Bovine galactosyltransferase: identification of a clone by direct immunological screening of a cDNA expression library.

122

The HNK-1 carbohydrate epitope is characteristically expressed on a series of cell adhesion molecules and also on some glycolipids in the nervous system over a wide range of species from insect to mammal. The HNK-1 epitope is involved in cell-cell and͞or cell-substrate interaction and recognition during the development of the nervous system. In this study, we isolated a novel glucuronyltransferase from rat brain, which is a key enzyme of the biosynthesis of the HNK-1 epitope on glycoproteins. Based on the partial amino acid sequences, we isolated cDNA encoding the glucuronyltransferase. The primary structure deduced from the cDNA sequence predicted a type II transmembrane protein with 347 amino acids and had no detectable similarity with any other proteins of known functions, including glucuronyltransferases of the liver and olfactory epithelium. Expression of a soluble recombinant form of the enzyme in COS-1 cells produced an active glucuronyltransferase. The selective expression of the glucuronyltransferase gene in the nervous system was consistent with the almost exclusive localization of the HNK-1 epitope in the nervous system. Transfection of the glucuronyltransferase cDNA into COS-1 cells induced not only expression of the HNK-1 epitope on the cell surface but also marked morphological changes of the cells, suggesting that the HNK-1 epitope associates with the cell-substratum interaction.

Section: Resultsmentioning

confidence: 99%

Cloning and functional expression of a novel glucuronyltransferase involved in the biosynthesis of the carbohydrate epitope HNK-1

Terayama

Oka²,

Seiki³

et al. 1997

122

“…A TC-32 cDNA library was made according to previously published procedures (10). Briefly, first-strand synthesis was accomplished using methyl mercury-denatured poly(A)+ RNA primed with oligo(dT) and murine leukemia virus reverse transcriptase (GIBCO/BRL).…”

mentioning

confidence: 99%

Ewing sarcoma 11;22 translocation produces a chimeric transcription factor that requires the DNA-binding domain encoded by FLI1 for transformation.

May

Gishizky

Lessnick

et al. 1993

518

390

The 11;22 chromosomal translocation specificaOly linked to Ewing sarcoma and primitive neuroectodermal tumor results in a chimeric molecule fusing the aminoterminal-encoding portion of the EWS gene to the carboxylterminal DNA-binding domain encoded by the FLII gene. We have isolated a fourth EWS-FLII fusion cDNA that is structurally distinct from the three forms previously described. To determine the transforming activity of this gene, alternative forms of the EWS-FLI1 fusion were transduced into NIH 3T3 Structural alteration or aberrant expression of transcription factors is also common in human malignancies but usually results through somatic genomic mutation (for reviews, see refs. 2 and 3). Karyotypic analyses have revealed a tumor-specific t(11;22)(q24;ql2) chromosomal translocation in 86% of both Ewing sarcoma and primitive neuroectodermal tumor (PNET), suggesting that the product of this rearrangement is necessary for the formation of both these malignancies (4,5 MATERIALS AND METHODSPNET cDNA Library Construction and Isolation of EWS-FL!) Chimeras. TC-32, a PNET tumor cell line containing the 11;22 translocation, was grown in RPMI medium/10% fetal calf serum, as described (5). Total RNA was harvested by lysis with guanidine isothiocyanate and purified over cesium chloride (9). Poly(A)+ RNA was obtained by using columns packed with oligo(dT)-cellulose (Collaborative Research) and used for construction of cDNA libraries.A TC-32 cDNA library was made according to previously published procedures (10). Briefly, first-strand synthesis was accomplished using methyl mercury-denatured poly(A)+ RNA primed with oligo(dT) and murine leukemia virus reverse transcriptase (GIBCO/BRL). Second-strand synthesis was done by using RNase H and polymerase I (GIBCO/ BRL), and synthesized products were purified over a Sephadex G100 column (Pharmacia). cDNAs were blunted by using T4 polymerase (GIBCO/BRL) and ligated to a molar excess of EcoRI adaptors (Invitrogen, San Diego). The adaptor ends were phosphorylated with T4 polynucleotide kinase (United States Biochemical), and cDNAs were fractionated over a 6% acrylamide gel. DNA species >600 bp were recovered from gel slices by electroelution, purified over

“…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)(2)(3). Molecular approaches have been utilized to study the various transferases and cDNAs for some transferases have been cloned (4)(5)(6)(7)(8)(9).…”

mentioning

confidence: 99%

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

Masibay

Qasba

1989

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...