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Originally published In Press as doi:10.1074/jbc.M002629200 on June 14, 2000

J. Biol. Chem., Vol. 275, Issue 33, 25308-25314, August 18, 2000
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Expression Cloning of a New Member of the ABO Blood Group Glycosyltransferases, iGb3 Synthase, That Directs the Synthesis of Isoglobo-glycosphingolipids*

Jeremy J. KeuschDagger §, Stephen M. ManzellaDagger , Kwame A. Nyame||, Richard D. Cummings||, and Jacques U. BaenzigerDagger

From the Dagger  Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110 and the || Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190

Received for publication, March 28, 2000, and in revised form, June 13, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The large array of different glycolipids described in mammalian tissues is a reflection, in part, of diverse glycosyltransferase expression. Herein, we describe the cloning of a UDP-galactose: beta -D-galactosyl-1,4-glucosylceramide alpha -1,3-galactosyltransferase (iGb3 synthase) from a rat placental cDNA expression library. iGb3 synthase acts on lactosylceramide, LacCer (Galbeta 1,4Glcbeta 1Cer) to form iGb3 (Galalpha 1,3Galbeta 1,4Glcbeta 1Cer) initiating the synthesis of the isoglobo-series of glycosphingolipids. The isolated cDNA encoded a predicted protein of 339 amino acids, which shows extensive homology (40-50% identity) to members of the ABO gene family that includes: murine alpha 1,3-galactosyltransferase, Forssman (Gb5) synthase, and the ABO glycosyltransferases. In contrast to the murine alpha 1,3-galactosyltransferase, iGb3 synthase preferentially modifies glycolipids over glycoprotein substrates. Reverse transcriptase-polymerase chain reaction revealed a widespread tissue distribution of iGb3 synthase RNA expression, with high levels observed in spleen, thymus, and skeletal muscle. As an indirect consequence of the expression cloning strategy used, we have been able to identify several potential glycolipid biosynthetic pathways where iGb3 functions, including the globo- and isoglobo-series of glycolipids.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

There are many diverse glycans found in nature that may be synthesized on different aglycone substrates, including proteins and lipids. The glycosphingolipids (GSL)1 are organized, based on their carbohydrate structure, into six major series in vertebrates: gangliosides, lacto-, neolacto-, muco-, isoglobo-, and globo-series GSL (1). The common precursor to these GSL is LacCer (Galbeta 1,4Glcbeta 1Cer). Recently, the vital importance of GSL has been demonstrated through the null mutation of glucosylceramide synthase (2), the first step in the synthesis of the majority of GSL. The significance of individual glycan structures within the major GSL series arising from LacCer remains to be established. To date, animals deficient in glycosyltransferases involved in the terminal modifications have not yielded the prominent or lethal phenotypes associated with the loss of the ability to synthesize core regions of glycans. Targeting null mutations in glycosyltransferases acting at key branch points in GSL biosynthesis will help reveal the contributions of the different series of GSL.

As a first step to this approach we have cloned Gb3 synthase and iGb3 synthase, two transferases that act on LacCer and initiate the synthesis of the globo- and isoglobo-series of GSL. Following the synthesis of Gb3 (Galalpha 1,4Galbeta 1,4Glcbeta 1Cer) or iGb3 (Galalpha 1,3Galbeta 1,4Glcbeta 1Cer) (boldface is used to show the linkage of saccharide transferred by Gb3 synthase and/or iGb3 synthase), sequential addition of GalNAc residues by Gb4 synthase and Gb5 synthase leads to the production of both Gb4 and Gb5 or iGb4 and iGb5, respectively. Using a mAb SMLDN1.1 that detects GalNAc on these downstream products, we have cloned Gb3 and iGb3 synthases from a rat placental cDNA expression library. In this paper we described the cloning of iGb3 synthase, an alpha 1,3-galactosyltransferase, that shares high homology with other glycosyltransferases in the histo-blood group ABO gene family but differs in its substrate specificity.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Rat Placental cDNA Library-- RNA was isolated from normal rat placental tissue, gestation day 18, and used to prepare a cDNA library with the mammalian expression vector pCDM8. This cDNA library, RPL18, was generously provided by Dr. P. Smith, University of Michigan.

