cerevisiae as active enzymes (33,34) and here we demonstrate that wild-type GnTI can also be expressed as an active enzyme. The GnTI C123R mutant showed no activity in the GnTI null background of yeast, directly demonstrating that a Cys 123 to Arg 123 substitution results in loss of GnTI activity.…”

Functional Expression of the Murine Golgi CMP-Sialic Acid Transporter in Saccharomyces cerevisiae

“…When the intact full-length human ␤-1,4-galactosyltransferase or ␣-2,3-sialyltransferase were expressed in S. cerevisiae, the active enzymes appeared to be retained in the endoplasmic reticulum (21). While the former studies (15,16) suggest that mammalian Golgi-targeting domains can be used in S. cerevisiae, the latter do not (21).…”

Exploring the Substrate Specificities of α‐2,6–and α‐2,3–Sialyltransferases using Synthetic Acceptor Analogues

“…Rat liver Gal@14)GlcNAc a-2,6-sialyltransferase (Boehringer Mannheim) had an activity of 0.25 U/ml; one unit (U) of enzyme activity is defined as the amount of enzyme catalyzing the transfer of 1 pmol NeuSAc/min using the incubation conditions as indicated below and 1 as an acceptor at a concentration of 2 mM. A crude preparation of recombinant full-length human liver Gal@ -4)GIcNAc a-2,6-sialyltransferase, with an activity of 3.5 niU/rnl when assayed with 2 mM 1, was isolated from a Saccharomyces cerevisiae cell culture as previously described [12]. This enzyme was tested without purification, since no interfering endogenous sialyltransferase activity is present in yeast cells.…”