Chinese hamster ovary (CHO) cells may express six beta 4-galactosyltransferases (beta 4GalTs). Consequences of the loss of functional beta 4GalT-1, beta 4GalT-6, or both in CHO glycosylation mutants.

Six beta4-galactosyltransferase (beta4GalT) genes have been cloned from mammalian sources. We show that all six genes are expressed in the Gat(-)2 line of Chinese hamster ovary cells (Gat(-)2 CHO). Two independent mutants termed Pro(-)5Lec20 and Gat(-)2Lec20, previously selected for lectin resistance, were found to have a galactosylation defect. Radiolabeled biantennary N-glycans synthesized by Pro(-)5Lec20 were proportionately less ricin-bound than similar species from parental CHO cells, and Lec20 cell extracts had a markedly reduced ability to transfer Gal to GlcNAc-terminating acceptors. Northern blot analysis revealed a severe reduction in beta4GalT-1 transcripts in Pro(-)5Lec20 cells. The Gat(-)2Lec20 mutant expressed beta4GalT-1 transcripts of reduced size due to a 311-base pair deletion in the beta4GalT-1 gene coding region. Northern analysis with probes from the remaining five beta4GalT genes revealed that Gat(-)2 CHO and Gat(-)2Lec20 cells express all six beta4GalT genes. Unexpectedly, the beta4GalT-6 gene is not expressed in either Pro(-)5 or Pro(-)5Lec20 cells. Thus, in addition to a deficiency in beta4GalT-1, Pro(-)5Lec20 cells lack beta4GalT-6. Nevertheless, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry data of N-glycans released from cellular glycoproteins showed that both the beta4GalT-1(-) (Gat(-)2Lec20) and beta4GalT-1(-)/beta4GalT-6(-) (Pro(-)5Lec20) mutants have a similar Gal deficiency, affecting neutral and sialylated bi-, tri-, and tetraantennary N-glycans. By contrast, glycolipid synthesis was normal in both mutants. Therefore, beta4GalT-1 is a key enzyme in the galactosylation of N-glycans, but is not involved in glycolipid synthesis in CHO cells. beta4GalT-6 contributes only slightly to the galactosylation of N-glycans and is also not involved in CHO cell glycolipid synthesis. These CHO glycosylation mutants provide insight into the variety of in vivo substrates of different beta4GalTs. They may be used in glycosylation engineering and in investigating roles for beta4GalT-1 and beta4GalT-6 in generating specific glycan ligands.

A novel 14-base-pair regulatory element is essential for in vivo expression of murine beta4-galactosyltransferase-I in late pachytene spermatocytes and round spermatids.

During murine spermatogenesis, beginning in late pachytene spermatocytes, the beta4-galactosyltransferase-I (beta4GalT-I) gene is transcribed from a male germ cell-specific start site. We had shown previously that a 796-bp genomic fragment that flanks the germ cell start site and contains two putative CRE (cyclic AMP-responsive element)-like motifs directs correct male germ cell expression of the beta-galactosidase reporter gene in late pachytene spermatocytes and round spermatids of transgenic mice (N. L. Shaper, A. Harduin-Lepers, and J. H. Shaper, J. Biol. Chem. 269:25165-25171, 1994). We now report that in vivo expression of beta4GalT-I in developing male germ cells requires an essential and previously undescribed 14-bp regulatory element (5'-GCCGGTTTCCTAGA-3') that is distinct from the two CRE-like sequences. This cis element is located 16 bp upstream of the germ cell-specific start site and binds a male germ cell protein that we have termed TASS-1 (transcriptional activator in late pachytene spermatocytes and round spermatids 1). The presence of the Ets signature binding motif 5'-GGAA-3' on the bottom strand of the TASS-1 sequence (underlined sequence) suggests that TASS-1 is a novel member of the Ets family of transcription factors. Additional transgenic analyses established that an 87-bp genomic fragment containing the TASS-1 regulatory element was sufficient for correct germ cell-specific expression of the beta-galactosidase reporter gene. Furthermore, when the TASS-1 motif was mutated by transversion, within the context of the original 796-bp fragment, transgene expression was reduced 12- to 35-fold in vivo.

