Lec1A Chinese hamster ovary cell mutants appear to arise from a structural alteration in N-acetylglucosaminyltransferase I

The biochemical and kinetic properties of UDP-GlcNAc:alpha-D-mannoside (GlcNAc to Man alpha 1,3) beta 1,2-N-acetylglucosaminyltransferase I (GlcNAc-TI) have been investigated in the Chinese hamster ovary glycosylation mutant Lec1A. Previous studies showed that, whereas Lec1A cells synthesize complex carbohydrates at levels consistent with partial GlcNAc-TI action, no GlcNAc-TI activity was detected in Lec1A cell-free extracts (Stanley, P., and Chaney, W. (1985) Mol. Cell. Biol. 5, 1204-1211). It is now reported that, under altered reaction conditions, GlcNAc-TI activity can be measured in Lec1A cell extracts. The GlcNAc-TI enzyme in Lec1A.2C has a pH optimum of 7.5 (compared with 6.25 for the parental enzyme) and apparent Km values for Man5GlcNAc2Asn and UDP-GlcNAc that are, respectively, 21- and 44-fold higher than the apparent Km values of GlcNAc-TI from parental Chinese hamster ovary cells. Two independent Lec1A mutants possess GlcNAc-TI activities with similarly altered biochemical and kinetic properties. In fact, under optimal assay conditions for each cell line, the level of GlcNAc-TI in Lec1A extracts is equal to that of parental Chinese hamster ovary cell extracts. Interestingly, the two glycosylation sites of the G glycoprotein of vesicular stomatitis virus are processed quite differently in Lec1A cells. The glycopeptide nearest the carboxyl-terminal appears to be a preferred substrate for the Lec1A GlcNAc-TI activity. The combined data suggest that the Lec1A mutation affects the gene that codes for GlcNAc-TI, giving rise to a structurally altered glycosyltransferase with different biochemical properties.     

Control of carbohydrate processing: the lec1A CHO mutation results in partial loss of N-acetylglucosaminyltransferase I activity.

Lec1 CHO cell glycosylation mutants are defective in N-acetylglucosaminyltransferase I (GlcNAc-TI) activity and therefore cannot convert the oligomannosyl intermediate (Man5GlcNAc2Asn) into complex carbohydrates. Lec1A CHO cell mutants have been shown to belong to the same genetic complementation group but exhibit different phenotypic properties. Evidence is presented that lec1A represents a new mutation at the lec1 locus resulting in partial loss of GlcNAc-TI activity. Structural studies of the carbohydrates associated with vesicular stomatitis virus grown in Lec1A cells (Lec1A/VSV) revealed the presence of biantennary and branched complex carbohydrates as well as the processing intermediate Man5GlcNAc2Asn. By contrast, the glycopeptides from virus grown in CHO cells (CHO/VSV) possessed only fully processed complex carbohydrates, whereas those from Lec1/VSV were almost solely of the Man5GlcNAc2Asn intermediate type. Therefore, the Lec1A glycosylation phenotype appears to result from the partial processing of N-linked carbohydrates because of reduced GlcNAc-TI action on membrane glycoproteins. Genetic experiments provided evidence that lec1A is a single mutation affecting GlcNAc-TI activity. Lec1A mutants could be isolated at frequencies of 10(-5) to 10(-6) from unmutagenized CHO cell populations by single-step selection, a rate inconsistent with two mutations. In addition, segregants selected from Lec1A X parental cell hybrid populations expressed only Lec1A or related lectin-resistant phenotypes and did not include any with a Lec1 phenotype. The Lec1A mutant should be of interest for studies on the mechanisms that control carbohydrate processing in animal cells and the effects of reduced GlcNAc-TI activity on the glycosylation, translocation, and compartmentalization of cellular glycoproteins.     

