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 LEC11 Chinese hamster ovary mutant synthesizes N-linked carbohydrates containing sialylated, fucosylated lactosamine units. Analysis by one- and two-dimensional 1H NMR spectroscopy

Glycoproteins synthesized by the Chinese hamster ovary cell mutants LEC11 and LEC12 carry the Lex determinant (Gal beta 1,4(Fuc alpha 1,3)GlcNAc), while those synthesized by LEC11 cells also carry the sialyl-Lex determinant (NeuAc alpha 2,3Gal beta 1,4(Fuc alpha 1,3)GlcNAc), and both mutants have been shown to possess a distinct alpha(1,3)-fucosyltransferase of the appropriate specificity to synthesize these determinants (Campbell, C., and Stanley, P. (1983) Cell 35, 303-309; Campbell, C., and Stanley, P. (1984), J. Biol. Chem. 259, 11208-11214; Howard, D. R., Fukuda, M., Fukuda, M. N., and Stanley, P. (1987) J. Biol. Chem. 262, 16830-16837). The LEC11 cells therefore provide a source of carbohydrates terminating in sialylated, fucosylated lactosamine, a relatively rare structure not previously characterized by 1H NMR spectroscopy when in association with an N-linked carbohydrate. In this paper we use a monoclonal antibody specific for Lex to show that the G glycoprotein of vesicular stomatitis virus (VSV) grown in LEC11 and LEC12 cells possesses the Lex determinant and that G from LEC11/VSV also possesses sialylated Lex. Biantennary carbohydrates purified from LEC11/VSV and LEC12/VSV were therefore used to examine the effects on the 1H NMR spectrum of the presence of alpha(1,3)-fucose residues on sialylated and unsialylated lactosamine units. Comparisons of one-dimensional spectra obtained at 500 MHz from LEC11/VSV and LEC12/VSV glycopeptides before and after neuraminidase treatment with spectra of biantennary carbohydrates lacking alpha(1,3)-fucose allowed the assignment of several new resonances. Resolution of certain signals and determinations of coupling constants were achieved by two-dimensional correlation spectroscopy (COSY) at 400 MHz and allowed the assignment of several more resonances in the one-dimensional spectrum.     

1H NMR spectroscopy of carbohydrates from the G glycoprotein of vesicular stomatitis virus grown in parental and Lec4 Chinese hamster ovary cells

Carbohydrate moieties derived from the G glycoprotein of Vesicular Stomatitis Virus (VSV) grown in parental Chinese hamster ovary (CHO) cells and the glycosylation mutant Lec4 have been analyzed by high-field ¹H NMR spectroscopy. The major glycopeptides of $\text{CHO}\text{VSV}$ and $\text{Lec4}\text{VSV}$ were purified by their ability to bind to concanavalin A-Sepharose. The carbohydrates in this fraction are of the biantennary, complex type with heterogeneity in the presence of α(2,3)-linked sialic acid and α(1,6)-linked fucose residues. A minor $\text{CHO}\text{VSV}$ glycopeptide fraction, which does not bind to concanavalin A-Sepharose but which binds to pea lectin-agarose, was also investigated by ¹H NMR spectroscopy. These carbohydrates are complex moieties which appear to contain N-acetylglucosamine in β(1,6) linkage. Their spectral properties are most similar to those of a triantennary complex oligosaccharide containing a 2,6-disubstituted mannose α(1,6) residue. Carbohydrates of this type are not found among the glycopeptides of VSV grown in the Lec4 CHO glycosylation mutant.     

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

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

Specific changes in the oligosaccharide moieties of VSV grown in different lectin-resistnat CHO cells.

The carbohydrate moieties of the G glycoprotein of vesicular stomatitis virus (VSV) grown in three distinct lectin-resistant (LecR) Chinese hamster ovary (CHO) cell lines have been compared by fine structural analysis of radiolabeled glycopeptides. The mutant WgaRIII, selected for resistance to wheat germ agglutinin (WGA), produces VSV containing G glycoprotein specifically lacking in sialic acid. The mutant PhaRI, selected for resistance to phytohemagglutinin (PHA) and previously shown to lack a particular glycoprotein N-acetyl-glucosaminyl-transferase activity, produces VSV containing G glycoprotein specifically lacking terminal N-acetylglucosamine-galactose-sialic acid sequences and possessing an increased number of mannose residues in the "core" region of its carbohydrate moieties. The mutant PhaRIConARII, a "double" mutant selected from PhaRI cells for resistance to concanavalin A (ConA), produces VSV containing G glycoprotein with a further alteration in the mannose residues of the "core" oligosaccharide region. We discuss the relevance of these findings to the mechanisms of glycoprotein biosynthesis in mammalian cells and to the biochemical bases of lectin resistance in CHO cells.     

