©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Substrate Specificity of 1,4-N-Acetylgalactosaminyltransferase in Vitro and in cDNA-transfected Cells
G/G SYNTHASE EFFICIENTLY GENERATES ASIALO-G IN CERTAIN CELLS (*)

(Received for publication, November 14, 1994; and in revised form, December 21, 1994)

Shuji Yamashiro (1) Masashi Haraguchi (1) Keiko Furukawa (1) Kogo Takamiya (1) Akihito Yamamoto (2) Yasuhiko Nagata (1) Kenneth O. Lloyd (3) Hiroshi Shiku (1) Koichi Furukawa (1)(§)

From the  (1)Department of Oncology, Nagasaki University School of Medicine, and the (2)Department of Prosthodontology, Nagasaki University School of Dentistry, Nagasaki, Japan 852, and the (3)Memorial Sloan-Kettering Cancer Center, New York, New York 10021

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The substrate specificity of beta1,4-N-acetylgalactosaminyltransferase has been analyzed using a fusion enzyme which consisted of the catalytic domain of the enzyme and the IgG binding domain of protein A, and also by extracts from cDNA transfectants. Both enzyme sources were capable of producing not only G and G, but also asialo-G, GalNAc-sialylparagloboside, and GalNAc-G from appropriate acceptors, although the efficiencies were at most 1-3% of those of G/G. The biological significance of these low specificities was studied with transient and stable transfectant cells. From the results of transient expression of the cDNA, asialo-G expression appeared to inversely correlate with G synthase levels in those lines. Consequently, G seemed to be preferentially synthesized when both G and lactosylceramide are available, and asialo-G is synthesized in the absence of G synthesis. However, the results of double immunostaining of CHO transfectants with anti-G and anti-asialo-G antibodies indicated that another factor may be involved in asialo-G synthesis. From the in vitro assay using mixed acceptors, it was concluded that the presence of certain levels of G might enhance the asialo-G synthesis. These results suggest that even acceptors showing low efficiencies in vitro might be used in certain cells depending on the availability of precursors, expression levels of other gangliosides, as well as the kinetic properties of the enzyme, and the compartmentation of the glycosylation machineries in the cells.


INTRODUCTION

Carbohydrate structures on glycoproteins and glycolipids are synthesized by the sequential addition of monosaccharides by glycosyltransferases. Since specific glycosyltransferases are considered to be needed for the individual combination of sugar donors, acceptors, and modes of linkage, 100150 or more different glycosyltransferases are required for synthesis of carbohydrate structures present in mammalian cells(1, 2) . The expression of these carbohydrate structures are regulated by the expression of each glycosyltransferase during differentiation, development, or malignant transformation. In order to further understand the regulatory mechanisms of carbohydrate expression, it is critical to establish the identity of each glycosyltransferase.

Recently we have cloned a cDNA of G/G synthase gene (beta1,4GalNAc-T)(^1)(3) . We successfully detected the GalNAc-T activity in the culture medium of cells transfected with a plasmid containing the catalytic domain of beta1,4GalNAc-T cDNA fused to the IgG binding domain of the protein A gene. In this study, substrate specificity of the beta1,4GalNAc-T was analyzed using this fusion enzyme (protA) as well as extracts from cDNA transfectants. As reported previously, major specificity of the enzyme was found in G and G(3) . However, very low but definite incorporation of GalNAc was also detected on G, lactosylceramide (LacCer), and sialylparagloboside (SPG). Despite low specificity in vitro, LacCer could be converted to asialo-G efficiently depending on the cell lines into which cDNAs were introduced. Regulatory factors governing the carbohydrate structures synthesized by this enzyme in cells were investigated.


MATERIALS AND METHODS

Glycolipids-The derivation of glycosphingolipids used as acceptors of enzyme reaction was as follows: G and G are purchased from Supelco Co. Inc. (Bellefonte, PA). G, G, and GalCer were from Sigma; LacCer, GlcCer, and G were from Snow Brand Milk Products Co. (Tokyo); G was obtained from BioCarb Chemicals (Lund, Sweden); N-glycolylneuraminic acid (NeuGc)-containing G was extracted and purified from horse red blood cells as described(4) ; N-acetylneuraminic acid (NeuAc)-containing SPG and NeuGc-type SPG were also purified from human and bovine red blood cells, respectively, as described previously (4) ; concentration of purified gangliosides was determined according to Warren(5) .

