Functional domains of bovine beta-1,4 galactosyltransferase.

A number of N- and C-terminal deletion and point mutants of bovine beta-1,4 galactosyltransferase (beta-1,4GT) were expressed in E. coli to determine the binding regions of the enzyme that interact with N-acetylglucosamine (NAG) and UDP-galactose. The N-terminal truncated forms of the enzyme between residues 1-129, do not show any significant difference in the apparent Kms towards NAG or linear oligosaccharide acceptors e.g. for chitobiose and chitotriose, or for the nucleotide donor UDP-galactose. Deletion or mutation of Cys 134 results in the loss of enzymatic activity, but does not affect the binding properties of the protein either to NAG- or UDP-agarose. From these columns the protein can be eluted with 15 mM NAG and 50 mM EDTA, like the enzymatically active protein, TL-GT129, that contains residues 130-402 of bovine beta-1,4GT. Also the N-terminus fragment, TL-GT129NAG, that contains residues 130-257 of the beta-1,4GT, binds to, and elutes with 15 mM NAG and 50 mM EDTA from the NAG-agarose column as efficiently as the enzymatically active TL-GT129. Unlike TL-GT129, the TL-GT129NAG binds to UDP-columns less efficiently and can be eluted from the column with only 15 mM NAG. The C-terminus fragment GT-257UDP, containing residues 258-402 of beta-1,4GT, binds tightly to both NAG- and UDP-agarose columns. A small fraction, 5-10% of the bound protein, can be eluted from the UDP-agarose column with 50 mM EDTA alone. The results show that the binding behaviour of N- and C-terminal fragments of beta-1,4GT towards the NAG- and UDP-agarose columns differ, the former binds preferentially to NAG-columns, while the latter binds to UDP-agarose columns via Mn2+.

Expression of deletion constructs of bovine beta-1,4-galactosyltransferase in Escherichia coli: importance of Cys134 for its activity.

Bovine beta-1,4-galactosyltransferase (beta-1,4-GT; EC 2.4.1.90) belongs to the glycosyltransferase family and as such shares a general topology: an N-terminal cytoplasmic tail, a signal anchor followed by a stem region and a catalytic domain at the C-terminal end of the protein. cDNA constructs of the N-terminal deleted forms of beta-1,4-GT were prepared in pGEX-2T vector and expressed in E. coli as glutathione-S-transferase (GST) fusion proteins. Recombinant proteins accumulated within inclusion bodies as insoluble aggregates that were solubilized in 5 M guanidine HCl and required an 'oxido-shuffling' reagent for regeneration of the enzyme activity. The recombinant beta-1,4-GT, devoid of the GST domain, has 30-85% of the sp. act. of bovine milk beta-1,4-GT with apparent Kms for N-acetylglucosamine and UDP-galactose similar to those of milk enzyme. Deletion analyses show that both beta-1,4-GT and lactose synthetase activities remain intact even in the absence of the first 129 residues (pGT-d129). The activities are lost when either deletions extend up to residue 142 (pGT-d142) or Cys134 is mutated to Ser (pGT-d129C134S). These results suggest that the formation of a disulfide bond involving Cys134 holds the protein in a conformation that is required for enzymatic activity.

Mutational analysis of the Golgi retention signal of bovine beta-1,4-galactosyltransferase.

