Lec32 is a new mutation in Chinese hamster ovary cells that essentially abrogates CMP-N-acetylneuraminic acid synthetase activity.

LEC29.Lec32 is a glycosylation mutant that was isolated from a selection of mutagenized Chinese hamster ovary (CHO) cells for lectin resistance. Compared with LEC29 CHO cells, the double mutant exhibited an unusually high sensitivity to the toxic lectin, ricin, indicating increased exposure of galactose residues on cell surface carbohydrates. Structural analysis of LEC29.Lec32 cellular glycoproteins showed a nearly complete lack of sialic acid residues. Genetic analysis demonstrated that the lec32 mutation is recessive and novel. Biochemical analysis showed that the mutant cells contained less than 5% of the cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) present in parental CHO cells (1.6 nmol/mg of cell protein). A sensitive radiochemical assay used to measure CMP-NeuAc synthetase activity showed that the properties of this enzyme in parental CHO cells were essentially identical to those of CMP-NeuAc synthetase in various mammalian tissues. However, no CMP-NeuAc synthetase activity was detected in LEC29.Lec32 extracts. Mixing experiments provided no evidence for an inhibitor in the mutant CHO cells, and two revertants, which expressed only the LEC29 phenotype, had normal CMP-NeuAc synthetase levels. The combined evidence indicates that the lec32 mutation resides in either the structural gene encoding CMP-NeuAc synthetase or in a gene that regulates the production of active enzyme.

A KB cell mutant lacking a sialyltransferase activity has been isolated by selecting for resistance to the cytotoxic effects of ultraviolet-inactivated Sendai virus (13), and numerous attempts have been made to obtain additional mammalian cell mutants, defective in any of the above steps, by selection for resistance to the sialic acid-binding and cytotoxic plant lectin, wheat germ agglutinin (WGA) (12, 14 -17). This latter strategy has primarily yielded mutants with defects in CMP-NeuAc transport, including three different Chinese hamster ovary (CHO) cell lines (12,18). Two other types of WGA-resistant mutant have either undergone derepression of a gene encoding an ␣(1,3)fucosyltransferase (19 -21) or alteration of a gene that leads to increased levels of CMP-NeuAc hydroxylase (22)(23)(24). These latter two phenotypes behave dominantly in somatic cell hybrids, whereas the three other WGA-resistant mutants exhibit a recessive phenotype.
In this report we describe the isolation and properties of LEC29.Lec32, a cell line obtained from a mutagenized population of CHO cells following selection with WGA. The dominant LEC29 phenotype is due to expression of an ␣(1,3)fucosyltransferase activity, apparently identical to that previously reported in LEC29 CHO cell extracts (21). The Lec32 phenotype is recessive and novel. We show in this paper that the lec32 mutation reduces CMP-NeuAc synthetase activity to undetectable levels and reduces NeuAc on glycoproteins and glycolipids by 95%.
To effect mutagenesis, a suspension culture of Pro Ϫ 5 CHO cells at 2 ϫ 10 5 cells/ml was treated with MNNG as described (28). After an expression time of 8 days, mutagenized cells were plated at 1 ϫ 10 6 cells/100-mm tissue culture dish in ␣ ϩ 10% FCS containing 13.3 g/ml WGA. Colonies present after 12 days were screened for the expression of the Lewis-X (Le x ) determinant on cell surface carbohydrates using the stage-specific embryonic antigen-1 (SSEA-1)/sheep red blood cell conjugate as described (29). Colonies that bound the SSEA-1/sheep red blood cell conjugate were picked and cloned by limiting dilution, and lectin resistance phenotypes were determined as described (21,26). Somatic cell hybridization analysis was performed by fusion of CHO cells using the polyethylene glycol/dimethyl sulfoxide method (26), and characterization of the resulting hybrids was as described (21).