Antibodies that recognize bisected complex N-glycans on cell surface glycoproteins can be made in mice lacking N-acetylglucosaminyltransferase III

The bisecting GlcNAc is transferred to complex or hybrid N-glycans by the action of N-acetylglucosaminyltransferase III (GlcNAc-TIII) encoded by the Mgat3 gene. CHO cells expressing mouse GlcNAc-TIII were shown by matrix-assisted laser desorption ionization (MALDI) mass spectrometry to produce mainly complex N-glycans with the predicted extra (bisecting) GlcNAc. In order to probe biological functions of the bisecting GlcNAc, antibodies that recognize this residue in the context of complex cell surface glycoconjugates were sought. The LEC10 gain-of-function Chinese hamster ovary (CHO) cell mutant that expresses GlcNAc-TIII and complex N-glycans with the bisecting GlcNAc was used to immunize Mgat3 ^+/+ and Mgat3 ^–/– mice. ELISA of whole sera showed that polyclonal antibodies that bound specifically to LEC10 cells were obtained solely from Mgat3 ^–/– mice. Fluorescence-activated cell cytometry of different CHO glycosylation mutants and western blotting after glycosidase treatments were used to show that anti-LEC10 cell antisera from Mgat3 ^–/– mice recognize cellular glycoproteins with complex N-glycans containing both a bisecting GlcNAc and Gal residues. The polyclonal antibody specificity was similar to that of the lectin E-PHA. IgM-depleted serum containing IgG and IgA antibodies retained full binding activity. Therefore Mgat3 ^–/– mice but not wild type mice can be used effectively to produce polyclonal antibodies that specifically recognize glycoproteins bearing complex N-glycans with a bisecting GlcNAc. Published in 2003.     

A novel glycosylation phenotype expressed by Lec23, a Chinese hamster ovary mutant deficient in alpha-glucosidase I.

Lec23 Chinese hamster ovary (CHO) cells have been shown to possess a unique lectin resistance phenotype and genotype compared with previously isolated CHO glycosylation mutants (Stanley, P., Sallustio, S., Krag, S. S., and Dunn, B. (1990) Somatic Cell Mol. Genet. 16, 211-223). In this paper, a biochemical basis for the lec23 mutation is identified. The carbohydrates associated with the G glycoprotein of vesicular stomatitis virus (VSV) grown in Lec23 cells (Lec23/VSV) were found to possess predominantly oligomannosyl carbohydrates that bound strongly to concanavalin A-Sepharose, eluted 3 sugar eq beyond a Man9GlcNAc marker oligosaccharide on ion suppression high pressure liquid chromatography, and were susceptible to digestion with jack bean alpha-mannosidase. Monosaccharide analyses revealed that the oligomannosyl carbohydrates contained glucose, indicating a defect in alpha-glucosidase activity. This was confirmed by further structural characterization of the Lec23/VSV oligomannosyl carbohydrates using purified rat mammary gland alpha-glucosidase I, jack bean alpha-mannosidase, and 1H NMR spectroscopy at 500 MHz. [3H]Glucose-labeled Glc3Man9GlcNAc was prepared from CHO/VSV labeled with [3H]galactose in the presence of the processing inhibitors castanospermine and deoxymannojirimycin. Subsequently, [3H]Glc2Man9GlcNAc was prepared by purified alpha-glucosidase I digestion of [3H]Glc3Man9GlcNAc. When these oligosaccharides were used as alpha-glucosidase substrates it was revealed that Lec23 cells are specifically defective in alpha-glucosidase I, a deficiency not previously identified among mammalian cell glycosylation mutants.     