Determination of beta1,4-galactosyltransferase enzymatic activity by capillary electrophoresis and laser-induced fluorescence detection.

We have developed a nonradioactive method to assay UDP-Gal:beta-d-GlcNAcbeta1,4-galactosyltransferase (beta4GalT-I) enzymatic activity. Capillary electrophoresis combined with laser-induced fluorescence detection (CE-LIF) was employed to provide a baseline separation of FITC-conjugated O-GlcNAc-containing substrate peptides and galactose-capped product peptides, while at the same time allowing a level of detection in the low attomole range (10(-18)). The addition of 2 mM hexamethylene diamine to the borate-based capillary electrophoretic buffer modulated the electroosmotic flow, resulting in optimum separation of the glycopeptide product from reactant. beta4GalT-I activity was dependent upon the addition of both manganese and UDP-galactose. Using this assay, we show that two beta4GalT-I constructs, predicted to localize to different intracellular compartments, are enzymatically active when expressed in vitro using a rabbit reticulocyte transcription-translation system. The high sensitivity of product detection by CE-LIF in combination with in vitro transcription-translation is applicable to the facile determination of the enzymatic activity of other newly cloned glycosyltransferases.

The increased level of beta1,4-galactosyltransferase required for lactose biosynthesis is achieved in part by translational control.

beta1,4-Galactosyltransferase (beta4GalT-I) participates in both glycoconjugate biosynthesis (ubiquitous activity) and lactose biosynthesis (mammary gland-specific activity). In somatic tissues, transcription of the mammalian beta4GalT-I gene results in a 4.1-kb mRNA and a 3.9-kb mRNA as a consequence of initiation at two start sites separated by approximately 200 bp. In the mammary gland, coincident with the increased beta4GalT-I enzyme level ( approximately 50-fold) required for lactose biosynthesis, there is a switch from the 4.1-kb start site to the preferential use of the 3.9-kb start site, which is governed by a stronger tissue-restricted promoter. The use of the 3.9-kb start site results in a beta4GalT-I transcript in which the 5'- untranslated region (UTR) has been truncated from approximately 175 nt to approximately 28 nt. The 5'-UTR of the 4.1-kb transcript [UTR(4.1)] is predicted to contain extensive secondary structure, a feature previously shown to reduce translational efficiency of an mRNA. In contrast, the 5'-UTR of the 3.9-kb mRNA [UTR(3.9)] lacks extensive secondary structure; thus, this transcript is predicted to be more efficiently translated relative to the 4.1-kb mRNA. To test this prediction, constructs were assembled in which the respective 5'-UTRs were fused to the luciferase-coding sequence and enzyme levels were determined after translation in vitro and in vivo. The luciferase mRNA containing the truncated UTR(3.9) was translated more efficiently both in vitro (approximately 14-fold) and in vivo (3- to 5-fold) relative to the luciferase mRNA containing the UTR(4.1). Consequently, in addition to control at the transcriptional level, beta4GalT-I enzyme levels are further augmented in the lactating mammary gland as a result of translational control.

The expanding beta 4-galactosyltransferase gene family: messages from the databanks.

From a systematic search of the UniGene and dbEST databanks, using human beta 4-galactosyltransferase (beta 4GalT-I), which is recognized to function in lactose biosynthesis, as the query sequence, we have identified five additional gene family members denoted as beta 4GalT-II, -III, -IV, -V, and -VI. Complementary DNA clones containing the complete coding regions for each of the five human homologs were obtained or generated by a PCR-based strategy (RACE) and sequenced. Relative to beta 4GalT-I, the percent sequence identity at the amino acid level between the individual family members, ranges from 33% (beta 4GalT-VI) to 55% (beta 4GalT-II). The highest sequence identity between any of the homologs is between beta 4GalT-V and beta 4GalT-VI (68%). beta 4GalT-II is the ortholog of the chicken beta 4GalT-II gene, which has been demonstrated to encode an alpha-lactalbumin responsive beta 4-galactosyltransferase (Shaper et al., J. Biol. Chem., 272, 31389-31399, 1997). As established by Northern analysis, beta 4GalT-II and -IV show the most restricted pattern of tissue expression. High steady state levels of beta 4GalT-II mRNA are seen only in fetal brain and adult heart, muscle, and pancreas; relatively high levels of beta 4GalT-VI mRNA are seen only in adult brain. When the corresponding mouse EST clone for each of the beta 4GalT family members was used as the hybridization probe for Northern analysis of murine mammary tissue, transcription of only the beta 4GalT-I gene could be detected in the lactating mammary gland. These observations support the conclusion that among the six known beta 4GalT family members in the mammalian genome, that have been generated through multiple gene duplication events of an ancestral gene(s), only the beta 4GalT-I ancestral lineage was recruited for lactose biosynthesis during the evolution of mammals.