Monoclonal Antibody Binding-The ability of the various cell lines to bind SSEA-1 and SNH3 mAbs was quantified as described (21). Briefly, ϳ10 6 cells were washed and resuspended in phosphate-buffered saline (PBS ϩϩ ) containing 2% bovine serum albumin (phosphate buffered saline/bovine serum albumin) in a volume of 200 l. After 1 h of incubation at 4°C in the presence or absence of the first antibody (4 g of SSEA-1 or SNH3), the cells were washed with phosphate buffered saline/bovine serum albumin and incubated for 1 h with second antibody (2 g of rabbit anti-mouse IgM or IgG). After washing, a final incubation of 1 h was performed in the presence of 100,000 cpm 125 Iprotein A. After two additional washes, the percentage of cpm bound was determined from the total cpm added per tube and the cpm bound to the washed cells.
Following incubation at 37°C for 1 h, the reaction was stopped by the addition of 1 ml of ice-cold distilled water. Protein product was precipitated with trichloroacetic acid onto a GF/C glass microfiber filter disc (Whatman, Maidstone, United Kingdom), washed 3 times with 5 ml of ice-cold 5% trichloroacetic acid and once with 5 ml of cold 95% ethanol, and counted with 4.5 ml of Ecolume.
CMP-NeuAc synthetase activity was determined initially by a thiobarbituric acid procedure (31), and subsequently, a radiochemical assay was used that was a modification of the method of Edwards and Frosch (32). Unless otherwise indicated, reaction mixtures contained 90 mM Tris-HCl (pH 9.0), 20 mM MgCl 2 , 5 mM CTP (adjusted to pH 7.0 with 1 M KHCO 3 ), 125 nmol of [ 14 C]NeuAc (560 cpm/nmol), and 5 l of extract containing 40 -140 g of protein. Incubation was at 37°C for 15 min, and the reaction was stopped by the addition of 1 ml of ice-cold water. The entire reaction mixture was loaded onto a 1-ml column of AG 1-X4 anion exchange resin (Cl Ϫ form), which was subsequently washed with 2 ml of water followed by 3 ml each of 0.05, 0.06, 0.07, 0.08, 0.15, 0.20, 0.25, and 0.30 M NaCl. Under these conditions, hydrolyzed NeuAc elutes with the water, NeuAc elutes with 0.05-0.08 M NaCl, and CMP-NeuAc elutes with 0.15-0.30 M NaCl (see Fig. 6). All eluates were mixed with 16 ml of Ecolume and counted on a Beckman LS6800 liquid scintillation counter.
Lectin Affinity Chromatography-Approximately 3.5 ϫ 10 6 cells in 10 ml of ␣ ϩ 10% FCS were grown in the presence of 100 Ci of [ 3 H]Glc or 100 Ci of [ 3 H]GlcNAc for 72 h. After three washes with PBS containing 1 mM CaCl 2 , 1 mM MgCl 2 , and 0.02% sodium azide (PBS ϩϩ ), extracts were prepared with 0.5 ml of 1% Nonidet P-40 in 1 mM Tris-HCl buffer, pH 8.5. The supernatant was removed and treated twice with Pronase at 55°C for 24 h. The resulting glycopeptides were purified on a column of Bio-Gel P-2 (1.5 ϫ 70 cm) and applied to a column of WGA-agarose (0.2 ϫ 20 cm) or of MAA-agarose (0.2 ϫ 10 cm). One-ml fractions were collected, mixed with 10 ml of Ecolume, and counted on a Beckman LS6800 liquid scintillation counter.
