Expression of bovine beta-1,4-galactosyltransferase cDNA in COS-7 cells.

Abstract

A bovine (-1,4-galactosyltransferase (GT; EC 2.4.1.90) cDNA in an Okayama-Berg vector, pLsGT, was constructed from a partial cDNA clone and a genomic fragment. We report that the cDNA sequence of pLsGT, in a transient expression assay in COS-7 cells, codes for an enzymatically active GT protein. There is an approximately 12-fold increase in the GT activity in pLsGT-transfected cells compared to cells transfected with the antisense bovine GT construct, pLasGT, or pSV2Neo or mock-transfected cells. The increased activity is correlated with the increase in bovine GT mRNA, which is distinguishable from COS GT mRNA with a 3'-end-specific probe of pLsGT. The expressed GT activity is modulated by a-lactalbumin, which changes the acceptor specificity to glucose to synthesize lactose. Polyclonal antibody raised against SDS/PAGE-purified bovine milk GT and a monoclonal antibody (mAb 4-10) directed against a synthetic peptide corresponding to the amino-terminal region of the protein encoded by pLsGT bind the expressed protein, and the resulting immunoprecipitates exhibit GT enzymatic activity.N-Acetylglucosamine 8-1,4-galactosyltransferase (GT; EC 2.4.1.90) is but one member of the glycosyltransferase family of enzymes. Many of these enzymes are involved in the synthesis and extension of oligosaccharide chains on glycoproteins and glycolipids, generating a series of disaccharide linkages (1-3). Molecular approaches have been utilized to study the various transferases and cDNAs for some transferases have been cloned (4-9).GT transfers galactose from UDP-galactose to N-acetylglucosamine (GlcNAc) to produce N-acetyllactosamine with a f8-1,4-glycosidic linkage. The cDNA for GT has been independently cloned by several laboratories from lactating bovine mammary gland (4), the bovine kidney cell line MDBK (5), human liver (6), F9 embryonal carcinoma cells (7), and the mouse mammary cell line c127 (8). All these clones show strong homology to each other. On the other hand the 5' sequence of human liver GT cDNA clone isolated by Humphreys-Beher et al. (10) does not exhibit homology to any of the reported GT cDNAs (4-8), in spite of the fact that the deduced partial amino acid sequence of the cDNA clone showed partial homology to the amino-terminal sequence of the bovine GT protein they isolated. This raises the question whether the described clones (4-8) do indeed code for active GT.To address this problem, we constructed a full-length bovine GT cDNA in an Okayama-Berg expression vector (pLsGT)*, transfected it into COS-7 cells, and obtained a 12-fold increase in GT enzymatic activity in the transfected COS-7 cells compared to mock-transfected cells. This GT activity is modulated by a-lactalbumin, which changes the acceptor specificity from GlcNAc to glucose. In addition, this GT activity is immunoprecipitable by a polyclonal antibody raised against SDS/PAGE-purified bovine milk GT and by a monoclonal antibody (mAb) directed against a synthetic peptide whose sequence was derived from the cDNA sequence that spans a portion o...

Golgi retention of a trans-Golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain.

“…The homogenate prepared from the transfected COS-1 cells with pGT hCG exhibited a GT activity of 35.9 x 10-3 units per mg of protein, while the homogenate prepared from the transfected COS-1 cells with vector alone showed a GT activity of 9.9 x 10-3 units per mg of protein. As the transfection efficiency in our experiments was 20-30%, this degree (3.6-fold) of overexpression is similar to the previously reported activity levels seen for COS cells transfected with GT cDNA (18,19 …”

Purification and characterization of recombinant human β1–4 galactosyltransferase expressed in Saccharomyces cerevisiae

“…This represents a drawback in the pursuit of our two long-term goals which concern the establishment of a cell system which may be instrumental in understanding the biogenesis of the Golgi apparatus and the topogenesis of Golgi enzymes and mass production of the enzyme to make it available as a tool for oligosaccharide synthesis in the context recently reviewed by Ichikawa et al (1992). In fact, the enzyme has already been expressed in COS-7 cells (Masibay and Qasba, 1989), COS-1 cells (Nakazawa et al, 1991), E. coli (Aoki et al, 1990;Chatterjee, 1991). We choose S. cerevisiae for heterologous expression for the following reasons.…”

“…While gal-T has been expressed in higher eukaryotic cells (Masibay and Qasba, 1989) and as a soluble enzyme from Escherichia coli (Aoki et al, 1990;Chatterjee, 1991), this work was designed to explore the possibility for expression and production of mammalian glycosyltransferases in yeast. As a first step towards this goal, we expressed the cDNA coding for the full-length, membrane-bound form of gal-T in Saccharomyces cerevisiae.…”

Proteins of the Golgi apparatus

“…cDNA sequences have been reported encoding the fulllength protein from murine, human, and bovine sources (Shaper et al, 1988;Nakazawa et al, 1988;Masri et al, 1988;D'Agostaro et al, 1989;Masibay and Qasba, 1989). Evidence supports the existence of mRNA species differing at the 5' end which permit initiation of translation at either of two methionine residues 12 amino acids apart at the Nterminus (Russo et al, 1990).…”