Carbohydrate heterogeneity of vesicular stomatitis virus G glycoprotein allows localization of the defect in a glycosylation mutant of CHO cells

The carbohydrate portion of the G glycoprotein of vesicular stomatitis virus (VSV) grown in CHO cells (CHO/VSV) has been fractionated on BioGelP6, concanavalin A-Sepharose, and pea lectin-agarose. The results suggest that, in addition to sialic acid and fucose heterogeneity, the asparagine-linked complex carbohydrate moieties of CHO/VSV also display branching heterogeneity. Although the majority of the glycopeptides bind to concanavalin A-Sepharose in a manner typical of certain biantennary carbohydrate structures, a significant proportion do not bind to the lectin. The latter behavior is typical of tri- or tetraantennary (branched) carbohydrate structures. The CHO/VSV glycopeptides which do not bind to concanavalin A-Sepharose separate into bound and unbound fractions on pea lectin-agarose suggesting that they include at least two different types of (branched) carbohydrate structures. Glycopeptides from the G glycoprotein of VSV grown in two, independently derived CHO glycosylation mutants which belong to complementation group 4 (Lec4 mutants) were examined in the same manner. In contrast to glycopeptides from CHO/VSV, glycopeptides from Lec4/VSV which passed through concanavalin A-Sepharose did not contain a component which subsequently bound to pea lectin-agarose. A glycopeptide fraction with these lectin-binding properties was also missing from cell surface glycopeptides derived from Lec4 cells. The combined results are consistent with the hypothesis that Lec4 CHO glycosylation mutants lack a glycosyltransferase activity responsible for the addition of a (branch) N-acetylglucosamine residue linked β1,6 to mannose.     

Binding of Clostridium botulinum C2 toxin to asparagine-linked complex and hybrid carbohydrates

Clostridium botulinum C2 toxin is a binary toxin composed of an enzymatic subunit (C2I) capable of ADP-ribosylating actin and a binding subunit (C2II) that is responsible for interaction with receptors on eukaryotic cells. Here we show that binding of C2 toxin depends on the presence of asparagine-linked carbohydrates. A recently identified Chinese hamster ovary cell mutant (Fritz, G., Schroeder, P., and Aktories, K. (1995) Infect. Immun. 63, 2334-2340) was found to be deficient in N-acetylglucosaminyltransferase I. C2 sensitivity of this mutant was restored by transfection of an N-acetylglucosaminyltransferase I cDNA. C2 toxin sensitivity was reduced after inhibition of alpha-mannosidase II. In contrast, Chinese hamster ovary cell mutants deficient in sialylated (Lec2) or galactosylated (Lec8) glycoconjugates showed an increase in toxin sensitivity compared with wild-type cells. Our results show that the GlcNAc residue linked beta-1,2 to the alpha-1,3-mannose of the asparagine-linked core structure is essential for C2II binding to Chinese hamster ovary cells.     

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.     

Mutations affecting the antigenic properties of hypoxanthine-guanine phosphoribosyl transferase in cultured Chinese hamster cells.

Cells of the mutant Chinese hamster strain RJK10 do not contain either hypoxanthine-guanine phosphoribosyl transferase activity (HGPRT) or protein that cross-reacts immunologically with HGPRT. HGPRT+ revertants have been isolated from RJK10 and those strains produce HGPRT with altered antigenic properties. HGPRT from the revertant cells is less reactive with anti-HGPRT serum than enzyme from the wild-type cells, and enzymes from the two sources are immunoprecipitated independently from mixtures of cell extracts. Thus one or more of the antigenic determinants present on Chinese hamster HGPRT are either missing or present in an altered form on HGPRT from revertants of RJK10. This indicates that RJK10 carries a mutation in the structural gene for HGPRT and that secondary mutations in the gene give rise to the revertants that produce the antigenically altered enzymes.     

Tyrosyl kinases acquired from anchorage-independent cells by a membrane-enveloped virus