To examine the role of the NH2-terminal region of the 402-residue-long beta-1,4-galactosyltransferase (beta-1,4-GT), a series of mutants and chimeric cDNA were constructed by polymerase chain reaction and transiently expressed in COS-7 cells, the enzyme activities were measured, and the protein was localized in the cells by subcellular fractionation or indirect immunofluorescence microscopy. We showed earlier that the deletion of the amino-terminal cytoplasmic tail and transmembrane domain from GT abolishes the stable expression of this protein in mammalian cells (Masibay, A.S., Boeggeman, E., and Qasba, P.K. (1992) Mol. Biol. Rep. 16, 99-104). Further deletion analyses of the amino-terminal region show that the first 21 amino acids of beta-1,4-GT are not essential for the stable production of the protein and are consistently localized in the Golgi apparatus. In addition, analysis of hybrid constructs showed that residues 1-25 of alpha-1,3-galactosyltransferase can functionally replace the beta-1,4-GT amino-terminal domain (residues 1-43). This fusion protein also showed Golgi localization. On the other hand, the alpha-2,6-sialyltransferase/beta-1,4-GT fusion protein (alpha-2,6-ST/beta-1,4-GT) needed additional COOH-terminal sequences flanking the transmembrane domain of the alpha-2,6-ST for stability and Golgi localization. Substitution of Arg-24, Leu-25, Leu-26, and His-33 of the beta-1,4-GT transmembrane by Ile (pLFM) or substitution of Tyr by Ile at positions 40 and 41 coupled with the insertion of 4 Ile residues at position 43 (pLB) released the mutant proteins from the Golgi and was detected on the cell surface. Our results show that (a) the transmembrane domains of beta-1,4-GT, alpha-1,3-galactosyltransferase, and alpha-2,6-ST, along with its stem region, all play a role in Golgi targeting and participate in a common mechanism that allows the protein to be processed properly and not be degraded in vivo; (b) increasing the length of the transmembrane domain overrides the Golgi retention signal and directs the enzyme to the plasma membrane; and (c) the length of the hydrophobic region of the transmembrane domain of beta-1,4-GT is an important parameter but is not sufficient by itself for Golgi retention.

Deletion analysis of the NH2-terminal region of beta-1,4-galactosyltransferase.

To determine the biological role, if any, of the NH2-terminal region of beta-1,4-galactosyltransferase (GT; EC 2.4.1.90), we constructed deletion mutants and expressed them in COS-7 cells. Each deletion construct was analyzed for enzymatic activity, protein production and mRNA transcription. All of the deletion mutants were transcribed to produce GT mRNA, but the GT protein was not detected in those constructs whose transmembrane (aa 14-42) domain was deleted. The results suggest that the transmembrane region is essential for the stability of the protein and perhaps contain sequences critical for the proper targeting of the molecule. The possible role of the NH2-terminal signal anchor domain in the in vivo regulation of GT is discussed.

Expression of beta-1,4-galactosyltransferase gene during 3T3 cell growth.

The beta-1,4-galactosyltransferase (GT; EC 2.4.1.90) is localized in the trans-cisternae of the Golgi apparatus where it catalyzes the transfer of galactose from UDP-galactose to the N-acetylglucosamine residue of secretory and membrane-bound glycoproteins. Given the potential role of GT in cell-cell interaction and the fact that numerous cell surface events occur during cell growth we studied the possible relationship between GT expression and 3T3 cell growth. The level of GT mRNA increases 3--4-fold 2 h after serum-stimulation of quiescent 3T3 cells. Protein biosynthesis inhibitors like cycloheximide and anisomycin superinduce GT mRNA expression. Concomitant with this increase is an observed rise in the level of GT protein as well as an increase in overall GT enzymatic activity. Antibody-binding studies and direct enzyme assays of intact cells, along with subcellular fractionation experiments indicate that there is an increase in both Golgi and cell surface-associated GT pools upon serum-stimulation of resting cells. We conclude that GT is a member of the cell-cycle dependent genes whose expression is growth regulated.

Identification and partial characterization of genes that are transactivated by different pathways in quiescent mouse cells stimulated with serum.