NeuAc Determinations-Washed cells (ϳ10 7 ) were extracted with 1% Nonidet P-40 as described above. Extracts were adjusted to 1 ml with water and 9 -10 ml of 90% ethanol was added to precipitate cellular glycoproteins. The precipitate was pelleted with low speed centrifugation (3000 rpm) at room temperature for 10 min, washed 3 times with 2-3 ml of 90% ethanol, resuspended in water, lyophilized, and resuspended in 1 ml of water. An aliquot (0.1 ml) from each sample was adjusted to 10 mM HCl and hydrolyzed at 100°C for 1 h. The hydrolyzate was subsequently evaporated to dryness, redissolved in 0.2 ml of water, passed through a Centrex filter (Schleicher and Schuell), and concentrated to 0.05 ml. About 25 l was analyzed by high performance anion exchange chromatography with pulsed electrochemical detection (HPAEC-PAD). A model PAD 2 detector (Dionex Corp., Sunnyvale, CA) and a CarboPac PA1 (4 ϫ 250-mm) pellicular anion exchange column equipped with a CarboPac guard column were used. Eluant 1 was 100 mM NaOH, eluant 2 was water, eluant 3 was 500 mM NaOH, and eluant 4 was 100 mM NaOH containing 1 M NaOAc. NeuAc, NeuGc, and CMP-NeuAc were eluted during 90 min using a gradient of 2-80% eluant 4. Elution for the first 10 min was isocratic with 2% eluant 4, followed by linearly increasing eluant 4 to 80% during the next 60 min and, finally, isocratic 80% eluant 4 for the last 20 min at a rate of 0.9 ml/min. The column was calibrated using standard monosaccharides, and the following pulse potentials and durations were used: E1 ϭ 0.05 V (t 1 ϭ 300 ms); E2 ϭ 0.65 V (t 2 ϭ 180 ms); E3 ϭ Ϫ0.65 V (t 3 ϭ 60 ms). Detection was with 1000 nm full scale. Postcolumn base addition was not used, as no base-line drift was observed with these detector settings.
Quantitation of Cytoplasmic CMP-NeuAc and NeuAc-Samples were prepared by a modification of the method described by Briles et al. (15). Briefly, frozen washed cell pellets from about 6 ϫ 10 7 cells were thawed in 1 ml of water and extracted with an equal volume of 100% ethanol. After sonication on ice for two 30-s pulses at an intensity setting of 4 on a VibraCell Sonicator (Sonics and Materials, Danbury, CT), debris was pelleted by centrifugation at 2000 rpm for 10 min at 4°C. Ethanol was removed by evaporation, the sample was resuspended in 0.5 ml of water, and insoluble material was removed by centrifugation at 4°C at 10,000 rpm for 10 min in a Biofuge A (Baxter/Scientific Products, McGaw Park, IL). The protein remaining in these samples (about 3.7-4.3 mg) was removed by passage through a small column of AG 50W-X2 (200 -400 mesh) H ϩ form ion exchange resin. Recovery was 71-74% of tracer [ 14 C]CMP-NeuAc added at the first step of the procedure. The samples were stored frozen at Ϫ20°C until thawed, passed through Centrex filter units, and analyzed at room temperature by HPAEC-PAD as described above. The standards used were NeuAc, NeuGc, CMP-NeuAc, and CMP-NeuGc.
Preparation of Nuclei-Nuclei were prepared by a modification of the method of Liu (33). Approximately 1 ϫ 10 7 washed Pro Ϫ 5 CHO cells were gently lysed in 0.15 ml of a solution containing 10 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 , 0.3 M sucrose, and 0.25% Nonidet P-40. The lysate was loaded onto a 4-ml cushion of 0.6 M sucrose and centrifuged for 10 min at 2000 rpm at 4°C in a Damon/IEC model DPR6000 centrifuge. After careful removal of the supernatant layer that contained the cytoplasmic contents, nuclei were aspirated from a band just above the 0.6 M sucrose cushion and used in enzyme assays. Microscopic examination confirmed the presence of nuclei and the virtual absence of intact cells.

Mutant Isolation and Lectin
Resistance-In a selection to obtain new glycosylation mutants expressing an ␣(1,3)fucosyltransferase, a population of 1 ϫ 10 7 MNNG-treated Pro Ϫ 5 CHO cells were incubated in 13.3 g/ml WGA, and surviving colonies were screened for cell surface expression of Le x (29) using the mAb SSEA-1, which binds the Le x determinant (Gal␤(1,4)-[Fuc␣(1,3)]GlcNAc␤1) (34). Colonies that bound SSEA-1 were found to express an ␣(1,3)fucosyltransferase activity and included the previously described mutants LEC29 and LEC30 (21) as well as novel variants. One new phenotype had an ␣(1,3)fucosyltransferase activity indistinguishable from that already described for the LEC29 mutant (21). However, this isolate was more resistant to WGA and had a markedly increased sensitivity to ricin, compared with LEC29 cells (Fig. 1). The enormous hypersensitivity to ricin suggested that a second mutation, unrelated to LEC29, had caused an increase in terminal galactose residues on cell-surface glycoproteins. This new mutation was designated lec32, and the mutant phenotype was termed LEC29.Lec32.