Tyrosyl kinase activity in vesicular stomatitis virus (VSV) acquired from host cells that differ in morphology was investigated. VSV grown in baby hamster kidney (BHK) cells with rounded morphology and a high efficiency of colony formation in soft agar (Rous sarcoma virus [RSV]- transformed and suspension BHK cells) was compared with VSV grown in BHK cells with a flattened morphology and lower efficiency of colony formation in soft agar (RSV-infected revertant and control BHK cells). Tyrosyl kinase activity measured with the substrates angiotensin II peptide or casein was found at 7-10-fold higher levels in virus released from the anchorage-independent BHK cells. Most of the VSV- associated tyrosyl kinases acquired from the RSV-transformed BHK cells reacted with antiserum to pp60src, whereas the activity acquired from the suspension BHK cells was unaffected by anti-src serum. The overall levels of tyrosyl kinase in subcellular fractions of the host BHK cells were also measured. Like the VSV released from them, the RSV- transformed cell extracts contained high levels. The suspension cells, however, contained the same low levels of tyrosyl kinase as was found in the control BHK cell extracts. Therefore, tyrosyl kinase was concentrated and acquired by VSV from the anchorage-independent suspension BHK cells. VSV-associated protein kinases acquired from other cell types followed a similar pattern. Tyrosyl kinase levels were high in VSV released from suspension cultures (Chinese hamster ovary and HeLa) and from virally transformed cells (Kirsten murine sarcoma virus-transformed rat kidney cells) and low in VSV released from an anchorage-dependent primary cell culture (chick embryo fibroblasts).     

Mutations affecting assembly of beta-tubulin localize to a region near the carboxyl terminus

The generation of Chinese hamster ovary cell lines that express assembly defective forms of beta-tubulin were isolated using selections based on reversion of conditional lethal or drug resistance phenotypes. Two such cell lines, D2 and 6H3, were chosen for further characterization because they contain beta-tubulin polypeptides that exhibit decreases in apparent molecular weight on two-dimensional gel electrophoresis. Analysis of the nucleic acid from these cell lines using both Southern and Northern procedures suggests a deletion in one of the beta-tubulin genes in each cell line. Localization of the missing sequence in D2 was first determined by tryptic peptide mapping by high performance liquid chromatography. Subsequently, the assignment was confirmed by constructing appropriate subclones of a wild type Chinese hamster ovary beta-tubulin cDNA for Southern analysis to demonstrate a failure to recognize characteristic hybridization patterns of the mutant tubulin gene. In the other revertant, 6H3, the deletion was detected on a Northern blot by differential hybridization of a 3' fragment of the cDNA to the beta-tubulin messages. The results indicate that D2 has an internal deletion whose approximate limits extend from amino acid residues 250 through 345. Cell line 6H3 has a deletion that begins near amino acid residue 330 and extends into the 3'-untranslated region of the gene.     

Mutants of Chinese hamster ovary cells with altered glucose-6-phosphate dehydrogenase activity

Two mutant clones of a Chinese hamster ovary cell line deficient in glucose-6-phosphate dehydrogenase (G6PD) activity have been characterized. In each case, there is evidence that a structural gene mutation has taken place. The first mutant produces 11% specific enzyme activity compared to wild-type parental cells, but this residual activity is much more heat sensitive than that of the wild type. The second mutant contains no residual activity, but a revertant was isolated that exhibits a partial restoration of G6PD activity with, again, an increased heat sensitivity. The selection of G6PD+ cells from G6PD- populations can be effected by exploiting the increased sensitivity of the latter to diamide, a compound that depletes the cell of reduced glutathione.     

Comparative study of the asparagine-linked sugar chains of natural human interferon-beta 1 and recombinant human interferon-beta 1 produced by three different mammalian cells

The asparagine-linked sugar chains of natural interferon-beta 1 secreted from human foreskin fibroblasts by poly I:poly C induction and of three recombinant human interferon-beta 1 produced by Chinese hamster ovary cells, mouse epithelial cells (C127), and human lung adenocarcinoma cells (PC8) were released quantitatively as oligosaccharides by hydrazinolysis followed by N-acetylation. After being reduced with either NaB3H4 or NaB2H4, their structures were comparatively analyzed. More than 80% of the sugar chains of natural interferon-beta 1 occur as biantennary complex-type sugar chains, approximately 10% of which contain N-acetyllactosamine repeating structure in their outer chain moieties. The remainders are 2,4- and 2,6-branched triantennary complex-type sugar chains. The sugar chains of the recombinant interferon-beta 1 derived from Chinese hamster ovary cells were very similar to those of its natural counterpart. In contrast, two other recombinant proteins contain quite different sugar chains. The protein derived from C127 cells contains complex-type sugar chains with the Gal alpha 1----3Gal beta 1----4GlcNAc group in their outer chain moieties. Their sialic acid residues occur solely as the Sia alpha 2----6Gal group, where Sia is sialic acid. In contrast, the sialic acid residues of other interferon-beta 1 occur as the Sia alpha 2----3Gal group only. A part of the sugar chains of the protein derived from PC8 cells contains bisecting N-acetylglucosamine residue in addition to the Gal alpha 1----3Gal beta 1----4GlcNAc group.     