Lec3 Chinese hamster ovary mutants lack UDP-N-acetylglucosamine 2-epimerase activity because of mutations in the epimerase domain of the Gne gene

Lec3 Chinese hamster ovary (CHO) cell glycosylation mutants have a defect in sialic acid biosynthesis that is shown here to be reflected most sensitively in reduced polysialic acid (PSA) on neural cell adhesion molecules. To identify the genetic origin of the phenotype, genes encoding different factors required for sialic acid biosynthesis were transfected into Lec3 cells. Only a Gne cDNA encoding UDP-GlcNAc 2-epimerase:ManNAc kinase rescued PSA synthesis. In an in vitro UDP-GlcNAc 2-epimerase assay, Lec3 cells had no detectable UDP-GlcNAc 2-epimerase activity, and Lec3 cells grown in serum-free medium were essentially devoid of sialic acid on glycoproteins. The Lec3 phenotype was rescued by exogenously added N-acetylmannosamine or mannosamine but not by the same concentrations of N-acetylglucosamine, glucosamine, glucose, or mannose. Sequencing of CHO Gne cDNAs identified a nonsense (E35stop) and a missense (G135E) mutation, respectively, in two independent Lec3 mutants. The G135E Lec3 mutant transfected with a rat Gne cDNA had restored in vitro UDP-GlcNAc 2-epimerase activity and cell surface PSA expression. Both Lec3 mutants were similarly rescued with a CHO Gne cDNA and with CHO Gne encoding the known kinase-deficient D413K mutation. However, cDNAs encoding the known epimerase-deficient mutation H132A or the new Lec3 G135E Gne mutation did not rescue the Lec3 phenotype. The G135E Gne missense mutation is a novel mechanism for inactivating UDP-GlcNAc 2-epimerase activity. Lec3 mutants with no UDP-GlcNAc 2-epimerase activity represent sensitive hosts for characterizing disease-causing mutations in the human GNE gene that give rise to sialuria, hereditary inclusion body myopathy, and Nonaka myopathy.     

Transfection of a human gene that corrects the Lec1 glycosylation defect: evidence for transfer of the structural gene for N-acetylglucosaminyltransferase I.

Chinese hamster ovary (CHO) glycosylation mutants provide an approach to cloning mammalian glycosyltransferases by transfection and gene rescue. In this paper, complementation of the lec1 CHO mutation by human DNA is described. Lec1 transfectants expressed human N-acetylglucosaminyltransferase I (GlcNAc-TI) activity and possessed common human DNA fragments. Cloning of GlcNAc-TI should therefore be possible.     

DNA-mediated transformation of N-acetylglucosaminyltransferase I activity into an enzyme deficient cell line

N-acetylglucosaminyltransferase I (GlcNAc-TI) catalyzes the first reaction in the conversion of ASN-linked cell surface oligosaccharides from a mannose-terminating structure to more complex carbohydrate structures. The mutant Chinese hamster ovary (CHO) cell line, Lec1, is deficient in this enzyme and, therefore, shows increased sensitivity to the lectin, Concanavalin A, which binds to the mannose-terminating oligosaccharides that accumulate on Lec1 cell surface glycoproteins. Spontaneous revertants of the Lec1 phenotype have never been observed. We report here the isolation of stable revertants of Lec1 cells to the parental CHO cell lectin-resistance phenotype after DNA-mediated transformation with human DNA. Both primary and secondary transformants express varying levels of GlcNAc-TI enzyme activity which was stable even when the cells were cultured in nonselective conditions. Human alu repeat DNA sequences are present in the primary transformants, but these sequences could not be detected in the secondary transformants.     

The Lec23 Chinese hamster ovary mutant is a sensitive host for detecting mutations in alpha-glucosidase I that give rise to congenital disorder of glycosylation IIb (CDG IIb)