Beta1,4-galactosyltransferase and lactose biosynthesis: recruitment of a housekeeping gene from the nonmammalian vertebrate gene pool for a mammary gland specific function.

Beta1,4-galactosyltransferase (beta4GalT-I) is a constitutively expressed trans-Golgi enzyme, widely distributed in vertebrates, which synthesizes the beta4-N-acetyllactosamine structure commonly found in glycoconjugates. In mammals beta4GalT-I has been recruited for a second biosynthetic function, the production of lactose; this function takes place exclusively in the lactating mammary gland. In preparation for lactose biosynthesis, beta4GalT-I enzyme levels are increased significantly. We show that mammals have evolved a two-step mechanism to achieve this increase. In step one there is a switch to the use of a second transcriptional start site, regulated by a stronger, mammary gland-restricted promoter. The transcript produced is distinguished from its housekeeping counterpart by the absence of approximately 180 nt of 5'-untranslated sequence. In step two, this truncated transcript is translated more efficiently, relative to the major transcript expressed in all other somatic tissues.

High efficiency reporter gene transfection of vascular tissue in vitro and in vivo using a cationic lipid-DNA complex.

Efficient transfection conditions for a number of human, rat and rabbit primary cells and established lines of vascular origin have been determined using a complex of a commercially available cationic lipid transfection agent (Tfx-50) and luciferase reporter plasmid constructs. The optimised conditions have also been successfully applied to rabbit carotid arteries in vivo and a series of human arteries in vitro. The most critical factors influencing the efficiency of gene transfection with this protocol are: DNA concentration; ratio of lipid reagent to DNA; transfection time and the presence or absence of serum. Immunohistochemical analysis shows that a high percentage of cells (approximately 30-80% dependent on lineage) were transfected under optimal conditions with minimal toxicity effects. Similar analyses performed on undamaged rabbit carotid vessels transfected in vivo and human arteries transfected in vitro show high-efficiency transfer and strong expression of the luciferase vector as demonstrated by reporter gene expression. The optimisation of gene transfer into vascular cells with this cationic lipid complex will be valuable for molecular studies of genes implicated in cardiovascular diseases and as a possible method of gene delivery with therapeutic intent.

The chicken genome contains two functional nonallelic beta1,4-galactosyltransferase genes. Chromosomal assignment to syntenic regions tracks fate of the two gene lineages in the human genome.

Two distinct but related groups of cDNA clones, CKbeta4GT-I and CKbeta4GT-II, have been isolated by screening a chicken hepatoma cDNA library with a bovine beta1,4-galactosyltransferase (beta4GT) cDNA clone. CKbeta4GT-I is predicted to encode a type II transmembrane glycoprotein of 41 kDa with one consensus site for N-linked glycosylation. CKbeta4GT-II is predicted to encode a type II transmembrane glycoprotein of 43 kDa with five potential N-linked glycosylation sites. At the amino acid level, the coding regions of CKbeta4GT-I and CKbeta4GT-II are 52% identical to each other and 62 and 49% identical, respectively, to bovine beta4GT. Despite this divergence in amino acid sequence, high levels of expression of each cDNA in Trichoplusia ni insect cells demonstrate that both CKbeta4GT-I and CKbeta4GT-II encode an alpha-lactalbumin-responsive, UDP-galactose:N-acetylglucosamine beta4-galactosyltransferase. An analysis of CKbeta4GT-I and CKbeta4GT-II genomic clones established that the intron positions within the coding region are conserved when compared with each other, and these positions are identical to the mouse and human beta4GT genes. Thus CKbeta4GT-I and CKbeta4GT-II are the result of the duplication of an ancestral gene and subsequent divergence. CKbeta4GT-I maps to chicken chromosome Z in a region of conserved synteny with the centromeric region of mouse chromosome 4 and human chromosome 9p, where beta4-galactosyltransferase (EC 2.4.1.38) had previously been mapped. Consequently, during the evolution of mammals, it is the CKbeta4GT-I gene lineage that has been recruited for the biosynthesis of lactose. CKbeta4GT-II maps to a region of chicken chromosome 8 that exhibits conserved synteny with human chromosome 1p. An inspection of the current human gene map of expressed sequence tags reveals that there is a gene noted to be highly similar to beta4GT located in this syntenic region on human chromosome 1p. Because both the CKbeta4GT-I and CKbeta4GT-II gene lineages are detectable in mammals, duplication of the ancestral beta4-galactosyltransferase gene occurred over 250 million years ago in an ancestral species common to both mammals and birds.