Cell Hybridization and Reversion Studies-Since a similar degree of ricin hypersensitivity had been previously observed in the recessive Lec2 CHO mutant (14), LEC29.Lec32 cells were fused to parental CHO cells and to Lec2 mutant cells for complementation analysis. The properties of the resulting hybrids resembled those of the dominant LEC29 phenotype (Table I). Two conclusions may be drawn from this result: 1) LEC29.Lec32 cells contain both a dominant mutation, which activates the expression of a previously quiescent ␣(1,3)fucosyltransferase gene, and a recessive mutation, whose expression causes the extreme hypersensitivity to ricin; and 2) this latter mutation belongs to a complementation group that is distinct from Lec2. Similar results (Table I) were obtained after fusion to Lec3, another NeuAc-deficient CHO mutant (14), indicating that the lec32 mutation is novel.
The existence of two glycosylation mutations in LEC29.Lec32 cells was further supported by a two-step reversion experiment in which LEC29.Lec32 cells were selected for resistance to ricin. When 0.4 ng/ml ricin was used, surviving colonies appeared at a rate of about 4 ϫ 10 Ϫ3 and retained the characteristics of LEC29 cells (i.e. the lec32 mutation had been reverted). When one of these revertants was exposed to 7 ng/ml ricin, surviving colonies appeared at a rate of 2 ϫ 10 Ϫ4 and were indistinguishable from parental CHO cells (i.e. both the LEC29 and lec32 mutations had been reverted). Attempts to isolate double revertants in a single step using the higher concentration of ricin were unsuccessful when 3 ϫ 10 6 cells were screened, as would be expected for an extremely rare event occurring at a predicted frequency of Յ 8 ϫ 10 Ϫ7 .
Monoclonal Antibody Binding Studies-An additional difference between LEC29 and LEC29.Lec32 cells was observed in antibody binding studies with SSEA-1 and SNH3, a mAb that has been reported to bind sialylated lactosamine with ␣(1,3)Fuc (35). Lec3.2.8.1 CHO cells, which do not bind either antibody because they do not produce complex or hybrid N-linked carbohydrates (27) were used as a negative control. The results in Fig. 2 show that LEC29 and LEC29.Lec32 cells bind similar amounts of the Le x -recognizing antibody, SSEA-1. However the binding of the SNH3 antibody was markedly reduced compared with parental CHO or LEC29 cells. As parental CHO cells do not possess an ␣(1,3)fucosyltransferase activity (20,25), the SNH3 antibody must recognize sialylated lactosamine units lacking fucose on CHO cells. The reduced binding of SNH3 by LEC29.Lec32 cells provides additional, indirect evidence that the lec32 mutation causes a loss of cell surface sialic acid residues.
Analysis of Cell Surface Glycoproteins and Glycolipids-Further evidence for a loss of NeuAc residues on LEC29.Lec32 cell surface glycoproteins was obtained by lectin affinity chromatography on WGA and MAA columns (Fig. 3). WGA recognizes ␣(2,3)and ␣(2,6)-linked terminal NeuAc residues (36, 37), while MAA is specific for ␣(2,3)-linked NeuAc residues (38). In both cases, species that bound to the column were present in parental CHO glycopeptides but were absent in glycopeptides from LEC29.Lec32 cells. To quantitate this difference, HPAEC-PAD analysis was performed using authentic NeuAc and cellular glycopeptides from parental and LEC29.Lec32 cells (Fig. 4). The NeuAc component was reduced in mutant cells to less than 5% of parental cell levels. Analysis of [ 3 H]mannosamine-labeled glycolipids showed the ratio of II 3 NeuAclactosylceramide (G M3 ) to lactosylceramide was 0.11 for mutant cells compared with 2.2 for parental CHO cells and a revertant line. 2 Therefore, the NeuAc content of glycolipids from LEC29.Lec32 cells was also reduced 95% compared with parental CHO cells.