Lec23 Chinese hamster ovary cells are defective in alpha-glucosidase I activity, which removes the distal alpha(1,2)-linked glucose residue from Glc(3)Man(9)GlcNAc(2) moieties attached to glycoproteins in the endoplasmic reticulum. Mutations in the human GCS1 gene give rise to the congenital disorder of glycosylation termed CDG IIb. Lec23 mutant cells have been shown to alter lectin binding and to synthesize predominantly oligomannosyl N-glycans on endogenous glycoproteins. A single point mutation (TCC to TTC; Ser to Phe) was identified in Lec23 Gcs1 cDNA and genomic DNA. Serine at the analogous position is highly conserved in all GCS1 gene homologues. A human GCS1 cDNA reverted the Lec23 phenotype, whereas GCS1 cDNA carrying the lec23 mutation (S440F in human) did not. By contrast, GCS1 cDNA with an R486T or F652L CDG IIb mutation gave substantial rescue of the Lec23 phenotype. Nevertheless, in vitro assays of each enzyme gave no detectable alpha-glucosidase I activity. Clearly the R486T and F652L GCS1 mutations are only mildly debilitating in an intact cell, whereas the S440F mutation largely inactivates alpha-glucosidase I both in vitro and in vivo. However, the S440F alpha-glucosidase I may have a small amount of alpha-glucosidase I activity in vivo based on the low levels of complex N-glycans in Lec23. A sensitive test for complex N-glycans showed the presence of polysialic acid on the neural cell adhesion molecule. The Lec23 Chinese hamster ovary mutant represents a sensitive host for detecting a wide range of mutations in human GCS1 that give rise to CDG IIb.     

Isolation of new CHO cell mutants defective in CMP-sialic acid biosynthesis and transport

Sialic acid is a sugar typically found at the N-glycan termini of glycoproteins in mammalian cells. Lec3 CHO cell mutants are deficient in epimerase activity, due to a defect in the gene that encodes a bifunctional UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). Sialic acid modification on the cell surface is partially affected in these cells. We have mutagenized Lec3 CHO cells and isolated six mutants (termed C2m) deficient in the cell surface expression of polysialic acid (PSA). Mutant C2m9 was partially defective in expression of cell-surface PSA and wheat germ agglutinin (WGA) binding, while in the other five mutants, both cell-surface PSA and WGA binding were undetectable. PSA expression was restored by complementation with the gene encoding the CMP-sialic acid transporter (CST), indicating that CST mutations were responsible for the phenotypes of the C2m cells. We characterized the CST mutations in these cells by Northern blotting and RT-PCR. C2m9 and C2m45 carried missense mutations resulting in glycine to glutamate substitutions at amino acids 217 (G217E) and 256 (G256E), respectively. C2m13, C2m39 and C2m31 had nonsense mutations that resulted in decreased CST mRNA stability, and C2m34 carried a putative splice site mutation. PSA and CD15s expression in CST-deficient Lec2 cells were partially rescued by G217E CST, but not by G256E CST, although both proteins were expressed at similar levels, and localized to the Golgi. These results indicate that the novel missense mutations isolated in this study affect CST activity.     

Mouse large can modify complex N- and mucin O-glycans on alpha-dystroglycan to induce laminin binding

The human LARGE gene encodes a protein with two putative glycosyltransferase domains and is required for the generation of functional alpha-dystroglycan (alpha-DG). Monoclonal antibodies IIH6 and VIA4-1 recognize the functional glycan epitopes of alpha-DG that are necessary for binding to laminin and other ligands. Overexpression of full-length mouse Large generated functionally glycosylated alpha-DG in Pro(-5) Chinese hamster ovary (CHO) cells, and the amount was increased by co-expression of protein:O-mannosyl N-acetylglucosaminyltransferase 1. However, functional alpha-DG represented only a small fraction of the alpha-DG synthesized by CHO cells or expressed from an alpha-DG construct. To identify features of the glycan epitopes induced by Large, the production of functionally glycosylated alpha-DG was investigated in several CHO glycosylation mutants. Mutants with defective transfer of sialic acid (Lec2), galactose (Lec8), or fucose (Lec13) to glycoconjugates, and the Lec15 mutant that cannot synthesize O-mannose glycans, all produced functionally glycosylated alpha-DG upon overexpression of Large. Laminin binding and the alpha-DG glycan epitopes were enhanced in Lec2 and Lec8 cells. In Lec15 cells, functional alpha-DG was increased by co-expression of core 2 N-acetylglucosaminyltransferase 1 with Large. Treatment with N-glycanase markedly reduced functionally glycosylated alpha-DG in Lec2 and Lec8 cells. The combined data provide evidence that Large does not transfer to Gal, Fuc, or sialic acid on alpha-DG nor induce the transfer of these sugars to alpha-DG. In addition, the data suggest that human LARGE may restore functional alpha-DG to muscle cells from patients with defective synthesis of O-mannose glycans via the modification of N-glycans and/or mucin O-glycans on alpha-DG.     

