A dominant mutation to ricin resistance in Chinese hamster ovary cells induces UDP-GlcNAc:glycopeptide beta-4-N-acetylglucosaminyltransferase III activity

A biochemical basis for the LEC10 mutant phenotype of Chinese hamster ovary cells has been identified. Independent LEC10 mutants, originally selected for resistance to the toxicity of ricin, have been shown to exhibit reduced binding of 125I-ricin at the cell surface. Although this is indicative of structural changes in cell-surface carbohydrates, labeling of plasma membranes with galactose oxidase/[3H]borohydride revealed no significant differences between mutant and parental cells. Alterations in the carbohydrates synthesized by LEC10 cells were, however, resolved by lectin-affinity chromatography of glycopeptides from the G glycoprotein of vesicular stomatitis virus (VSV) grown in LEC10. LEC10/VSV glycopeptides contain a fraction which is not bound to concanavalin A-Sepharose but is strongly retarded on E-PHA (erythroagglutinin from Proteus vulgaris)-agarose. In contrast, CHO/VSV glycopeptides or those from a LEC 10 revertant (R.LEC 10/VSV) do not contain carbohydrates with these properties. High-field 1H NMR spectroscopy of the novel LEC10/VSV carbohydrates showed that they are complex, biantennary structures containing N-acetylglucosamine in beta(1,4)-linkage to the beta-linked core mannose residue. The presence of these structures correlates with the expression of the enzyme responsible for the addition of this "bisecting" GlcNAc residue, UDP-GlcNAc:glycopeptide beta-4-N-acetylglucosaminyltransferase III (GlcNAc-TIII). Parental Chinese hamster ovary cells and the LEC10 revertant possess no detectable GlcNAc-TIII activity. The combined evidence suggests that the LEC10 mutation induces the expression of the GlcNAc-TIII enzyme in Chinese hamster ovary cells.     

Two distinct UDP-galactose: 2-acetamido-2-deoxy-D-glucose 4 beta-galactosyltransferases in porcine trachea

In previous studies on glycosyltransferase activities in porcine trachea, we demonstrated the presence of two galactosyltransferases which transfer galactose from UDP-galactose to N-acetylglucosamine (Sheares, B.T. and Carlson, D.M. (1983) J. Biol. Chem. 258, 9893-9898). One enzyme, UDP-galactose:N-acetylglucosamine 3 beta-galactosyltransferase, synthesized galactosyl-beta 1,3-N-acetylglucosamine while the other, UDP-galactose:N-acetylglucosamine 4 beta-galactosyltransferase, synthesized galactosyl-beta 1,4-N-acetylglucosamine. A third galactosyltransferase has now been demonstrated utilizing a solubilized membrane preparation from pig trachea, which also synthesizes galactosyl-beta 1,4-N-acetylglucosamine as determined by gas-liquid chromatography and Diplococcus pneumoniae beta-galactosidase treatment. This new UDP-galactose:N-acetylglucosamine 4 beta-galactosyltransferase is distinct from the lactose synthetase A protein in that it does not bind to alpha-lactalbumin-agarose or to N-acetylglucosamine-agarose. The enzyme is separable from the UDP-galactose:N-acetylgalactosaminyl-mucin 3 beta-galactosyltransferase by affinity chromatography on asialo ovine submaxillary mucin adsorbed to DEAE-Sephacel. This newly discovered 4 beta-galactosyltransferase binds to UDP-hexanolamine-Sepharose and is partially separated from UDP-galactose:N-acetylglucosamine 3 beta-galactosyltransferase by Sephacryl S-200 gel filtration chromatography. Neither high concentrations of N-acetylglucosamine (200 mM) nor alpha-lactalbumin inhibits the incorporation of galactose into galactosyl-beta 1,4-N-acetylglucosamine by this enzyme.     