Intracellular Pools of NeuAc and CMP-NeuAc-In order to determine whether the lec32 mutation affected the synthesis of NeuAc or CMP-NeuAc, soluble cytoplasmic extracts prepared from both the parental and mutant cells were analyzed by HPAEC-PAD (Fig. 5). The standards used were NeuAc, NeuGc, CMP-NeuAc, and CMP-NeuGc and eluted at approximately 22, 46, 73, and 90 min, respectively. Only the profile for CMP-NeuAc is shown. The LEC29.Lec32 mutant cells were found to contain 10-fold more free NeuAc than the parental CHO cells. By contrast, LEC29.Lec32 cells contained less than 1% of parental cell levels of CMP-NeuAc (or breakdown products derived from it). This difference was extremely significant, and 2 W. Young, unpublished observations.

TABLE I Complementation analysis
Cells were fused, hybrids were selected, and lectin resistance phenotypes were determined as described under "Experimental Procedures." Hybrids from each cross were karyotyped and shown to be pseudotetraploid. The numbers following the names of the cytotoxic lectins indicate-fold sensitivity (S) or -fold resistance (R) in comparison with the levels observed with the Gat Ϫ 2 cell line (Ϫ). (R) indicates a reproducible resistance of Ͻ2-fold. The number of independent hybrids tested for lectin resistance was Ն2 for each cross. The ability of the cells to bind to the SSEA-1 antibody was measured by the SSEA-1/sheep red blood cell assay described by Howard et al. (25), and the number of hybrids tested was Ն100 colonies.

Cell line or cells fused
Phenotype Lectin resistance Anti-SSEA-1 binding Ricin WGA the combined data suggested that the lec32 mutation affects the synthesis of CMP-NeuAc from NeuAc and CTP. CMP-NeuAc Synthetase Activity in CHO Cells-The synthesis of CMP-NeuAc may be investigated using a thiobarbituric acid assay (31). With this assay, parental CHO cell extracts had an activity corresponding to slightly less than twice the background of the assay (ϳ5.1 nmol/min/mg protein). In order to reliably measure low levels of enzyme activity, a radiochemical assay with [ 14 C]NeuAc as substrate was used (see "Experimental Procedures"). Unreacted [ 14 C]NeuAc was separated from CMP-[ 14 C]NeuAc by elution of an anion exchange resin column with a step-wise gradient of NaCl (Fig. 6)  peak but also caused considerable breakdown of NeuAc, as evidenced by an increase in counts eluting with the initial water washes (data not shown). Attempts to reduce the number of steps in the elution procedure by using different salt concentrations and elution volumes failed to adequately separate substrate and product.
CMP-NeuAc synthetase activity has been characterized in various mammalian tissues (39,40), and the activity in CHO cells was optimized for comparison. The optimum pH for the CHO enzyme was found to be 8.5-9.0 in the presence of Mg 2ϩ (Fig. 7) and 7.0 with Mn 2ϩ . However, when Mn 2ϩ was used at pH values above 8.0 under conditions that minimized the formation of Mn(OH) 2 , a similar optimum to that observed with Mg 2ϩ (i.e. pH 9.0) was found, although the levels of activity were only half those observed with Mg 2ϩ (data not shown). Highest levels of activity were obtained when the Mg 2ϩ concentration was at least 20 mM (Fig. 7). Divalent cations other than Mn 2ϩ were unable to effectively substitute for Mg 2ϩ , although some activity (10% of that with Mg 2ϩ ) was obtained in the presence of Fe 2ϩ (Table II). As reported for the rat liver enzyme (40), Cu 2ϩ and Zn 2ϩ were inhibitory when added with Mg 2ϩ .