Cell Culture-- Chinese hamster ovary (CHO) cells were grown in Ham's F-12 medium with 10% fetal bovine serum ± 2 µM 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol, HCl (DL-threo-PPPP, Calbiochem, CA), an inhibitor of glucosylceramide synthase (3).

SMLDN1.1 mAb Binding Specificity-- The generation and characterization of this SMLDN1.1 mAb has been described previously (4). Subsequent analysis of SMLDN1.1 binding characteristics with additional carbohydrate structures indicates that the presence of a single terminal GalNAc residue in either an alpha  or beta  linkage is sufficient for recognition. We have observed that SMLDN1.1 mAb efficiently recognizes the terminal GalNAc residues in both Gb4 (GalNAcbeta 1,3) and Gb5 (GalNAcalpha 1,3GalNAcbeta 1,3).

Expression Cloning of a Rat iGb3 Synthase cDNA-- RPL18 plasmid cDNA was isolated from an overnight culture of transformed Escherichia coli MC1061/P3 cells selected in 50 µg/ml ampicillin and 10 µg/ml tetracycline. Ten 100-mm plates of CHO cells (2.5 × 106 cells per plate) were cotransfected with 10 µg of RPL18 plasmid cDNA and 3 µg of pSVE1-PyE plasmid, harboring the polyoma large T cDNA (a kind gift from Dr. Minoru Fukuda, The Burnham Institute), using Lipofectamine (Life Technologies, Inc.). The pCDM8 vector requires the polyoma large T antigen for high levels of replication, but unlike the library vector the pSVE1-PyE plasmid is neither amplified nor selected in subsequent enrichment steps, because it lacks the supF gene (5). Cells were harvested using 0.02% EDTA 60 h post-transfection, stained with SMLDN1.1 mAb, and processed for flow cytometry (FACS). Plasmid cDNA was recovered, from the FACS-sorted cells, by Hirt extraction (6) and amplified in E. coli MC1061/P3. Two further rounds of transfection and FACS enrichment were performed. Plasmid cDNA obtained from the third FACS sort was transformed into E. coli MC1061/P3, and dilutions were plated. Plaque lifts from these dilution plates were used to prepare more plasmid cDNA for transfection into CHO cells and screened with the SMLDN1.1 mAb via FACS. A positive plate with 590 colonies was divided into 12 smaller pools of 50 colonies, which were subsequently screened for SMLDN1.1 binding. This process was repeated on the reactive smaller pool until a single plasmid was isolated whose expression in CHO cells conferred reactivity with SMLDN1.1 mAb. We refer to this cDNA clone as iGb3 synthase.

DNA Sequencing-- Plasmid cDNA was isolated from clones of interest using Qiagen columns. The cDNA insert was sequenced using T7, a pCDM8 reverse primer and gene-specific primers, in a reaction mix containing ABI Big-Dye terminators with AmpliTaq DNA polymerase according to the manufacturer's instructions (PE Applied Biosystems). Reaction products were run on an ABI 310 sequencer. Complete insert sequence was obtained in both directions and compared against sequences held in GenBankTM using BLAST (NCBI). Additional analysis was performed using Geneworks (version 2.5, Oxford Molecular, UK) and the transmembrane prediction program TMHMM (7).

Site-directed Mutagenesis-- The 198DVD200 sequence in the iGb3 cDNA clone was mutated to 198AVA200 by inverse PCR using high fidelity KlenTaq LA-polymerase mix (Sigma) and completely overlapping primers containing the point mutations (underlined), in the forward direction: 5'-CTATGTGTTCTGCCTGGCCGTGGCCCAGTACTTCAGCGG-3' and reverse direction: 5'-CCGCTGAAGTACTGGGCCACGGCCAGGCAGAACACATAG-3'. Following thermocycling and digestion of parental DNA template with DpnI, MC1061/P3 cells were transformed and mini-preps sequenced to verify the desired mutations.