Ehrlich ascites tumor cell UDP-Gal:N-acetyl-D-glucosamine beta(1,4)-galactosyltransferase. Purification, characterization, and topography of the acceptor-binding site

A UDP-Gal:N-acetylglucosamine beta(1,4)-galactosyltransferase which catalyzes the synthesis of beta-D-Gal(1,4)-D-GlcNAc units has been purified 17,560-fold from Ehrlich tumor cells to apparent electrophoretic homogeneity. The enzyme appears to be a monomeric protein with Mr = 56,000-58,000. Enzymatic activity requires the presence of MnCl2, is stimulated by detergent, and exhibits a pH optimum at 6.9. The Km values for GlcNAc and UDP-Gal are 1.89 and 0.046 mM, respectively. The Ehrlich cell beta-galactosyltransferase acts efficiently on glycoproteins and glycolipids terminating in GlcNAc, but is inactive toward glycoconjugates possessing terminal GalNAc units. The oligosaccharides beta-D-GlcNAc(1,3)-D-Gal and beta-D-GlcNAc(1,3)[beta-D-GlcNAc(1,6)]-D-Gal are good acceptors for the beta-galactosyltransferase from Ehrlich cells, suggesting that the enzyme may participate in the biosynthesis of i/I structures. In addition, other linear and branched sugars presenting GlcNAc residues at their nonreducing termini also act as acceptors for the enzyme. The activity of Ehrlich cell beta-galactosyltransferase both in the presence and absence of alpha-lactalbumin has been studied using a series of derivatives of Glc and GlcNAc which were substituted at various positions of the pyranose ring. This study has provided a map of the molecular contacts necessary for enzymatic activity in the presence and in the absence of alpha-lactalbumin.     

UDP-GalNAc:NeuAc alpha 2,3Gal beta-R (GalNAc to Gal) beta 1,4-N-acetylgalactosaminyltransferase responsible for the Sda specificity in human colon carcinoma CaCo-2 cell line

The UDP-GalNAc:NeuAc alpha 2,3Gal beta-R (GalNAc to Gal) beta-1,4-N-acetylgalactosaminyltransferase (beta 1,4GalNAc-transferase) is the enzyme responsible for the addition of the immunodominant sugar of the Sda isto-blood group determinant. In humans the enzyme is mainly expressed in the large intestine. We screened nine human colorectal carcinoma cell lines (SW-948, SW-948 FL, SW-480, SW-48, SW-1417, COLO-205, LOVO, HT-29 and CaCo-2) in order to ascertain the occurrence of beta 1,4GalNAc-transferase. Only in CaCo-2 cells the glycosyltransferase was detected and the activity increased with the degree of enterocytic differentiation. Nevertheless, in highly differentiated CaCo-2 cells the activity was thirty times lower than that found in cells detached from normal colon mucosa. These results support the notion that the expression of beta 1,4GalNAc-transferase is a marker of the colonic cell maturation.     

Activation of two new alpha(1,3)fucosyltransferase activities in Chinese hamster ovary cells by 5-azacytidine.

Several mammalian alpha(1,3)fucosyltransferases (alpha[1,3]Fuc-T) that synthesize carbohydrates containing alpha(1,3)fucosylated lactosamine units have been identified. Although Chinese hamster ovary (CHO) cells do not express alpha(1,3)Fuc-T activity, the rare mutants LEC11 and LEC12, isolated after mutagenesis or DNA transfection, each express an alpha(1,3)Fuc-T that may be distinguished by several criteria. Two new CHO mutants possessing alpha(1,3)Fuc-T activity (LEC29 and LEC30) have now been isolated after treatment of a CHO cell population with 5-azacytidine (5-AzaC), ethylnitrosourea (ENU), or 5-AzaC followed by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Like LEC12, both mutants possess an N-ethylmaleimide-resistant alpha(1,3)Fuc-T activity that can utilize a variety of acceptors and both express the Lewis X (Lex) determinant (Gal beta[1,4](Fuc alpha[1,3])GlcNAc beta 1)) but not the sialyl alpha(2,3)Lex determinant on cell-surface carbohydrates. However, LEC29 and LEC30 may be distinguished from LEC11 and LEC12, as well as from each other, on the basis of their unique patterns of lectin resistance and their abilities to bind the VIM-2 monoclonal antibody that recognizes carbohydrates terminating in NeuNAc alpha(2,3)Gal beta(1,4)GlcNAc beta(1,3)Gal beta(1,4)(Fuc alpha[1,3])GlcNAc beta and also by the different in vitro substrate specificities and kinetic properties of their respective alpha(1,3)Fuc-T activities. The combined data provide good evidence that the LEC29 and LEC30 alpha(1,3)Fuc-Ts are novel transferases encoded by distinct gene products.     

