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.     

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.     

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.     

Independent Lec1A CHO glycosylation mutants arise from point mutations in N-acetylglucosaminyltransferase I that reduce affinity for both substrates. Molecular consequences based on the crystal structure of GlcNAc-TI

A key enzyme in regulating the maturation of N-linked glycans is UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GlcNAc-TI, EC 2.4.1.101). Lec1 CHO cells lack GlcNAc-TI activity and synthesize only the oligomannosyl class of N-glycans. By contrast, Lec1A CHO mutants have weak GlcNAc-TI activity due to the reduced affinity of GlcNAc-TI for both the UDP-GlcNAc and Man(5)GlcNAc(2)Asn substrates. Lec1A CHO mutants synthesize hybrid and complex N-glycans, albeit in reduced amounts compared to parental CHO cells. In this paper, we identify two point mutations that gave rise to the Lec1A phenotype in three independent Lec1A CHO mutants. The G634A mutation in Lec1A.2C converts an aspartic acid to an asparagine at amino acid 212, disrupting a conserved DXD motif (E(211)DD(213) in all GlcNAc-TIs) that makes critical interactions with bound UDP-GlcNAc and Mn(2+) ion in rabbit GlcNAc-TI. The C907T mutation in Lec1A.3E and Lec1A.5J converts an arginine conserved in all GlcNAc-TIs to a tryptophan at amino acid 303, altering interactions that are important in stabilizing a critical structural element in rabbit GlcNAc-TI. Correction of each mutation by site-directed mutagenesis restored their GlcNAc-TI activity and lectin binding properties to parental levels. The effect of the two amino acid changes on GlcNAc-TI catalysis is discussed in relation to the crystal structure of rabbit GlcNAc-TI complexed with manganese and UDP-GlcNAc.     

A subclass of cell surface carbohydrates revealed by a CHO mutant with two glycosylation mutations

A novel lectin-resistance phenotype was displayed by a LEC10 Chinese hamster ovary (CHO) cell mutant that was selected for resistance to the erythroagglutinin, E-PHA. Biochemical and genetic analyses revealed that the phenotype results from the expression of two glycosylation mutations, LEC10 and lec8. The LEC10 mutation causes the appearance of N-acetylglucosaminyltransferase III (GlcNAc-TIII) activity and the production of N-linked carbohydrates with a bisecting GlcNAc residue. The lec8 mutation inhibits translocation of UDP-Gal into the Golgi lumen and thereby dramatically reduces galactosylation of all glycoconjugates. This reduction in galactose addition does not, however, cause Lec8 mutants to be very resistant to the galactose-binding lectin, ricin. By contrast, the double mutant LEC10.Lec8 behaved like a LEC10 mutant and was highly resistant to ricin. Based on structural studies of cellular glycopeptides as well as glycopeptides of the G glycoprotein of vesicular stomatitis virus grown in mutant cells, it appears that the ricin resistance of LEC10.Lec8 cells is due to the presence of a small number of Gal residues on branched, N-linked carbohydrates that also carry the bisecting GlcNAc residue. Labelling of N-linked cellular carbohydrates with [3H]galactose was found to occur at a low level for a wide spectrum of cellular glycoproteins in independent Lec8 mutants. Studies of the LEC10.Lec8 mutant have, therefore, led to the identification of a subset of structures that are acceptors for Gal when intra-Golgi UDP-Gal levels are limiting. This mutant also illustrates the potential for regulating cell surface recognition by carbohydrate-binding proteins by altering the expression of a single glycosyltransferase such as GlcNAc-TIII.     

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.     

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.     

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.     

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.     

Truncated, inactive N-acetylglucosaminyltransferase III (GlcNAc-TIII) induces neurological and other traits absent in mice that lack GlcNAc-TIII

N-Acetylglucosaminyltransferase III (GlcNAc-TIII), the product of the Mgat3 gene, transfers the bisecting GlcNAc to the core mannose of complex N-glycans. The addition of this residue is regulated during development and has functional consequences for receptor signaling, cell adhesion, and tumor progression. Mice homozygous for a null mutation at the Mgat3 locus (Mgat3(Delta)) or for a targeted mutation in the Mgat3 gene (previously called Mgat3(neo), but herein renamed Mgat3(T37) because the allele generates inactive GlcNAc-TIII of approximately 37 kDa) were found to exhibit retarded progression of liver tumors. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of neutral N-glycans from kidneys revealed no significant differences, and both mutants showed the expected lack of N-glycan species with an additional GlcNAc. However, the two mutants differed in several biological traits. Mgat3(T37/T37) homozygotes in a mixed or 129(SvJ) background were retarded in growth rate and exhibited an altered leg clasp reflex, an altered gait, and defective nursing behavior. Pups abandoned by Mgat3(T37/T37) mothers were rescued by wild-type foster mothers. None of these Mgat3(T37/T37) traits were exhibited by Mgat3(Delta/Delta) mice or by heterozygous mice carrying the Mgat3(T37) mutation. Similarly, no dominant-negative effect was observed in Chinese hamster ovary cells expressing truncated GlcNAc-TIII in the presence of wild-type GlcNAc-TIII. However, compound heterozygotes carrying both the Mgat3(T37) and Mgat3(Delta) mutations exhibited a marked leg clasp reflex, indicating that in the absence of wild-type GlcNAc-TIII, truncated GlcNAc-TIII causes this phenotype. The Mgat3 gene was expressed in brain at embryonic day 10.5 and thereafter and in neurons of adult cerebellum. The mutant Mgat3 gene was also highly expressed in Mgat3(T37/T37) brain. This may be the basis of the unexpected neurological phenotype induced by truncated, inactive GlcNAc-TIII in the mouse.     

cea-kil operon of the ColE1 plasmid.

We isolated a series of Tn5 transposon insertion mutants and chemically induced mutants with mutations in the region of the ColE1 plasmid that includes the cea (colicin) and imm (immunity) genes. Bacterial cells harboring each of the mutant plasmids were tested for their response to the colicin-inducing agent mitomycin C. All insertion mutations within the cea gene failed to bring about cell killing after mitomycin C treatment. A cea- amber mutation exerted a polar effect on killing by mitomycin C. Two insertions beyond the cea gene but within or near the imm gene also prevented the lethal response to mitomycin C. These findings suggest the presence in the ColE1 plasmid of an operon containing the cea and kil genes whose product is needed for mitomycin C-induced lethality. Bacteria carrying ColE1 plasmids with Tn5 inserted within the cea gene produced serologically cross-reacting fragments of the colicin E1 molecule, the lengths of which were proportional to the distance between the insertion and the promoter end of the cea gene.     

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.