FACS Analysis-- Cells were harvested using 0.02% EDTA. After washing in phosphate-buffered saline (PBS), pH 7.2, cells were counted and resuspended at 107 cells/ml in ice-cold PBS/1% BSA. Typically 106 cells were incubated with 100 µl of mouse IgM mAb, SMLDN1.1, diluted to 5 µg/ml in blocking buffer (PBS/1% BSA/5% normal goat serum), for 45 min on ice. Cells were washed in PBS/1% BSA and incubated for 30 min on ice with goat anti-mouse IgM-FITC (Jackson ImmunoResearch Laboratories, PA) diluted 1:200 in blocking buffer. After washing, cells were resuspended in PBS and read directly on the FACS Calibur (Becton Dickinson) and analyzed using CellQuest version 3.1 software. Other mAbs, lectins, and secondary antibodies used in FACS analysis included rat IgM anti-Forssman mAb M1/22.21, used at 1:100; anti-SSEA-3 mAb MC631 (both reagents kindly provided by Dr. D. Haslam, Washington University, St. Louis, MO); anti-CD77 mAb, clone 38-13 diluted 1:5 (Biodesign International); Griffonia simplicifolia lectin I-B4-biotin, used at 10 µg/ml with 0.1 mM CaCl2 (Vector Laboratories); and streptavidin-FITC (PharMingen).

Assay of alpha 1,3GalT Activity-- Cell extracts from transfected CHO cells were prepared and assayed as described previously except 100 mM sodium cacodylate, pH 6.8 was used in place of MES buffer (8). Supernatants from the cultured cells were concentrated 10-fold, using Microcon-10 (Millipore) mini-spin columns, prior to assaying a 50-µl fraction. The reaction mixture contained the following in a final volume of 100 µl: 100 mM sodium cacodylate-HCl, pH 6.8, 0.5% Triton X-100, 5 mM ATP, protease inhibitors (50 µg/ml turkey egg white trypsin inhibitor, 20 µg/ml leupeptin, 20 µg/ml antipain, 20 µg/ml pepstatin, 20 µg/ml chymotrypsin, and 0.115 trypsin inhibitor units/ml aprotinin, Sigma), 250 µM UDP-galactose, UDP-[3H]galactose (800 cpm/pmol) (American Radiolabeled Chemicals, Inc.), 15 mM MnCl2, 1 mM LacCer (Sigma). Reactions were started with the addition of 15- or 50-µg membrane fractions or 50 µl of concentrated culture supernatant and incubated for 90 min or overnight at 37 °C. The enzyme assay was linear over these times. Reaction products were isolated by reverse-phase chromatography using Sep-Pak C18 cartridges (Waters, MA) and analyzed by thin layer chromatography (TLC) using silica gel aluminum-backed plates (Whatman) developed in chloroform:methanol:water (65:25:4, v/v). After drying, the plates were sprayed with En3Hance (NEN Life Science Products) then exposed to Kodak BioMax MR x-ray film at -80 °C. Neutral glycolipid standards from porcine blood (Calbiochem) were included, and this portion of the plate was cut-off and sprayed with orcinol.

Metabolic Labeling of CHO Cells-- The day after transfection, cells were labeled with either 35 µCi/ml [3H]Gal or [3H]GlcNAc (NEN Life Science Products) for a further 24-48 h. Cells were scraped off the culture plate, and glycolipids were extracted in chloroform/methanol as described previously (8).