Expression of UDP-N-acetylgalactosamine: beta-galactose beta 1,4-N-acetylgalactosaminyltransferase in functionally defined T-cell clones.

To measure UDP-N-acetylgalactosamine: beta-galactose beta 1,4-N-acetylgalactosaminyltransferase (beta 1,4-GalNActransferase) in crude cell and tissue extracts we designed an assay containing UDP-[3H]N-acetylgalactosamine as donor and biotinylated human glycophorin A as an acceptor. After incubation the labelled acceptor was separated by the use of avidin-agarose from extract-derived endogenous acceptors. This assay permitted one to measure specifically the beta 1,4-GalNActransferase in crude extracts. This glycosyltransferase has previously been shown to be involved in the biosynthesis of Vicia villosa (hairy winter vetch)-lectin (VV)-binding sites of the murine cytotoxic T-cell line B6.1. Since VV-binding sites are a distinct marker for the cytotoxic subclass of murine T-lymphocytes, we used this assay to determine enzyme levels in a panel of functionally defined murine T-cell clones. Non-cytolytic T-cell lines generally have low activity, whereas most cytotoxic lines have high levels of activity. However, one cytotoxic T-cell line does not express the enzyme, although it has large numbers of VV-binding sites. This suggests the existence of another type of VV-binding sites which is independent of the beta 1,4-GalNActransferase in some cytotoxic-T-lymphocyte lines. The enzyme was also assayed in a variety of other tissues and found to have a very high activity in the intestine but a low activity in most other tissues. This was in considerable contrast with the ubiquitously high expression of UDP-GalNAc:peptide alpha 1-GalNActransferase. Therefore, the beta 1,4-GalNActransferase seems to be regulated during differentiation.     

Transfection of a human alpha-(1,3)fucosyltransferase gene into Chinese hamster ovary cells. Complications arise from activation of endogenous alpha-(1,3)fucosyltransferases

In order to isolate a human gene encoding an alpha-(1,3)fucosyltransferase (alpha-(1,3)Fuc-T), genomic DNA from HL-60 cells was transfected by several methods into Chinese hamster ovary (CHO) cells. Colonies expressing alpha-(1,3)Fuc-T activity were identified by their ability to bind a monoclonal antibody (anti-SSEA-1) that recognizes the carbohydrate product of alpha-(1,3)Fuc-T action. CHO cells do not express alpha-(1,3)Fuc-T activity but contain at least two, silent alpha-(1,3)Fuc-T genes previously identified by their activation in the rare, dominant mutants LEC11 and LEC12. These CHO enzymes were shown to be distinguishable from the alpha-(1,3)Fuc-T activity of HL-60 cells by the latter's comparative inability to transfer fucose to paragloboside and fetuin. Based on these criteria, only 11 isolates from more than 70 putative transfectants examined were found to stably express an alpha-(1,3)Fuc-T activity typical of HL-60 cells. Genomic DNA from two of these isolates was used to generate five independent secondary transfectants with HL-60-like alpha-(1,3)Fuc-T activity. Southern analysis revealed a common DNA fragment that hybridized to an Alu probe in each secondary, providing evidence that a human alpha-(1,3)Fuc-T gene had been transfected. However, in all transfection experiments, isolates that expressed alpha-(1,3)Fuc-T activities similar to CHO-encoded enzymes were also obtained. Several lines of evidence indicated that these cells arose from activation of endogenous CHO alpha-(1,3)Fuc-T genes as a consequence of DNA transfection. These false positives complicated the identification of transfectants expressing a human alpha-(1,3)Fuc-T gene and represent an important consideration in experiments to transfect other glycosyltransferase genes.     