Cell Culture and Establishment of Transfectants

Mouse melanoma line B16 (B78) and fibroblast cell line L cell were donated by Dr. A. Albino at the Sloan-Kettering Cancer Center (New York) and were maintained in Dulbecco's minimum essential medium supplemented with 7.5% fetal bovine serum. MeWo was from Dr. L. J. Old at the Sloan-Kettering Cancer Center. Rat-1 was a rat fibroblast line provided by Dr. Kondo in Ohsaka University. CHO K1 was obtained from RIKEN cell bank (Tsukuba, Japan). Stable transfectants of beta1,4GalNAc-T gene were prepared by co-transfection with beta1,4GalNAc-T cDNA (pM2T1-1) and pSV2neo as described previously(3) . After selection of neoresistant colonies, G-positive clones were picked up by immunofluorescence assay and used for limiting dilution cloning.

Flow Cytometry

Cell surface expression of glycolipid antigens was analyzed by flow cytometry with FACScan (Becton-Dickinson) as described(3) . G was analyzed using mAb 10-11 ((6) ) which was kindly donated by Dr. P. O. Livingston at the Sloan-Kettering Cancer Center. Human mAb KM966 was also used as an anti-G mAb, which was a generous gift from Dr. N. Hanai at the Kyowa Hakko Research Institute. Asialo-G expression was detected by mAb 2D4 ((7) ) which was obtained from American Type Culture Collection.

Construction of pM2T1-1/PROTA

The SmaI-ScaI fragment of pM2T1-1 extending from the 3` side of the transmembrane region to the 3`-uncoding region (coding 505 amino acids) was purified from agarose gel using GeneClean II (BIO 101 Inc., La Jolla, CA) and inserted into the unique EcoRI site of pPROTA vector (8) (a generous gift from Dr. R. Breathnach, at INSERM/CNRS, Cédex, France), after blunt-ended with the Klenow fragment DNA polymerase I. One clone, named pM2T1-1/PROTA with correct orientation was obtained by confirming the DNA sequence across the vector and insert junction.

Expression and Purification of the Soluble Enzyme

B78 or COS7 cells were transfected with pM2T1-1/PROTA or pPROTA alone by the DEAE-dextran method(3) . Regular culture medium was replaced by serum-free ITS (insulin, transferrin, and selenious acid) medium (Collaborative Research) on the next day, and the culture medium was daily collected at day 25 after transfection. Collected medium was concentrated 100-fold using Molcut-L (Nihon Millipore Ltd., Yonezawa, Japan) and dialyzed against 0.1 M sodium cacodylate-HCl (pH 7.2). It was stored in a frozen tube at -80 °C without significant loss of the activity. A nonrelevant mouse IgG (mAb 229 reactive with nm23, IgG2a subclass) was purified and conjugated to Sepharose 4B (Pharmacia, Uppsala, Sweden) according to the manufacturer's instruction, and packed in a small column. The concentrated culture supernatant was applied twice to the column and unbound proteins were removed by washing rigorously with phosphate-buffered saline. Bound fusion protein was stored in 25% glycerol of cacodylate buffer at -80 °C until used.

beta1,4GalNAc-T Enzyme Assay

The enzyme activity of beta1,4GalNAc-T was measured basically according to the method previously described(9) . The membrane fractions of cells were prepared as described by Thampoe et al.(10) . Briefly, cells were lysed using a nitrogen cavitation apparatus. Nuclei were removed by low centrifugation and supernatant was centrifuged at 105,000 times g for 1 h at 4 °C. The pellet was resuspended in ice-cold cacodylate-HCl buffer. The reaction mixture contained in a volume of 50 µl: 100 mM sodium cacodylate-HCl (pH 7.2), 10 mM MnCl(2), 0.3% Triton CF-54 (Sigma), 325 µM G (for G synthesis), 400 µM UDP-GalNAc (Sigma), UDP-[^14C]GalNAc (3.5 times 10^5 dpm) (DuPont NEN), 10 mM CDP-choline (Kojin Co., Tokyo), and membranes containing 200 µg of protein. This mixture was incubated at 37 °C for 2 h. The products were isolated by C18 Sep-Pak cartridge (Waters, Millford, MA) and analyzed by thin layer chromatography (TLC) and fluorography as described(11) .

alpha2,3-Sialyltransferase Assay

alpha2,3-Sialyltransferase activity was determined as described previously(9, 10) . Briefly, the reaction mixture contained in a volume of 50 µl: 50 mM sodium cacodylate-HCl (pH 6.0), 0.3% Triton CF-54, 5 mM MgCl(2), 500 µM LacCer, 500 µM CMP-NeuAc (Sigma), CMP-[^14C]NeuAc (3 times 10^5 dpm, 1200 dpm/nmol) (DuPont NEN), and membranes containing 200 µg of protein. The reaction products were applied to a Sep-Pak C18 column and analyzed as described for the beta1,4GalNAc-T assay.