Glycolipid Digestion with Exoglycosidases-- Glycolipid extracts (approximately 1-10 µg or 30,000 cpm) were isolated over a Sep-Pak C18 cartridge and separated by HPLC using an Iatrobead column (kindly provided by Dr. R. Schnaar, John Hopkins School of Medicine) as described (9). Pooled peak fractions were digested overnight at 37 °C with exoglycosidases in the presence of 0.05% sodium taurodeoxycholate in a final volume of 10 µl. The glycosidases used included alpha -galactosidase (green coffee bean, Calbiochem); alpha 1,3-galactosidase (recombinant Escherichia coli, Calbiochem); beta 1,3,4,6-galactosidase (bovine testes, Calbiochem); alpha -N-acetylgalactosaminidase (chicken liver, Oxford GlycoSciences, UK); and beta -N-acetylhexosaminidase (Streptomyces plicatus recombinant in E. coli, New England BioLabs). The digested samples were re-isolated using a Sep-Pak C18 cartridge prior to separation on TLC as described above.

Expression of iGb3 Synthase in Rat Tissues Using RT-PCR-- Tissues were taken from a 6-month-old female Long Evans rat and snap-frozen in liquid nitrogen. These tissues were homogenized in Trizol (Life Technologies), and total RNA was purified according to the manufacturer's instructions. The quality and quantity of RNA was verified by gel electrophoresis. First strand cDNA synthesis was prepared from 1-5 µg of RNA using the reverse transcriptase enzyme, Superscript II, and oligo(dT) primer (Life Technologies). Gene-specific primer pairs were used in the PCR to yield an expected product size of 780 base pairs. Glyceraldehyde-3-phosphate dehydrogenase gene-specific primers were used as a control. Products were analyzed by gel electrophoresis.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of a Rat iGb3 Synthase cDNA Clone-- Phenotypic cloning has been used to isolate many glycosyltransferase genes, which are typically expressed at low levels in cells (10). The screening strategy used in this paper relies on the specificity of the SMLDN mAb, which recognizes terminal GalNAc residues. Positive clones identified in this cloning approach are involved in the cell surface expression of a GalNAc epitope. Despite expecting the isolated clone to express GalNAc transferase activity, no such activity was detected. These results were initially confusing until it became apparent that our readout signal, GalNAc expression, was in fact the product of a key upstream biosynthetic step. Thus, the isolated clone synthesizes iGb3, a product that Gb4 and Gb5 synthases, enzymes endogenous to the CHO host recipient cells, convert to iGb4 and iGb5, respectively. Using this expression cloning procedure, the iGb3 synthase cDNA (accession number AF248543) and later Gb3 synthase cDNA (accession number AF248544, see Keusch et al., companion paper (35)) were isolated from the rat placental library, RPL18.

cDNA Sequence Analysis-- Sequencing of the iGb3 synthase cDNA clone revealed an insert of 1496 base pairs. Translation from the first methionine in the open reading frame predicts a protein of 339 amino acids (Fig. 1). The TMHMM transmembrane prediction program (7) identifies a single transmembrane domain of 19 amino acids with a type II transmembrane topology typical of Golgi glycosyltransferases (11). Other hydrophobic regions are also apparent, including two stretches near the N terminus (residues 15-29 and 34-58) but are not predicted to be membrane spanning by the TMHMM program. Two N-linked glycosylation consensus sequences are present in the stem region. Within the putative catalytic domain there is a 198DVD200 sequence. Mutation of the DXD motif in other glycosyltransferases results in the loss of enzyme activity (12).


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  		Fig. 1.   cDNA sequence analysis of rat iGb3 synthase. The deduced amino acid sequence in the single-letter code is shown above the nucleotide sequence. The putative transmembrane domain is underlined, and two potential N-linked glycosylation sites are boxed. The 198DXD200 motif is indicated by three black dots.

iGb3 Synthase Belongs to the Histo-blood Group ABO Gene Family-- BLAST (NCBI) sequence analysis identified significant homology (39% identity) at the amino acid level between iGb3 synthase and members of the ABO glycosyltransferases, including alpha 1,3-galactosyltransferase (alpha 1,3GalT), Forssman (Gb5) synthase, and blood group A and B synthases (Fig. 2). The identity is as high as 51% in the putative catalytic domain with three conserved cysteines and the DXD motif. All members of this gene family catalyze the transfer of a UDP-sugar in an alpha 1,3-linkage to a beta -linked Gal/GalNAc. iGb3 synthase represents the third distinct alpha 1,3-galactosyltransferase in the ABO gene family.