Glycomics Profiling of Chinese Hamster Ovary Cell Glycosylation Mutants Reveals N-Glycans of a Novel Size and Complexity

Identifying biological roles for mammalian glycans and the pathways by which they are synthesized has been greatly facilitated by investigations of glycosylation mutants of cultured cell lines and model organisms. Chinese hamster ovary (CHO) glycosylation mutants isolated on the basis of their lectin resistance have been particularly useful for glycosylation engineering of recombinant glycoproteins. To further enhance the application of these mutants, and to obtain insights into the effects of altering one specific glycosyltransferase or glycosylation activity on the overall expression of cellular glycans, an analysis of the N-glycans and major O-glycans of a panel of CHO mutants was performed using glycomic analyses anchored by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. We report here the complement of the major N-glycans and O-glycans present in nine distinct CHO glycosylation mutants. Parent CHO cells grown in monolayer versus suspension culture had similar profiles of N- and O-GalNAc glycans, although the profiles of glycosylation mutants Lec1, Lec2, Lec3.2.8.1, Lec4, LEC10, LEC11, LEC12, Lec13, and LEC30 were consistent with available genetic and biochemical data. However, the complexity of the range of N-glycans observed was unexpected. Several of the complex N-glycan profiles contained structures of m/z ∼13,000 representing complex N-glycans with a total of 26 N-acetyllactosamine (Galβ1–4GlcNAc)_n units. Importantly, the LEC11, LEC12, and LEC30 CHO mutants exhibited unique complements of fucosylated complex N-glycans terminating in Lewis^x and sialyl-Lewis^x determinants. This analysis reveals the larger-than-expected complexity of N-glycans in CHO cell mutants that may be used in a broad variety of functional glycomics studies and for making recombinant glycoproteins.     

Temporally specific involvement of cell surface beta-1,4 galactosyltransferase during mouse embryo morula compaction

Cell surface beta-1,4 galactosyltransferase (GalTase) is shown to mediate intercellular adhesions between embryonal carcinoma (EC) cells and specifically during late morula compaction in the preimplantation mouse embryo. Monospecific anti-GalTase IgG raised against affinity-purified bovine beta-1,4 GalTase recognizes F9 EC cell GalTase as judged by immunoprecipitation and inhibition of GalTase activity, as well as by immunoprecipitation of a single 52 kd metabolically labeled membrane protein. Anti-GalTase IgG inhibits cell adhesions between EC cells, dissociates compacted mouse morulae, and inhibits blastocyst formation. Anti-GalTase IgG specifically inhibits cell adhesions during late morula compaction, coincident with a peak of surface GalTase activity as determined by direct enzyme assay. On EC cells, GalTase activity can be proteolytically released from intact cells, and is localized by indirect immunofluorescence to areas of intercellular contact, consistent with its proposed role in cell adhesion. Beta-1,4 GalTase is the first cell adhesion molecule identified that participates during late morula compaction, subsequent to uvomorulin function.     

The GDP-fucose:N-acetylglucosaminide 3-alpha-L-fucosyltransferases of LEC11 and LEC12 Chinese hamster ovary mutants exhibit novel specificities for glycolipid substrates

Previous studies have shown that the GDP-fucose:N-acetylglucosaminide 3-alpha-L-fucosyltransferase (alpha (1,3) fucosyltransferase (Fuc-T)) activities expressed by the Chinese hamster ovary cell mutants LEC11 (Fuc-TI) and LEC12 (Fuc-TII) are different enzymes and indicated that Fuc-TI might act on sialylated lactosamine sequences (Campbell, C., and Stanley, P. (1984) J. Biol. Chem. 259, 11208-11214). In this paper we show that CSLEX-1, a monoclonal antibody specific for NeuNac alpha (2,3)Gal beta (1,4)(Fuc alpha (1,3))GlcNAc beta 1 sequences, bound to LEC11 cells but not to LEC12 cells. Direct evidence that Fuc-TI could act on sialylated substrates was sought with a series of glycolipid acceptors. Optimal assay conditions in crude cell extracts were determined with nLc4, a glycolipid which accepted fucose with both Fuc-TI and Fuc-TII to generate the Lex antigenic determinant. The two enzymes differed in their detergent sensitivities, pH optima, Mn2+ requirements, and apparent Km values for nLc4. When sialylated glycolipids were examined as substrates, Fuc-TI added fucose to IV3NeuNAcnLc4 but not to IV6NeuNAcnLc4, whereas Fuc-TII was unable to utilize either glycolipid as a substrate. Further studies showed that Fuc-TI and Fuc-TII possess novel specificities for glycolipids containing two lactosamine sequences as potential fucose acceptors. Fuc-TI exhibited good activities with VI3NeuNAcnLc6 and VI6NeuNAcnLc6 whereas Fuc-TII had very low activity with both substrates. Glycosidase digestions of the labeled products showed that Fuc-TI added fucose primarily to the internal N-acetylglucosamine of both glycolipids. The same preference for the internal N-acetylglucosamine was shown by Fuc-TI when nLc6 was the acceptor. In contrast, Fuc-TII preferred to transfer fucose to the external acceptor site of nLc6, consistent with the low activities of Fuc-TII with sialylated nLc6 derivatives. Thus the two enzymes preferentially add fucose to different N-acetylglucosamines in the same substrate, nLc6. This indicates that the biosynthetic pathway for fucosylation of polylactosamine sequences in glycolipids and glycoproteins will vary depending upon the particular alpha (1,3)fucosyltransferase present.     