Transcriptional regulation of murine beta1,4-galactosyltransferase in somatic cells. Analysis of a gene that serves both a housekeeping and a mammary gland-specific function.

beta1,4-Galactosyltransferase (beta4-GT) is a constitutively expressed enzyme that synthesizes the beta4-N-acetyllactosamine structure in glycoconjugates. In mammals, beta4-GT has been recruited for a second biosynthetic function, the production of lactose which occurs exclusively in the lactating mammary gland. In somatic tissues, the murine beta4-GT gene specifies two mRNAs of 4. 1 and 3.9 kilobases (kb), as a consequence of initiation at two different start sites approximately 200 base pairs apart. We have proposed that the region upstream of the 4.1-kb start site functions as a housekeeping promoter, while the region adjacent to the 3.9-kb start site functions primarily as a mammary gland-specific promoter (Harduin-Lepers, A., Shaper, J. H., and Shaper, N. L. (1993) J. Biol. Chem. 268, 14348-14359). Using DNase I footprinting and electrophoretic mobility shift assays, we show that the region immediately upstream of the 4.1-kb start site is occupied mainly by the ubiquitous factor Sp1. In contrast, the region adjacent to the 3.9-kb start site is bound by multiple proteins which include the tissue-restricted factor AP2, a mammary gland-specific form of CTF/NF1, Sp1, as well as a candidate negative regulatory factor that represses transcription from the 3.9-kb start site. These data experimentally support our conclusion that the 3.9-kb start site has been introduced into the mammalian beta4-GT gene to accommodate the recruited role of beta4-GT in lactose biosynthesis.

Gangliosides are neuronal ligands for myelin-associated glycoprotein.

Nerve cells depend on specific interactions with glial cells for proper function. Myelinating glial cells are thought to associate with neuronal axons, in part, via the cell-surface adhesion protein, myelin-associated glycoprotein (MAG). MAG is also thought to be a major inhibitor of neurite outgrowth (axon regeneration) in the adult central nervous system. Primary structure and in vitro function place MAG in an immunoglobulin-related family of sialic acid-binding lactins. We report that a limited set of structurally related gangliosides, known to be expressed on myelinated neurons in vivo, are ligands for MAG. When major brain gangliosides were adsorbed as artificial membranes on plastic microwells, only GT1b and GD1a supported cell adhesion of MAG-transfected COS-1 cells. Furthermore, a quantitatively minor ganglioside expressed on cholinergic neurons, GQ1b alpha (also known as Chol-1 alpha-b), was much more potent than GT1b or GD1a in supporting MAG-mediated cell adhesion. Adhesion to either GT1b or GQ1b alpha was abolished by pretreatment of the adsorbed gangliosides with neuraminidase. On the basis of structure-function studies of 19 test glycosphingolipids, an alpha 2,3-N-acetylneuraminic acid residue on the terminal galactose of a gangliotetraose core is necessary for MAG binding, and additional sialic acid residues linked to the other neutral core saccharides [Gal(II) and GalNAc(III)] contribute significantly to binding affinity. MAG-mediated adhesion to gangliosides was blocked by pretreatment of the MAG-transfected COS-1 cells with anti-MAG monoclonal antibody 513, which is known to inhibit oligodendrocyte-neuron binding. These data are consistent with the conclusion that MAG-mediated cell-cell interactions involve MAG-ganglioside recognition and binding.