The optimum temperature over the range tested (22-52°C) was 37°C (data not shown). The reaction was essentially linear for at least 60 min and at protein concentrations from about 40 -360 g/reaction (Fig. 8). Other reports have indicated that (at least for the partially purified mammalian enzyme) sulfhydryl reagents, such as dithiothreitol, are stabilizing or stimulatory (39,40). The inclusion of 2.5 mM dithiothreitol in our standard reaction increased CMP-NeuAc synthetase activity by about 30%. It was also observed that concentrations of CTP above 5 mM inhibited activity but that the addition of 0.5 nmol of unlabeled CMP-NeuAc to a reaction mixture had no effect. Kinetic experiments performed under optimal conditions (see "Experimental Procedures") but in the absence of dithiothreitol gave apparent K m values of about 0.34 mM for NeuAc and 1.3 mM for CTP. In each case, the value falls within the range of those reported for the CMP-NeuAc synthetase from other mammalian sources (39 -41).
Since both the E. coli (42) and rat liver (40) CMP-NeuAc synthetase enzymes are known to be sensitive to NEM, the CHO enzyme was tested. It was found that, whereas very low levels of NEM were slightly stimulatory, the addition of 1 mM NEM to the reaction mixture reduced the CHO activity to about 12%, while the activity of the purified E. coli enzyme was only reduced to 88% under these conditions (Fig. 9).
The mammalian CMP-NeuAc synthetase has been reported by many authors to be localized to the nucleus (39,40,(43)(44)(45)(46). Nuclei prepared from parental CHO cells were found to contain at least 70% of the total extractable activity. Our usual extraction buffer containing 0.15 M NaCl, 25% glycerol, and 1% Nonidet P-40 solubilized 85-95% of total cellular activity. Reextraction of the pelleted material with the same extraction buffer released the remaining activity. Increasing the detergent concentration to 2% did not significantly increase the amount of enzyme activity initially solubilized.
LEC29.Lec32 Cells Lack Detectable CMP-NeuAc Synthetase Activity-Initial results with the thiobarbituric acid assay in-

TABLE II
CHO CMP-NeuAc synthetase activity with various cations CMP-NeuAc synthetase was assayed as described under "Experimental Procedures" except that all cations were used at a final concentration of 10 mM and the pH was 9 or 7 as indicated. Values of 100% correspond to specific activities of 3.0 nmol/min/mg protein (for pH 9 with Mg 2ϩ ) and 1.3 nmol/min/mg protein (for pH 7 with Mn 2ϩ ). Results from one experiment are shown; similar results were obtained in two other experiments. dicated that LEC29.Lec32 cells had no CMP-NeuAc synthetase activity, and this was confirmed with the more sensitive radiochemical method (Fig. 6). LEC29.Lec32 cell-free extracts contained no detectable activity (Յ0.2 nmol/min/mg protein), while extracts from the parental Pro Ϫ 5 cells, as well as those from two independently isolated revertants, contained 3.7-4.0 nmol/ min/mg protein. Similar results were obtained when assays were run at room temperature for longer time periods (data not shown), making it unlikely that a temperature-sensitive mutation has occurred. Mixing of equal amounts of parental and mutant cell extracts yielded about 103% of the expected levels of activity (  (47), the lec32 mutation described in this paper is the first to result in the loss of CMP-NeuAc synthetase activity. Analysis of LEC29.Lec32 glycolipids and cell-surface glycopeptides has demonstrated a reduction of 95% in NeuAc content. Furthermore, the cellular pools of CMP-NeuAc are greatly reduced, while that of free NeuAc is increased about 10-fold in mutant cells compared with parental CHO cells. This latter observation may be the result of two different mechanisms: 1) the lack of CMP-NeuAc synthetase activity should result in a build-up of unused NeuAc substrate; and 2) the deficiency of CMP-NeuAc will lead to release of a previously reported feed-back inhibition of UDP-N-acetylglucosamine 2-epimerase (48), the enzyme responsible for the synthesis of N-acetylmannosamine, a precursor in the synthesis of NeuAc.