Extraction of 11 beta-hydroxysteroid dehydrogenase from rat liver microsomes by detergents

In these studies our goal was to solubilize the microsomal enzyme, 11 beta-hydroxysteroid dehydrogenase (11-HSD) as the first step in its purification. Enzyme was extracted from rat liver microsomes with representative detergents (Zwittergents, Tritons, modified sterols). Oxidation-reduction (O-R) ratios of extracts varied with detergent used and ranged from 0.18 (CHAPS) to 3.8 (Zwittergent 3-14) relative to a ratio of 1.7 in intact microsomes. All detergents solubilized 11-HSD using lack of sedimentation during high speed centrifugation as criterion. With Triton DF-18 and Triton X-100, optimum extraction of 11-HSD occurred in the detergent-protein ratio range of 0.1 to 0.2 O-R ratios decreased with increased Triton X-100, but were constant as Triton DF-18 was varied. The pH optimum of enzyme extraction was 9 at a detergent-protein ratio of 0.05 and 7.5-8.0 at a ratio of 0.2. Sodium chloride increased enzyme extraction by detergents; in the absence of detergent, salt extracted protein, but not enzyme. In aqueous solution at 0 degrees C or -15 degrees C, microsomal 11-oxidation activity rose within 24 h, then decreased; reductase activity consistently decreased. Oxidation and reduction activities were inversely related in the microsomal bound enzyme. No relationship between these activities appeared in detergent-solubilized enzymes. Possible mechanisms to account for the unexpected behavior of this enzyme are discussed.     

Regulatory mutations in CHO cells induce expression of the mouse embryonic antigen SSEA-1

Two rare and dominant mutants of Chinese hamster ovary (CHO) cells, LEC11 and LEC12, express the mouse embryonic antigen SSEA-1. Parental CHO cells and the revertants, LEC11.R9 and LEC12.R10, do not express this antigen as detected by a sensitive radioimmunoassay with a monoclonal antibody to SSEA-1. The presence of the SSEA-1 determinant correlates with the apparent de novo expression of specific N-acetylglucosaminide alpha(1,3)fucosyltransferase activities not detected in parental or revertant cell extracts. Several differences in the enzymes substrate specificities and their products have been identified. The combined data suggest that LEC11 and LEC12 mutants result from regulatory mutations affecting different fucosyltransferase genes.     

Deficient uridine diphosphate-N-acetylglucosamine:glycoprotein N-acetylglucosaminyltransferase activity in a clone of Chinese hamster ovary cells with altered surface glycoproteins.