Neuraminidase Treatment

Enzyme reaction products were dried in small tubes and dissolved in 50 µl of 0.1 M acetate buffer (pH 6.0). Neuraminidase from Clostridium perfringens (Worthington) or neuraminidase from Vibrio cholerae (Calbiochem Corp.) was added (final concentration, 0.5 unit/ml) and incubated for 1 h at 37 °C. The reaction was stopped by adding 4 volumes of chloroform/methanol (1:1) and the organic phase was removed by partition, and used for TLC.

Transient Expression and Flow Cytometric Analysis

For the transient expression, cells were transfected with pM2T1-1 using the DEAE-dextran method(3) . Expression of G and asialo-G was examined by flow cytometry as described above.

Determination of Optimal Condition for Asialo-G Synthase Assay

For investigation of optimal pH, sodium cacodylate buffers containing MnCl(2) and Triton CF-54 were prepared by adjusting the pH to the indicated values. In order to determine the best cation for the enzyme assay, MgCl(2), MnCl(2), CaCl(2), and CuSO(4) were dissolved in the cacodylate buffer with Triton CF-54, and adjusted to pH 7.2 with NaOH prior to using for the assay. Different detergents (Triton X-100, Tween 20, deoxycholate, and Triton CF-54) were tested at appropriate concentrations. The membrane fraction of a MeWo transfectant, C7, expressing a high level of beta1,4GalNAc-T was used as an enzyme source.

Extraction of Glycolipids, TLC, and TLC Immunostaining

Glycolipids were extracted as described previously (12) . Briefly, lipids were extracted by chloroform/methanol 2:1, 1:1, then 1:2 sequentially. Glycolipids were isolated by Florisil column after acetylation, then neutral and acidic fractions were separated by DEAE-Sephadex (A-50) column chromatography. TLC and TLC immunostaining were performed as described previously(12) . Bands in TLC were analyzed by Nu200 CCD Camera System (Photometrics, Tucson, AZ) and IPLab Gel (Signal Analytics Corp., Vienna, VA). MAb M2590 was used for detection of G(13) and mAb 81-87 (donated by Dr. D. Scheinberg in Sloan-Kettering Cancer Center) was used for LacCer(14) .

Enzyme Kinetics Assay

K(m) values of the beta1,4GalNAc-T for G and LacCer in the cDNA transfected cells or the protA were investigated under the standard assay conditions. In order to analyze the preferred use of G or LacCer as the enzyme acceptors, and to investigate the effect of gangliosides on the synthesis of asialo-G, G, G, or G were mixed with LacCer in various ratios and used as acceptors in the enzyme assay.


RESULTS

Initial Analysis of GalNAc-T in Soluble Enzyme and Cell Extracts

In order to demonstrate that the product of the putative beta1,4GalNAc-T cDNA we recently isolated (3) really catalyzes the synthesis of G and/or G, the predicted catalytic domain, containing the carboxyl-terminal 505 amino acids, was fused to a secreted form of the IgG-binding domain of Staphylococcus aureus protein A in the mammalian expression vector pPROTA (8) (Fig. 1A).


Figure 1: Expression of soluble beta1,4GalNAc-T fused with protein A. A, construction of the fusion enzyme. SmaI-ScaI fragment of pM2T1-1 was inserted in the EcoRI site of pPROTA as described under ``Materials and Methods.'' B, detection of G synthase activity in the culture medium of B78 transfected with pM2T1-1/PROTA. Inset shows the products (G) of the enzyme assay at the time points indicated.