Impact of laparoscopic cholecystectomy on surgical training.

All cholecystectomies in a single health district were studied during a 5-year period spanning the introduction of laparoscopic cholecystectomy (LC). The number of LCs increased from 2 (1.3%) in year 3 to 86 (56%) in year 5. The number of operative cholangiograms and explorations of the common bile duct performed both fell substantially. The age distribution did not change significantly during the study period, but the percentage of females undergoing cholecystectomy increased. The percentage of trainee operations remained constant in those Firms performing only open cholecystectomy (OC), but fell from 67% to 9% in those adopting LC. An increase in annual cholecystectomy rate was seen with the laparoscopic surgeons, with a corresponding fall for those surgeons performing only OC. There was a threefold increase in the percentage of operations performed privately from years 2 to 5, with 73% being laparoscopic in year 5. The consequences for training of the introduction of LC must be addressed.

The gene encoding murine alpha 1,3-galactosyltransferase is expressed in female germ cells but not in male germ cells.

An essential step in murine fertilization is the binding of acrosome-intact sperm to specific O-linked glycans on zona pellucida glycoprotein 3 (ZP3). While there is agreement on the primary role of O-linked glycans in sperm-ZP3 binding, there is a striking lack of consensus on both the terminal monosaccharide(s) required for a functional binding site and the cognate protein on the sperm cell surface that recognizes this glycan. Much current debate centers on the essential role of nonreducing terminal N-acetyl-glucosaminyl or alternatively, alpha-galactosyl residues, to form a functional sperm binding ligand. Relevant to this debate, we demonstrated that alpha 1,3-galactosyltransferase (alpha 3-GT), which adds nonreducing terminal alpha-galactosyl residues to glycans, is not expressed in murine spermatocytes or spermatids. The objectives of this study were to determine whether alpha 3-GT is expressed in female germ cells and to compare the pattern of expression of two other terminal glycosyltransferases, beta 1,4-galactosyltransferase (beta 4-GT) and alpha 2,6-sialyltransferase (alpha 6-ST), between male and female germ cells. Total RNA was isolated from growing oocytes obtained from 15-day-old animals, fully grown oocytes, and eggs as well as spermatogonia, spermatocytes, and spermatids. The presence of alpha 3-GT, beta 4-GT, and alpha 6-ST mRNAs was analyzed by an RT-PCR-based assay. Our data demonstrate that the alpha 3-GT gene is expressed in female germ cells, but not in male germ cells. In contrast, both beta 4-GT and alpha 6-ST are expressed during oogenesis and spermatogenesis. This differential expression of alpha 3-GT in female germ cells is consistent with the model of sperm-egg binding in which a nonreducing terminal alpha-galactosyl residue is required for a functional determinant on ZP3 and with our hypothesis that the biological significance for the suppression of alpha 3-GT expression in male germ cells is to prevent sperm-sperm aggregation.

Male germ cell expression of murine beta 4-galactosyltransferase. A 796-base pair genomic region, containing two cAMP-responsive element (CRE)-like elements, mediates male germ cell-specific expression in transgenic mice.

In murine somatic cells, transcription of the single gene encoding beta 4-galactosyltransferase results in two transcripts of 4.1 and 3.9 kilobases (kb), as a consequence of the use of two transcriptional start sites that are located on exon one separated by 200 base pairs (bp). In early male germ cell development, spermatogonia use only the 4.1-kb start site to yield a transcript that is identical to its somatic cell counterpart. As these cells enter meiosis, there is a switch from the use of this somatic cell start site to the exclusive use, beginning in pachytene spermatocytes, of a male germ cell-specific start site. Germ cell-specific transcripts are distinguished from their somatic counterparts by an additional approximately 560 nucleotides of 5'-untranslated sequence that is located immediately upstream and contiguous with the transcriptional start site defined for the 4.1-kb mRNA (Harduin-Lepers, A., Shaper, N.L., Mahoney, J.A., and Shaper, J.H. (1992) Glycobiology 2, 361-368). This observation predicts the use of a different upstream male germ cell-specific promoter. In this study we show that a 796-bp fragment containing 543 bp of genomic sequence upstream of the germ cell specific transcriptional start site and 253 bp of flanking downstream sequence, directs expression of the reporter gene, beta-galactosidase, exclusively to the pachytene spermatocytes and round spermatids of transgenic mice. This pattern of cell type-specific expression of the transgene is comparable with that of the endogenous beta 4-galactosyltransferase gene.