The Lec4A CHO glycosylation mutant arises from miscompartmentalization of a Golgi glycosyltransferase

Two CHO glycosylation mutants that were previously shown to lack N-linked carbohydrates with GlcNAc beta 1,6Man alpha 1,6 branches, and to belong to the same genetic complementation group, are shown here to differ in the activity of N-acetylglucosaminyltransferase V (GlcNAc-TV) (UDP-GlcNA: alpha 1,6mannose beta-N-acetylglucosaminyltransferase V). One mutant, Lec4, has no detectable GlcNAc-TV activity whereas the other, now termed Lec4A, has activity equivalent to that of parental CHO in detergent cell extracts. However, Lec4A GlcNAc-TV can be distinguished from CHO GlcNAc-TV on the basis of its increased sensitivity to heat inactivation and its altered subcellular compartmentalization. Sucrose density gradient fractionation shows that the major portion of GlcNAc-TV from Lec4A cells cofractionates with membranes of the ER instead of Golgi membranes where GlcNAc-TV is localized in parental CHO cells. Other experiments show that Lec4A GlcNAc-TV is not concentrated in lysosomes, or in a post-Golgi compartment, or at the cell surface. The altered localization in Lec4A cells is specific for GlcNAc-TV because two other Lec4A Golgi transferases cofractionate at the density of Golgi membranes. The combined data suggest that both lec4 and lec4A mutations affect the structural gene for GlcNAc-TV, causing either the loss of GlcNAc-TV activity (lec4) or its miscompartmentalization (lec4A). The identification of the Lec4A defect indicates that appropriate screening of different glycosylation-defective mutants should enable the isolation of other mammalian cell trafficking mutants.     

Two Chinese hamster ovary glycosylation mutants affected in the conversion of GDP-mannose to GDP-fucose

A biochemical basis for the pea and lentil lectin resistance of two Chinese hamster ovary (CHO) cell mutants, Lec13 and Lec13A, was investigated. Studies of the G glycopeptides of vesicular stomatitis virus grown in the mutants indicated that Lec13 cells essentially lack the ability to add fucose to complex carbohydrates while Lec13A cells synthesize significant proportions of fucosylated, complex moieties. However, both mutants were known to be reverted to lectin sensitivity by growth in L-fucose, making them similar to the mouse lymphoma mutant, PLR1.3, which is defective in the conversion of GDP-mannose to GPD-fucose [M. L. Reitman, I. S. Trowbridge, and S. Kornfeld (1980) J. Biol. Chem. 255, 9900-9906]. Optimal conditions for the production of GDP-fucose from GDP-mannose by CHO cytosol were found to occur at pH 8 in the presence of 7.5 microM GDP-mannose, 15 mM Mg2+, 0.2 mM NAD+, 0.2 mM NADPH, 10 mM niacinamide, 5 mM ATP, and 50 mM Tris-HCl. Under these conditions, Lec13 cytosol produced no detectable GDP-fucose nor GDP-sugar intermediates while Lec13A cytosol produced significant quantities of both. Mixing experiments with Lec13 cytosol identified the first enzyme of the conversion pathway (GDP-mannose 4,6-dehydratase, EC 4.2.1.47) as the site of the block. In addition to being markedly reduced, the Lec13A 4,6-dehydratase activity was relatively insensitive to changes in pH in comparison to the activity in parental cytosol, suggesting that Lec13A cells might possess a structurally altered GDP-mannose 4,6-dehydratase enzyme.     