When B78 melanoma cells were transfected with pM2T1-1/PROTA, the conditioned media showed increasing activity of G synthase as expected and reached plateau levels at 3 days after transfection (Fig. 1B), whereas transfectants with pPROTA or pM2T1-1/CDM8 demonstrated no or very low levels of the enzyme activity, respectively. In contrast to transfectants with pM2T1-1/CDM8, B78 transfected with pM2T1-1/PROTA showed no surface expression of G (data not shown). COS7 cells transfected with pM2T1-1/PROTA also produced secreted enzyme (data not shown). In order to exclude the possible effects of contaminants in the culture medium, the concentrated medium was affinity-purified using an IgG-Sepharose column. For this purpose, culture medium from COS7 transfectants were used because they contained higher levels of enzyme activity than those from B78 transfectants.

Using protA, the substrate specificity of soluble beta1,4GalNAc-T was examined and compared with the enzyme activity of SK-MEL-31 (a highly expressing melanoma line, (11) ) extracts and extracts from a stable MeWo transfectant. As shown in Table 1A, these sources of enzyme demonstrated very similar substrate specificities, i.e. very high activity with NeuAc- and NeuGc-type G and NeuAc-G as substrates. In addition, they showed very low but definite activity using LacCer, G, and SPG as substrates. The possibility that the asialo-G was a product formed by an endogenous neuraminidase in the melanoma cells was examined by adding a melanoma extract to the enzyme assay; no effect was observed (data not shown).



GalNAc-SPG Synthesis by the beta1,4GalNAc-T

The ability of beta1,4GalNAc-T to use SPGs was examined in more detail and the activity was compared to the beta1,4GalNAc-T from stomach, since Dohi et al.(15) reported the presence of a similar enzyme for GalNAc-SPG synthesis in normal stomach. Both (NeuAc) SPG and (NeuGc)SPG were used as acceptors in assays with either C7 or protA (Fig. 2). When treated by neuraminidase without detergent, the products corresponding to GalNAc-SPG were stable, whereas G bands, which were apparently generated from G contaminating the substrates, were completely converted to G. When membrane extracts from normal stomach tissue were examined, GalNAc-SPGs with both type of sialic acids were produced (Fig. 2C). In the products from normal stomach extracts, no G bands were seen, indicating that this beta1,4GalNAc-T is distinct from G/G synthase.


Figure 2: Transfer of GalNAc onto SPG by the extracts from pM2T1-1 transfectant cells and the fusion protein protA. A, TLC of the enzyme products with a MeWo transfectant line, C7. Both (NeuAc)SPG and (NeuGc)SPG showed generation of GalNAc-SPG. These components were resistant to neuraminidase treatment, whereas G, apparently formed from G contaminating the SPG samples, was converted to G. B, results with the purified protA. G synthesis was demonstrated in this assay also. C, synthesis of GalNAc-SPG with extracts from normal stomach. GalNAc-SPG showed similar migration and sensitivity to neuraminidase as in A and B, although no other bands were detected in this assay.



Similar Assay Conditions Are Required for the Maximal Enzyme Activity to Synthesize G and Asialo-G

As Pohlentz et al.(16) had reported that G/G synthase and asialo-G synthase were identical whereas we found that LacCer was used at only 1-2% the rate of G, we suspected that the conditions of enzyme assay used in our study might not be optimal for asialo-G synthesis although they may be suitable for G/G synthesis. Therefore, various kinds of pH, metal ions, and detergents were used for synthesis of asialo-G using C7 cell extracts as an enzyme source. The optimal pH was 7.2, and Mn was the most effective metal ion for asialo-G synthesis (data not shown). Triton CF-54 was the best among detergents tried. These results were basically the same as for G synthesis, indicating that our assay conditions were suitable for both enzyme activities(9) .

Comparison of Affinities of G and LacCer as Acceptors for beta1,4GalNAc-T

In order to examine the basis for difference in the activity of beta1,4GalNAc-T with G and LacCer, we analyzed the enzyme kinetics for the reaction using G and LacCer as acceptors and cell extracts as the enzyme source. As shown in Table 1B, G showed very high acceptor activity for beta1,4GalNAc-T in comparison to LacCer. Although K(m) with LacCer was smaller than that with G, the apparent V(max) with G was much higher than that with LacCer by more than 2 orders of magnitude. We were unable to determine kinetic data for G using the protA because of nonlinear data. The V(max)/K(m) ratio with G was about 10 times higher than with LacCer, providing a reasonable explanation for the results of the substrate specificity analysis.