Measurement of beta-galactosyltransferase activity in cell extracts with an ELISA-based assay

An enzyme-linked immunosorbent assay (ELISA)-based glycosyltransferase assay has been used to measure UDP-Gal:N-acetylglucosamine beta-1,4-galactosyl-transferase (EC 2.4.1.38) activity in detergent extracts of chinese hamster ovary (CHO) cells. LEC11 cells (a mutant of the CHO cell line, Pro -5), which are known to express a complex array of carbohydrate structures, were used to develop the assay for use with whole cell extracts. A detergent-solubilized preparation of the enzyme from whole cells was used to convert the substrate, lactotriglycosylceramide, to the product, neolactotetraglycosylceramide. The monoclonal antibody, 1B2, which specifically binds to the Gal beta 1-4GlcNAc epitope, was used in an ELISA to identify and quantify the product. The enzyme activity in the preparations was found to be similar to that obtained by conventional radioactive assay methods. The beta-galactosyltransferase found in LEC11 cell detergent extracts exhibited an absolute requirement for the nucleotide sugar and MnCl2. The activity of the enzyme was also strictly dependent on the presence of exogenous glycolipid acceptor. When Triton X-114 was used to solubilize the LEC11 beta-galactosyltransferase, activity was found in both the hydrophilic and the hydrophobic phases, suggesting the presence of two forms of the enzyme. The ELISA-based assay was used to compare beta-1,4-galactosyltransferase activity in detergent extracts of four CHO cell lines: Pro-5, Lec1, LEC11, and LEC12 and in detergent-solubilized microsomes from human leukemia cells. The results from this study demonstrate the utility of the ELISA-based assay for measuring glycosyltransferase activity in detergent-solubilized whole cells and microsome preparations.     

Asn-linked oligosaccharides in lectin-resistant tumor-cell mutants with varying metastatic potential

MDW4, a wheat germ agglutinin-resistant mutant of the metastatic murine tumor MDAY D2 has previously been shown to be poorly metastatic when injected intravenously and non-metastatic when injected subcutaneously into syngeneic mice. W4EB8, a Bandeiraea simplicifolia (BSII) lectin-selected subline of MDW4 has previously been shown to be intermediate between that of MDAY-D2 and MDW4 cell for sensitivity to lectin and metastatic phenotype when injected intravenously into mice. The Asn-linked oligosaccharides from MDAY-D2, MDW4 and W4EB8 cells were released enzymatically with peptide N-glycosidase, reduced with tritiated sodium borohydride and fractionated by Concanavalin-A--Sepharose affinity chromatography and high-performance liquid chromatography (HPLC). Structures of the major fractions were determined by a combination of glycosidase digestion and sizing, gas-liquid chromatography/mass spectrometry and by proton nuclear magnetic resonance. Wild-type and mutant cells processed high-mannose-type structures to biantennary (GlcNAc)2(Man)3(GlcNAc)2. In MDAY-D2 cells this structure was processed further to sialylated tetra-antennary complex with polylactosamine-containing antennae terminating in either sialic acid or alpha 1-3-linked galactose. MDW4 cells had four or five times more (GlcNAc)2(Man)3(GlcNAc)2 than MDAY-D2 cells and a major component of tri-antennary (GlcNAc)3(Man)3(GlcNAc)2 (i.e. 2,2,6-substituted tri-mannosyl core) that was not found in wild-type cells. The partial revertant, W4EB8 had intermediate levels of mutant (GlcNAc)3(Man)3(GlcNAc)2 and sialylated complex-type carbohydrates. The results indicate that a shift in expression from incomplete complex type to sialylated tri/tetra-antennary complex-type carbohydrates in tumor cell may enhance the metastatic potential of tumor cells in the experimental metastasis assay. In addition, somatic cell hybridization analysis indicated that the defect in MDW4 cells was identical to that of the Chinese hamster ovary mutant Lec8: a deficiency in UDP-galactose transport into the golgi.