We have reported the isolation of a clone (termed 15B) of Chinese hamster ovary (CHO) cells which are deficient in certain plant lectin-binding sites and have decreased amounts of sialic acid, galactose, and N-acetylglucosamine in its membranes (Gottlieb et al. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 1078-1082). This study demonstrates that extracts of 15B cells, in contrast to the parent cell line, do not transfer N-acetylglucosamine residues from UDP-GlcNAc to certain glycopeptide and glycoprotein acceptors containing terminal nonreducing alpha-linked mannose residues. The decreased enzyme activity could not be accounted for by the presence of inhibitors, altered pH, or Mn2+ requirements of the glycosyltransferase or increased N-acetylglucosaminidase activity in the extracts. The finding that the 15B cell extracts have significant but reduced N-acetylglucosaminyltransferase activity toward a degraded orosomucoid acceptor suggests that these cells have a selective loss of one of several specific N-acetylglucosaminyltransferases which are present in the parent CHO cells. The sialyl- and galactosyltransferase activities of 15B and parent CHO cells are comparable. Parent CHO and 15B cells were grown in radioactive glucosamine to label the membrane glycoproteins. Solubilization of these glycoproteins and passage over a Ricinus communis agglutinin I (RCA I) Sepharose affinity column revealed that no labeled 15B glycoprotein material bound, whereas 50 percent of the CHO membrane glycoproteins bound and could be eluted with the haptene lactose, demonstrating that 15B cells are virtually devoid of membrane oligosacharides capable of binding to the RCA I lectin. The 15B membrane glycoproteins exhibited a marked shift toward glycoprotein species of lower molecular weight when examined by gel electrophoresis in sodium dodecyl sulfate. It is proposed that this shift in the mobility of the 15B membrane glycoproteins results from a decreased glycosylation of a number of membrane glycoproteins relative to their counterparts in CHO cells. The deficient N-acetylglucosaminyltransferase activity in 15B cells can account for the decreased glycosylation of the 15B cell membrane glycoproteins.     

Inhibition of chondroitin and heparan sulfate biosynthesis in Chinese hamster ovary cell mutants defective in galactosyltransferase I

We have isolated five Chinese hamster ovary cell mutants defective in galactosyltransferase I (UDP-D-galactose:xylose beta-1,4-D-galactosyltransferase) and studied the effect of p-nitrophenyl-beta-D-xyloside supplementation on glycosaminoglycan biosynthesis in the mutant cells. Assays of galactosyltransferase I showed that the mutants contained less than 2% of the enzyme activity present in wild-type cells, and enzyme activity was additive in mixtures of mutant and wild-type cell extracts, suggesting that the mutations most likely defined the structural gene encoding the enzyme. Cell hybridization studies showed that the mutations in all five strains were recessive and that the mutants belonged to the same complementation group. The mutants contained wild-type levels of xylosyltransferase (UDP-D-xylose:core protein (serine) beta-D-xylosyltransferase), lactose synthase (UDP-D-galactose:N-acetyl-glucosaminide beta-1,4-D-galactosyltransferase), and lactosylceramide synthase (UDP-D-galactose:glucosylceramide beta-1,4-D-galactosyltransferase). Their sensitivity to lectin-mediated cytotoxicity was virtually identical to that of the wild-type, indicating that there were no gross alterations in glycoprotein or glycolipid compositions. Anion-exchange high performance liquid chromatography of 35S-glycosaminoglycans from one of the galactosyltransferase I-deficient mutants showed a dramatic reduction in both heparan sulfate and chondroitin sulfate, demonstrating that galactosyltransferase I is responsible for the formation of both glycosaminoglycans in intact cells. Surprisingly, the addition of 1 mM-p-nitrophenyl-beta-D-xyloside, a substrate for galactosyltransferase I, restored glycosaminoglycan synthesis in mutant cells. This finding suggested that another galactosyltransferase, possibly lactose synthase, can transfer galactose to xylose in intact cells.     

Microplate diffusion assay for screening of beta-glucanase-producing microorganisms

A method is described to screen fungal strains rapidly for overexpression of extracellular beta-1,4-endoglucanase in the presence of high levels of sugar compounds. The semi-quantitative assay utilizes microplates in a 96-well format and an azurine dye covalently cross-linked (AZCL) chromogenic substrate. The digestion of AZCL-hydroxyethyl-beta-1,4-endoglucanase results in the release of a blue dye directly proportional to the amount of enzyme activity present in the sample. Sample absorbance was read at 590 nm. and the enzyme activity was determined by reference to a standard curve. The results from the microplate diffusion assay were similar to the results derived from the Ostazin Brilliant Red-hydroxyethyl cellulose assay. The technique described allowed the rapid comparison and screening beta-1,4-glucanase activity directly in spent fungal supernatant, from cultures grown in potato dextrose broth. The method could also be easily adapted for the screening of the presence of other activities such as beta-1,3-glucanase activity by using either AZCL-beta-glucan or AZCL-pachyman in place of the AZCL-hydroxyethyl-cellulose. This assay could be used to measure supernatant within an activity range of 0.1-2 U/mL