Glycolipid Products in Cells Transiently Transfected with beta1,4GalNAc-T cDNA

The expression of glycolipids in cell lines transiently transfected with beta1,4GalNAc-T cDNA was examined by flow cytometry. Five cell lines derived from mouse melanoma (B78), fibroblast (L cell), rat fibroblast (Rat-1), human melanoma (MeWo), and Chinese hamster ovary (CHO) were used because of the lack of beta1,4GalNAc-T gene expression and high efficiency of transfection. Transfected B78, MeWo, and Rat-1 showed G expression but no asialo-G (Table 2). On the other hand, transfected L cells expressed only asialo-G and transfected CHO cells expressed both G and asialo-G, in a ratio of 16:1.



The glycolipids in the cell lines were also extracted and analyzed by TLC and TLC immunostaining, with a special focus on the levels of precursors present. All cell lines, except for L cells, contained high levels of G (Table 2). On the other hand, L cells showed no ganglioside bands as detected by resorcinol spraying. In the neutral fractions, all the cell lines showed doublet bands corresponding to LacCer; its identity was confirmed by TLC immunostaining with mAb 81-87 (data not shown) with various intensities. When the levels of G synthase activity in these 5 cell lines were measured, B78, MeWo, and Rat-1 showed very high activity as expected, whereas CHO and L cell showed very low or no G synthase activity (Table 2).

Glycolipid Composition of Stable Transfectant Cell Lines

Glycolipids were extracted from B78 and L cells and their stable transfectants and analyzed by TLC. As shown in Fig. 3, the majority of LacCer was converted to asialo-G in L cells. The B78 transfectant showed a very faint line corresponding to asialo-G and the LacCer band was reduced. As for the ganglioside composition, the majority of G in B78 was converted to G in the transfectants. No ganglioside bands were seen either in the parent L cell or in its transfectant.


Figure 3: Changes of glycolipid components in cells before and after expression of stably transfected beta1,4GalNAc-T cDNA. Acidic glycolipids (G and G) and neutral glycolipids (CMH, CDH, CTH, and asialo-G) of B78 and L cell were detected using a resorcinol spray and orcinol spray, respectively. Solvents for TLC were chloroform/methanol, 2.5 N NH(4)OH (60:35:8) for acidic fraction, and chloroform/methanol/H(2)O (60:35:8) for neutral. Relative intensities of bands in each fraction were analyzed as described under ``Materials and Methods.'' CTH includes glycolipids migrating at the CTH region.



Two-dimensional Flow Cytometry of CHO Transfectants with Anti-G and Anti-asialo-G MAbs

In order to examine whether the G positive CHO transfected cells were a population distinct from asialo-G positive cells, double staining of bulk CHO transfectants with anti-G and anti-asialo-G mAbs was performed. Contrary to expectation, almost all asialo-G positive cells belonged to the G positive population as shown in Fig. 4.


Figure 4: Double staining of G and asialo-G in CHO transfected with pM2T1-1. CHO cells were co-transfected with pM2T1-1 and pSV2neo, then selected with G418. Neo-resistant cells were applied for flow cytometry by staining with mAb 2D4 plus fluorescein isothiocyanate-conjugated anti-mouse IgM, and human mAb KM966 plus phycoerythrin-conjugated protein A. A, results of transfectant cells. B, parent cells.



Efficiency of Asialo-G Synthesis Was Affected by Coexisting Gangliosides

To investigate the preferential use of G and LacCer by beta1,4GalNAc-T, we performed the enzyme assay using variable mixtures of the two substrates. G synthesis increased as the G/LacCer ratio increased (Fig. 5). Unexpectedly, asialo-G synthesis also increased as the G/LacCer ratio increased, although there was a big difference in the amounts of synthesized G and asialo-G. These results suggested that the presence of G might enhance the asialo-G synthesis, or that the LacCer preparation might contain neuraminidase activity which may convert G to asialo-G. The latter possibility was excluded by an experiment in which LacCer was added to the labeled G in the assay mixture (data not shown). The effect of G or G on asialo-G synthesis was then examined. As shown in Fig. 6, the presence of G markedly enhanced the synthesis of asialo-G, whereas G had less effect. With higher amounts of G or G, the efficiency of asialo-G synthesis from LacCer was diminished. These results suggest that the presence of G or G at appropriate levels may enhance the asialo-G synthesis by beta1,4GalNAc-T.