Characterization of two cis-regulatory regions in the murine beta 1,4-galactosyltransferase gene. Evidence for a negative regulatory element that controls initiation at the proximal site.

The beta 1,4-galactosyltransferase (beta 1,4-GT) gene is unusual in that it specifies two mRNAs in somatic cells of 3.9 and 4.1 kilobases (kb). These two transcripts arise as a consequence of initiation at two different sets of start sites that are separated by approximately 200 base pairs. Translation of each mRNA results in the predicted synthesis of two related protein isoforms that differ only in the length of their NH2-terminal cytoplasmic domain (Russo, R.N., Shaper, N. L., and Shaper, J.H. (1990) J. Biol. Chem. 265, 3324-3331). In this study we show that the cellular requirements for beta 1,4-GT correlate with the transcriptional start site used. In cells and tissues that express low transcript levels, the 4.1-kb transcriptional start site is apparently used exclusively. Increased transcription from the 4.1-kb start site plus low levels of transcription from the 3.9-kb start site result in the intermediate beta 1,4-GT transcript levels that are found in almost all somatic cell types. However, in mid- to late pregnant and lactating mammary glands very high transcript levels are observed, which correlate with the predominant use of the 3.9-kb transcriptional start site. To identify the cis-acting elements that regulate the use of the two transcriptional start sites, we constructed a series of beta 1,4-GT/CAT hybrids and carried out transient transfection assays using mouse L cells and Drosophila SL2 cells. These studies have delineated both a distal and proximal regulatory region just upstream of the 4.1- and 3.9-kb transcriptional start sites, respectively. In addition, a negative cis-acting regulatory region was identified that represses transcription from the proximal site. These results suggest a model of transcriptional regulation in which the distal region functions as a housekeeping promoter while the proximal region functions as a mammary cell-specific promoter. Differential initiation from the two promoters is a mechanism for regulation of beta 1,4-GT enzyme levels. The predictions from this model are consistent with the conclusion that both protein isoforms are functionally equivalent resident trans-Golgi membrane-bound enzymes.

Immunocytochemical localization of beta 1,4 galactosyltransferase in epithelial cells from bovine tissues using monoclonal antibodies.

Post-embedding immunocytochemistry was employed to investigate the distribution of UDP-galactose:N-acetylglucosamine galactosyltransferase (beta 1,4-GT) in epithelial cells from various bovine organs. Several well characterized monoclonal antibodies previously demonstrated to recognize distinct polypeptide epitopes within the primary structure of beta 1,4-GT were applied to thin sections from tissues embedded in Lowicryl K4M, followed by the protein A-gold technique. Immunoreactivity was observed in the Golgi apparatus of epithelial cells from intestine, thymus and trachea. No immunoreactivity was observed in other intracellular structures, including rough endoplasmic reticulum, nuclear envelope and goblet cell mucus droplets. Within the Golgi apparatus, the staining was restricted to several cisternae in the trans region, with most portions of the trans-Golgi network appearing unlabelled. However, in thymic epithelial-reticular cells trans-Golgi network portions resembling classical GERL elements were stained by the antibodies. Thus, although immunoreactivity was subcompartmentalized within the Golgi apparatus in all epithelial cell types examined, the extent of staining within the trans-Golgi network was variable. Immunoreactivity was not detected at the plasma membrane (ecto-galactosyl-transferase), except in the case of a subpopulation of tracheal cells that resemble brush cells. These results suggest that in the epithelial cells examined, the subcompartmental distribution of beta 1,4-GT within the Golgi apparatus is maintained across different types of epithelial cell organization. Moreover, no evidence for a general epithelial cell ecto-galactosyltransferase could be discerned with these reagents.