A single point mutation resulting in an adversely reduced expression of DPM2 in the Lec15.1 cells

Mammalian dolichol-phosphate-mannose (DPM) synthase consists of three subunits, DPM1, DPM2, and DPM3. Lec15.1 Chinese hamster ovary cells are deficient in DPM synthase activity. The present paper reports that DPM1 cDNA from wild type and Lec15.1 CHO cells were found to be identical, and transfection with CHO DPM1 cDNA did not reverse the Lec15.1 phenotype. Neither did a chimeric cDNA containing the complete hamster DPM1 open reading frame fused to the Saccharomyces cerevisiae DPM1 C-terminal transmembrane domain. In contrast, Lec15.1 cells were found to have a single point mutation G29A within the coding region of the DPM2 gene, resulting in a glycine to glutamic acid change in amino acid residue 10 of the peptide. Moreover, mutant DPM2 cDNA expressed a drastically reduced amount of DPM2 protein and poorly corrects the Lec15.1 cell phenotype when compared with wild type CHO DPM2 cDNA (G(29) form).     

Characterization of the galactose-specific binding activity of a purified soluble Entamoeba histolytica adherence lectin.

We studied galactose (Gal)-specific binding of the soluble purified 260-kDa Entamoeba histolytica adherence protein to glycosylation deficient Chinese hamster ovary (CHO) cell mutants. Our goal was to further define the lectin's functional activity and carbohydrate receptor specificity. The adherence protein was purified by acid elution from an immunoaffinity column; however, exposure of the surface membrane lectin on viable trophozoites to identical acid pH conditions had no effect on carbohydrate binding activity. Saturable Gal-specific binding of soluble lectin to parental CHO cells was demonstrated at 4 degrees C by radioimmunoassay; the dissociation coefficient (Kd) was 2.39 x 10(-8) M-1 with 5.97 x 10(4) lectin receptors present per CHO cell. Gal-specific binding of lectin to Lec2 CHO cell mutants, which have increased N- and O-linked terminal Gal residues on cell surface carbohydrates, was increased due to an enhanced number of receptors (2.41 x 10(5)/cell) rather than a significantly reduced dissociation constant (4.93 x 10(-8) M-1). At 4 degrees C, there was no measurable Gal-specific binding of the adherence protein to the Lec1 and 1dlD.Lec1 CHO mutants, which contain surface carbohydrates deficient in terminal Gal residues. Binding of lectin (20 micrograms/ml) to CHO cells was equivalent at 4 degrees C and 37 degrees C and unaltered by adding the microfilament inhibitor, Cytochalasin D (10 micrograms/ml). Gal-specific binding of the lectin at 4 degrees C was calcium independent and reduced by 81% following adsorption of only 0.2% of the lectin to CHO cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased tumorigenicity correlates with expression of altered cell surface carbohydrates in Lec9 CHO cells.

To investigate a role for surface carbohydrates in cellular malignancy, 15 different glycosylation-defective CHO cell mutants were examined for their tumorigenic and metastatic capacities after subcutaneous injection into nude mice. Most of the glycosylation mutants displayed similar or slightly decreased tumorigenicity compared with parental CHO cells. Neither parental CHO cells nor any of the mutants were observed to metastasize. However, independent isolates of one mutant type, Lec9, showed a dramatic reduction in tumor formation. The altered carbohydrates expressed at the surface of Lec9 cells appeared to be responsible for their loss of tumorigenicity, because revertants for lectin resistance were able to form tumors, and a double mutant (Lec9.Lec1) that expressed a Lec1 glycosylation phenotype also formed tumors. Finally, Lec9 cells were able to form tumors in gamma-irradiated nude mice, suggesting that recognition by an irradiation-sensitive host cell(s) was responsible for their reduced tumorigenicity in untreated nude mice.     