Figure 5: Asialo-G and G synthesis are not competitive in in vitro assays. A, TLC of the reaction products of protA enzyme (22 µg of fusion protein-IgG complex). Mixtures of G and LacCer at indicated molar ratios were used as acceptors. Separated products were analyzed by TLC with solvent of chloroform, methanol, 0.22% CaCl(2) (55:45:10). B, plots of the bands shown in A.




Figure 6: Effects of gangliosides on asialo-G synthesis. LacCer was mixed with G, G, or G at the indicated molar ratios and used as acceptors. Inset demonstrates the asialo-G bands (arrows) synthesized in the presence of G (a) or G (b). Molar ratios of ganglioside to LacCer of the individual points were 5:95 (lane 1), 20:80 (lane 2), 50:50 (lane 3), and 83:17 (lane 4) to make a total concentration 325 µM. These results were plotted for G (), G (&cjs3409;), and GM1 () addition. Only the points at the right end were 5 times the amount (415/17 molar ratio) of G or G at the point of 83:17.




DISCUSSION

The data presented in this study support the identification of the cDNA previously reported (3) as coding for the UDP-GalNAc:G/G beta1,4-N-acetylgalactosaminyltransferase gene and show that the enzyme efficiently synthesizes G and G in the presence of appropriate acceptors. We also investigated whether the enzyme could synthesize related compounds, i.e. asialo-G, GalNAc-G, and GalNAc-SPG. Although the purified enzyme could synthesize all three products, the efficiency of the reaction was only 1-3% of that for G/G formation. Changing the assay conditions did not improve the efficiency. Hashimoto et al.(17) reported that a GalNAc-T purified from mouse liver also preferentially used G and G as substrates. This enzyme preparation had only trace (<2%) levels of activity with LacCer, SPG, and G. As Pohlentz et al.(16) had reported that G, G, and asialo-G were produced by the same enzyme in extracts of rat liver, we examined the specificity of the enzyme in more detail and determined whether these minor reactions were biologically significant in cultured cells.

The synthesis of GalNAc-SPG by melanoma GalNAc-T can be compared with the activity of the enzyme from normal stomach extracts previously studied by Dohi et al.(15) . These investigators showed that the stomach enzyme synthesizes the NGM-1 antigen, which may be related to the Sd^a blood group specificity. A cDNA coding for the latter structure has recently been cloned and is clearly different from beta1,4GalNAc-T studied here(18) . Moreover, we show (Fig. 2), that in contrast to our beta1,4GalNAc-T, the stomach enzyme does not synthesize G from G, again suggesting that it may be a separate enzyme.

Whether a separate enzyme is also responsible for asialo-G synthesis is not clear. It appears that even though the G/G synthase use LacCer very inefficiently as a substrate in vitro, it is able to produce substantial amounts of asialo-G in certain cells. Analysis of the glycolipid content of cells transiently and stably transfected with beta1,4GalNAc-T cDNA showed that synthesis of G or asialo-G was highly influenced by the precursors available in the cells. Thus, asialo-G, and no G, was produced in transfected L cells. Since high expression of asialo-G was observed even in transiently transfected cells, the high level should reflect the actual rate of synthesis and not be due to the slow accumulation of asialo-G in the cells. L cells have no G; moreover, in all the transfected cell lines studied, levels of alpha2,3-sialyltransferase were inversely correlated with expression of asialo-G. These results show that the levels of appropriate precursors (G or LacCer) influence the propensity of the cell to produce G or asialo-G. Transfected CHO cells, in contrast to transfected L cells, produced both asialo-G and G. This result may be explained by the synthesis of smaller amounts of G in CHO cells than in the other cell lines studied (except L cells). The preference of beta1,4GalNAc-T for G over LacCer as substrate also influences the ratio of G/asialo-G produced by a cell. This enzyme had a lower K(m) and lower V(max) for LacCer than it did for G (Table 1B). The low V(max)/K(m) value for LacCer in comparison to G as substrate observed in vitro is consistent with the cell line ganglioside composition data. These data show that the ability of a cell to synthesize asialo-G is determined by G levels as well as the preferential use of G over LacCer by beta1,4GalNAc-T. G levels, in turn, are controlled by alpha2,3-sialyltransferase levels and possibly by other factors(19) .