We have identified four cDNA clones, cl-1, cl-5, cl-15, and cl-16, that represent genes induced by serum in resting mouse 3T3 cells. Partial sequence analysis of the four cDNAs indicated that cl-15 corresponds to the mouse beta-actin gene. Comparison of the DNA sequences of the other three clones with the sequence data bank (Genbank) showed little homology to other known DNA sequences and thus represent novel genes. The level of the mRNAs corresponding to the four genes began to increase in resting cells following serum stimulation, reached a peak between 5 h and 8 h and then started to decline. Inhibitors of transcription diminished the induction of the mRNAs corresponding to the four genes. Cycloheximide and anisomycin had little effect on the induction of beta actin mRNA while the induction of the other three genes was suppressed by the same inhibitors. 12-O-Tetradecanoylphorbol-13-acetate and the calcium ionophore A23187 enhanced the expression of the cl-16 mRNA while epidermal growth factor, fibroblast growth factor, or insulin enhanced the expression of cl-1- and cl-5-specific transcripts. The level of beta-actin mRNA was elevated in resting cells by epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate and to a lesser extent by fibroblast growth factor, insulin, and dibutyryl cyclic AMP-elevating agents. Pertussis toxin, an inhibitor of the action of G proteins, did not significantly suppress the activation of the four genes by serum. However, 2-aminopurine, a protein kinase inhibitor, suppressed the induction of the four transcripts in serum-stimulated cells. The possible pathways involved in the activation of these genes in resting cells are discussed.

Genetic relatedness among animal rotaviruses.

The genomic relatedness among representative rotavirus strains was examined by employing cross-hybridization techniques. Single stranded (ss) RNA prepared by in vitro transcription of purified rotavirus particles and labeled with either 32P or 125I was hybridized to denatured genomic, double stranded (ds) RNAs. The hybrids formed were analyzed by polyacrylamide gel electrophoresis (PAGE) or by testing their sensitivity to digestion with single strand specific nuclease (S-1 nuclease). A relatively high degree of genomic homology was found to exist among several bovine rotavirus strains obtained from different geographical areas. Similarly, a high degree of homology was found between two different simian rotavirus strains, and also between two porcine strains. The human Wa strain exhibited a low degree of genomic homology with simian, bovine and canine strains whereas a higher level of homology was detected between the human Wa strain and the porcine strains. The observed RNA sequence divergences of rotaviruses isolated from different animal species are in agreement with the restricted host range of these viruses and their known antigenic differences and suggest a divergent evolution of their genomes.

Genetic relatedness among human rotaviruses.

The genetic relatedness of 81 clinical rotavirus isolates to the human rotavirus prototype strains Wa (subgroup 2, serotype 1) and DS-1 (subgroup 1, serotype 2) was examined by RNA hybridization techniques. Labeled single-stranded (+) transcripts of Wa or DS-1 virus were incubated with denatured genomic rotaviral RNAs, and the resulting hybrids were subjected to gel electrophoresis and autoradiography. Nineteen of the specimens contained subgroup 1 rotavirus with a "short" RNA migration pattern. These viruses were found to be closely related to the DS-1 strain and were associated with illness of short duration. The remaining 62 isolates belonged to subgroup 2 and exhibited a "long" RNA migration pattern. Fifty-four of these isolates exhibited significant hybridization with the Wa strain probe. Four isolates yielded multiple hybrid bands with the Wa probe but also possessed at least one gene segment homologous to the DS-1 strain. The remaining four subgroup 2 rotaviruses did not exhibit significant homology in the form of labeled hybrid bands when tested with either the Wa or DS-1 probe. These findings suggest that most clinical rotavirus isolates belong to one of two human rotavirus "families" defined as Wa-like or DS-1-like. Our observations also suggest that reassortment occurs in vivo between rotaviruses belonging to the two human rotavirus "families" and that there are one or more additional families of human rotavirus.

A dot hybridisation assay for detection of rotavirus.

A "dot" hybridisation technique for the detection of rotavirus in stools and other biological materials is described. The assay is based on the in-situ hybridisation of labelled single-stranded RNA probes, obtained by in-vitro transcription of rotavirus particles, to heat-denatured rotavirus RNA immobilised on nitrocellulose membranes. The method is highly specific and allows for the detection of as little as 8 pg of viral RNA. Its use for the detection of rotavirus in stool suspensions and rectal swabs obtained from children with diarrhoea may facilitate epidemiological studies of rotavirus gastroenteritis.