Co-transformation of Lec 1 CHO cells with N-acetylglucosaminyltransferase 1 activity and a selectable marker

In animal cells, the enzyme alpha(1,3)-mannoside-beta(1,2)-N-acetylglucosaminyltransferase I (GlcNAc-TI, EC.2.4.1.101) catalyzes the addition of N-acetylglucosamine to the ASN-linked Man GlcNAc oligosaccharide. The Chinese hamster ovary (CHO) mutant cell line Lec1 is deficient in this enzyme activity and, therefore, accumulates mannose-terminating cell surface ASN-linked oligosaccharides. Consequently, Lec1 cells are sensitive to the cytotoxic effects of the mannose-binding lectin Concanavalin A (Con A). Lec1 cells were co-transformed with human DNA from A431 cells and eukaryotic expression plasmids containing the bacterial neo gene by calcium phosphate/DNA-mediated transformation. Co-transformants were selected for resistance to Con A and G-418. DNA from a primary co-transformant was purified and used to transform Lec1 cells, resulting in secondary co-transformants. Both primary and secondary co-transformants exhibited in vitro GlcNAc-TI-specific enzyme activity. DNA gel blot analysis indicated that secondary co-transformants contained both human and neo sequences.     

Molecular cloning and characterization of cDNA coding for beta1, 2N-acetylglucosaminyltransferase I (GlcNAc-TI) from Nicotiana tabacum.

In plants as well as in animals beta1, 2N-acetylglucosaminyltransferase I (GlcNAc-TI) is a Golgi resident enzyme that catalyzes an essential step in the biosynthetic pathway leading from oligomannosidic N-glycans to complex or hybrid type N-linked oligosaccharides. Employing degenerated primers deduced from known GlcNAc-TI genes from animals, we were able to identify the cDNA coding for GlcNAc-TI from a Nicotiana tabacum cDNA library. The complete nucleotide sequence revealed a 1338 base pair open reading frame that codes for a polypeptide of 446 amino acids. Comparison of the deduced amino acid sequence with that of already known GlcNAc-TI polypeptides revealed no similarity of the tobacco clone within the putative cytoplasmatic, transmembrane, and stem regions. However, 40% sequence similarity was found within the putative C-terminal catalytic domain containing conserved single amino acids and peptide motifs. The predicted domain structure of the tobacco polypeptide is typical for type II transmembrane proteins and comparable to known GlcNAc-TI from animal species. In order to confirm enzyme activity a truncated form of the protein containing the putative catalytic domain was expressed using a baculovirus/insect cell system. Using pyridylaminated Man(5)- or Man(3)GlcNAc(2)as acceptor substrates and HPLC analysis of the products GlcNAc-TI activity was shown. This demonstrates that the C-terminal region of the protein comprises the catalytic domain. Expression of GlcNAc-TI mRNA in tobacco leaves was detected using RT-PCR. Southern blot analysis gave two hybridization signals of the gene in the amphidiploid genomes of the two investigated species N. tabacum and N.benthamiana.     

Reduction in Golgi apparatus dimension in the absence of a residential protein,N-acetylglucosaminyltransferase V

Various proteins are involved in the generation and maintenance of the membrane complex known as the Golgi apparatus. We have used mutant Chinese hamster ovary (CHO) cell lines Lec4 and Lec4A lacking N-acetylglucosaminyltransferase V (GlcNAcT-V, MGAT5) activity and protein in the Golgi apparatus to study the effects of the absence of a single glycosyltransferase on the Golgi apparatus dimension. Quantification of immunofluorescence in serial confocal sections for Golgi α-mannosidase II and electron microscopic morphometry revealed a reduction in Golgi volume density up to 49 % in CHO Lec4 and CHO Lec4A cells compared to parental CHO cells. This reduction in Golgi volume density could be reversed by stable transfection of Lec4 cells with a cDNA encoding Mgat5. Inhibition of the synthesis of β1,6-branched N-glycans by swainsonine had no effect on Golgi volume density. In addition, no effect on Golgi volume density was observed in CHO Lec1 cells that contain enzymatically active GlcNAcT-V, but cannot synthesize β1,6-branched glycans due to an inactive GlcNAcT-I in their Golgi apparatus. These results indicate that it may be the absence of the GlcNAcT-V protein that is the determining factor in reducing Golgi volume density. No dimensional differences existed in cross-sectioned cisternal stacks between Lec4 and control CHO cells, but significantly reduced Golgi stack hits were observed in cross-sectioned Lec4 cells. Therefore, the Golgi apparatus dimensional change in Lec4 and Lec4A cells may be due to a compaction of the organelle.