Another factor that can apparently influence the ability of GalNAc-T to utilize LacCer as substrate is the level of other gangliosides, particularly G, in the cell. This was demonstrated in CHO transfected cells in which asialo-G was detected only in G-expressing cells. The presence of G in certain concentrations, but not G, also enhanced asialo-G synthesis in vitro. From these data, it is also suggested that G stimulates its own synthesis as well as asialo-G. If this is true, it may explain the sigmoid reaction curve and nonlinear double reciprocal plots obtained in the kinetics of protA for G (data not shown). This fact would also be consistent with the sigmoidal character of Fig. 5B, upper panel. The mechanism for the effect is at present not known. We suspect that the presence of G has an effect in modifying the affinity of beta1,4GalNAc-T, but this effect would have to be concentration-dependent. It is also possible, but unlikely, that G is simply stabilizing the protA enzyme during the reaction.

The glycolipid composition of cells may also be determined by the differential compartmentation of the various glycosyltransferases in the biosynthetic machinery of the cell. The possibility for differential compartmentation of these enzymes is suggested by organelle fractionation studies in which alpha2,3-sialyltransferase was recovered in different gradient regions containing cis-Golgi cisternae, from other sialyltransferase existing in trans-Golgi-containing fractions(20) . Thus, the preferential use of G may also be because alpha2,3-sialyltransferase is located in an earlier compartment than GalNAc-T and may utilize most of the LacCer before it can reach the GalNAc-T compartment. The use of brefeldin A also suggested that alpha2,3-sialyltransferase was located within the Golgi stacks (brefeldin A resistant) while GalNAc-T was located beyond the brefeldin A block (brefeldin A sensitive)(21, 22) .

In brief, factors that determine ganglioside composition in cells are quite complex, but glycosyltransferase levels, substrate specificities of the enzymes, and the levels of precursors and other glycolipids in the cell clearly play important roles. Fig. 7summarizes the ganglioside expression in the cell lines studied and the effect of transfection with the beta1,4GalNAc-T gene on the ganglioside patterns. These studies suggest that even acceptors showing low efficiencies with the enzyme might be used in certain cells. The existence of other GalNAc-T species which would preferentially glycosylate LacCer or other gangliosides containing a similar terminal Gal residue cannot, however, be excluded. On-going studies to knock-out the beta1,4GalNAc-T gene in either mice or cultured cells should clarify these issues.


Figure 7: Ganglioside patterns in cell lines before and after transfection with beta1,4GalNAc-T cDNA.




FOOTNOTES

*
This work was supported by a Grant-in-Aid for Scientific Research of Priority Areas from the Ministry of Education, Science and Culture of Japan, and a Grant-in-Aid of Nissan Foundation, and U. S. Public Health Service Grant CA 60680. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 958-47-2111; Fax: 958-49-3695.

(^1)
The abbreviations used are: GalNAc-T, N-acetylgalactosaminyltransferase; protA, GalNAc-T fusion protein with IgG binding domain of protein A; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid; LacCer, lactosylceramide; SPG, sialylparagloboside; mAb, monoclonal antibodies; CHO, Chinese hamster ovary; TLC, thin layer chromatography. Ganglioside nomenclature is based on that of Svennerholm(23) : G, NeuAc (or NeuGc)alpha2,3Galbeta1,4Glc-Cer; G, GalNAcbeta1,4(NeuAcalpha2,3)Galbeta1,4Glc-Cer; G, NeuAcalpha2,8NeuAcalpha2,3Galbeta1,4Glc-Cer; G, GalNAcbeta1,4(NeuAcalpha2,8NeuAcalpha2,3)Galbeta1,4GlcCer; G, NeuAcalpha2,3Galbeta1,4GalNAcbeta1,4(NeuAcalpha2,8NeuAcalpha2,3)Galbeta1,4Glc-Cer; sialyl-paragloboside, NeuAc or NeuGcalpha2,3Galbeta1,4GlcNAcbeta1,3Galbeta1,4Glc-Cer; other glycolipids were: asialo-G (gangliotriaosylceramide), GalNAcbeta1,4Galbeta1,4Glc-Cer; lactosylceramide or CDH (ceramide dihexoside), Galbeta1,4Glc-Cer; CMH (ceramide monohexoside), Glc-Cer; CTH (ceramide trihexoside), Galalpha1,4Galbeta1,4Glc-Cer.


ACKNOWLEDGEMENTS

We are grateful to Drs. T. Maita and T. Hayashibara for valuable discussion. We thank Dr. R. Breathnach for pPROTA, and Dr. N. Hanai for mAb KM966. We also thank T. Shimomura for excellent technical assistance and Y. Nakaji for secretarial help.