The properties of CMP-NeuAc synthetase in cell-free extracts prepared from parental CHO cells appear to be nearly identical to those previously described for CMP-NeuAc synthetase studied in other mammalian tissues (39 -41). In addition, the CHO CMP-NeuAc synthetase is predominantly localized to the nucleus, as observed for the mammalian enzyme from other sources (39,40,(43)(44)(45)(46) Molecular biological approaches to the study of CMP-NeuAc synthetase have thus far been confined to the enzyme from bacterial sources such as E. coli (31,49,50) and Neisseria meningitidis (32). In both cases, the studies were aimed at understanding the steps involved in the synthesis of bacterial virulence factors, such as capsular polysaccharides, which contain large amounts of ␣(2,8)-linked NeuAc residues, or providing a source of enzyme for the synthesis of large amounts of CMP-NeuAc. The nature of the lec32 mutation is currently unknown and is the subject of further investigation. Critical reagents, such as a molecular probe for the mammalian CMP-NeuAc synthetase gene and an antibody that specifically recognizes the enzyme must be developed. Attempts to detect the CHO enzyme with an antibody directed against E. coli CMP-NeuAc synthetase were unsuccessful. 3 Transfection experiments aimed at correcting the defect in LEC29.Lec32 cells using two different mammalian expression vectors containing the cloned E. coli gene (kindly supplied by Dr. Willie Vann), were also negative (data not shown). The fact that small amounts of NeuAc are present on both glycoproteins and glycolipids of LEC29.Lec32 cells suggests that CMP-NeuAc syn-3 W. Vann, personal communication. under the standard reaction conditions described under "Experimental Procedures." In the absence of NEM, 100% activity for the CHO extract was 3.01 nmol/min/mg protein, and for the E. coli enzyme, it was 46,167 nmol/min/mg protein.

TABLE III
Mixing experiment CMP-NeuAc synthetase was assayed as described under "Experimental Procedures." For the mixing experiment, 5 l of each extract was assayed separately, or equal amounts (2.5 l) of extract from the two cell types were mixed together. The activities shown are for equivalent amounts of protein (about 70 -90 g/reaction). thetase is not completely inactivated by the lec32 mutation. This would be consistent with a mutation in the coding region of the CMP-NeuAc synthetase gene that affects the activity, stability, or localization of the enzyme. It is also possible that the lec32 mutation involves a mutation in an upstream control element, a gene encoding a positive or negative transcriptional regulatory factor, or a gene rearrangement, all of which could allow small amounts of enzyme to be synthesized. Although a partial NH 2 -terminal amino acid sequence has been reported for the CMP-NeuAc synthetase from rat liver (40), there have, to date, been no reports of the cloning of a mammalian gene that encodes this enzyme. As an alternative approach, an expression cloning strategy will be used to correct the defect in LEC29.Lec32 cells by transfection of a cDNA library. This should result in the cloning of a CMP-NeuAc synthetase cDNA or a cDNA encoding a factor that regulates CMP-NeuAc synthetase expression.
As for any enzyme involved in the regulation and synthesis of carbohydrate ligands for cell adhesion molecules, further studies of CMP-NeuAc synthetase may be of considerable importance. The nuclear localization of this enzyme (which has long puzzled investigators) provides yet another incentive for probing both the range of its normal cellular functions and the regulation of the gene that encodes this enzyme. It has been proposed that the compartmentalization of this enzyme may be required to protect its product from the action of a cytoplasmic, membrane-bound hydrolase (39). Recent work involving the glycosylation of nuclear proteins (51) has suggested that a sialyltransferase, whose activity is critical for the normal functions of the various nuclear proteins that never transit the Golgi apparatus, may exist in the nucleus. This theory is supported by a report in which five glycosyltransferases, including a sialyltransferase, were found to be associated with rat liver nuclei (52). In this case, the nuclear localization of the enzyme responsible for producing CMP-NeuAc would clearly be of functional importance.