REFERENCES

  1. Beyer, T. A., Sadler, J. E., Rearick, J. I., Paulson, J. C., and Hill, R. L. (1981) Adv. Enzymol. Relat. Areas Mol. Biol. 52, 23-175 [Medline] [Order article via Infotrieve]
  2. Paulson, J. C., and Colley, K. J. (1989) J. Biol. Chem. 264, 17615-17618 [Free Full Text]
  3. Nagata, Y., Yamashiro, S., Yodoi, J., Lloyd, K. O., Shiku, H., and Furukawa, K. (1992) J. Biol. Chem. 267, 12082-12089 [Abstract/Free Full Text]
  4. Furukawa, K., Chait, B. T., and Lloyd, K. O. (1988) J. Biol. Chem. 263, 14939-14947 [Abstract/Free Full Text]
  5. Warren, L. (1959) J. Biol. Chem. 234, 1971-1975 [Free Full Text]
  6. Natoli, E. J., Livingston, P. O., Pukel, C. S., Lloyd, K. O., Wiegandt, H., Szalay, J., Oettgen, H. F., and Old, L. J. (1986) Cancer Res. 46, 4116-4120 [Abstract]
  7. Young, W. W., Jr., MacDonald, E. M. S., Nowinski, R. C., and Hakomori, S. (1979) J. Exp. Med. 150, 1008-1019 [Abstract]
  8. Sanchez-Lopez, R., Nicholson, R., Gesnel, M.-C., Matrisian, L. M., and Breathnach, R. (1988) J. Biol. Chem. 263, 11892-11899 [Abstract/Free Full Text]
  9. Ruan, S., and Lloyd, K. O. (1992) Cancer Res. 52, 5725-5731 [Abstract]
  10. Thampoe, I. J., Furukawa, K., Vellvé, E., and Lloyd, K. O. (1989) Cancer Res. 49, 6258-6264 [Abstract]
  11. Yamashiro, S., Ruan, S., Furukawa, K., Tai, T., Lloyd, K. O., Shiku, H., and Furukawa, K. (1993) Cancer Res. 53, 5395-5400 [Abstract]
  12. Furukawa, K., Clausen, H., Hakomori, S., Sakamoto, J., Look, K., Lundblad, A., Mattes, M. J., and Lloyd, K. O. (1985) Biochemistry 24, 7820-7826 [Medline] [Order article via Infotrieve]
  13. Hirabayashi, S., Hamaoka, A., Matsumoto, M., Matsubara, T., Tagawa, M., Wakabayashi, S., and Taniguchi, M. (1985) J. Biol. Chem. 260, 13328-13333 [Abstract/Free Full Text]
  14. Kalisiak, A., Oosterwijk, E., Minniti, J. G., Old, L. J., and Scheinberg, D. A. (1991) Glycoconj. J. 8, 55-62 [Medline] [Order article via Infotrieve]
  15. Dohi, T., Ohta, S., Hanai, N., Yamaguchi, K., and Ohshima, M. (1990) J. Biol. Chem. 265, 7880-7885 [Abstract/Free Full Text]
  16. Pohlentz, G., Klein, D., Schwarzmann, G., Schmitz, D., and Sandhoff, K. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7044-7048 [Abstract]
  17. Hashimoto, Y., Sekine, M., Iwasaki, K., and Suzuki, A. (1993) J. Biol. Chem. 268, 25857-25864 [Abstract/Free Full Text]
  18. Smith, P. L., and Lowe, J. B. (1994) J. Biol. Chem. 269, 15162-15171 [Abstract/Free Full Text]
  19. Deutscher, S. L., Nuwayhid, N., Stanley, P., Briles, E. I. B., and Hirschberg, C. B. (1984) Cell 39, 295-299 [Medline] [Order article via Infotrieve]
  20. Trinchera, M., and Ghidoni, R. (1989) J. Biol. Chem. 264, 15766-15769 [Abstract/Free Full Text]
  21. van Echten, G., Iber, H., Stotz, H., Takatsuki, A., and Sandhoff, K. (1990) Eur. J. Cell Biol. 51, 135-139 [Medline] [Order article via Infotrieve]
  22. Young, W. W., Jr., Lutz, M. S., Mills, S. E., and Lechler-Osborn, S. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6838-6842 [Abstract]
  23. Svennerholm, L. (1963) J. Neurochem. 10, 613-623 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.