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  		Fig. 2.   ClustalW alignment of histo-blood group ABO gene family members. The amino acid sequences are from rat iGb3 synthase (iGb3 S, accession number AF248543), murine alpha 1,3-galactosyltransferase (a1-3 GalT, accession number M26925), dog Forssman synthase (FS, accession number U66140), and human blood group B glycosyltransferase (B-Trans, accession number AF134414). Boxed regions indicate areas of homology, with conserved residues in light shading, and absolute identity in bold. Dashes depict gaps inserted to optimize the alignment. The 198DXD200 motif is indicated by three black dots.

Expression of iGb3 Synthase in CHO Cells Leads to the Synthesis of the Isoglobo-series GSL-- FACS analysis shows that CHO cells transfected with iGb3 synthase cDNA express high levels of terminal GalNAc on their cell surface. In contrast, CHO cells transfected with the pCDM8 vector did not express terminal GalNAc. The highest reactivity in the iGb3 synthase cDNA transfectants is seen using the SMLDN1.1 mAb, with approximately 70% of the transfected cells expressing terminal GalNAc (Fig. 3). This positive staining is predominantly due to glycolipids, because reactivity to the SMLDN1.1 mAb is lost when transfectants are cultured in the presence of PPPP, an inhibitor of the major series of GSL (3). Apart from GM3, parent CHO cells do not synthesize any GSL beyond LacCer (13). Therefore, we were surprised to see that transfection of iGb3 synthase cDNA into CHO cells results in strong reactivity with the anti-Forssman (Gb5) glycolipid mAb (Fig. 3). Forssman glycolipid, Gb5 (GalNAcalpha 1,3GalNAcbeta 1,3Galalpha 1,4Galbeta 1,4Glcbeta 1Cer), and iGb5 (GalNAcalpha 1,3GalNAcbeta 1,3Galalpha 1,3Galbeta 1,4Glcbeta 1Cer), represent the terminal structures in the globo- and isoglobo-series GSL, respectively (14, 15). Transfection of Gb5 synthase cDNA into parent CHO cells does not result in Gb5 or iGb5 expression due to the absence of precursor substrates (35). Therefore, we reasoned that the iGb3 synthase cDNA is able to initiate the synthesis of isoglobo-GSL in CHO cells. Gb4 synthase and Gb5 synthase but not Gb3 synthase nor iGb3 synthase are endogenous to CHO cells. Thus, the de novo production of iGb3 in CHO cells, transfected with the iGb3 synthase cDNA, provides substrate for the sequential addition of beta 1,3- and alpha 1,3GalNAc moieties by Gb4 and Gb5 synthases, respectively. Assuming similar expression levels, a catalytically inactive form of iGb3 synthase produced by mutating the 198DVD200 sequence to 198AVA200 does not result in synthesis of any isoglobo-GSL (Fig. 3). Transfection of CHO cells with iGb3 synthase cDNA results in the synthesis of isoglobo-GSL but not globo-GSL (Gb3 core structures) as indicated by the lack of reactivity with anti-CD77/Gb3 (Galalpha 1,4Galbeta 1,4Glcbeta 1Cer) (Fig. 3) or anti-SSEA-3 (Galbeta 1,3GalNAcbeta 1,3Galalpha 1,4Galbeta 1,4Glcbeta 1Cer) mAbs (data not shown).


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  		Fig. 3.   Cell surface expression of isoglobo-GSL in CHO cells transfected with iGb3 synthase cDNA is inhibited by PPPP. CHO cells transfected with either empty pCDM8 vector, iGb3 synthase cDNA, murine alpha 1,3GalT cDNA, or mutant 198AVA200 iGb3 synthase cDNA were cultured in the absence (thick line) or presence of PPPP (thin line) and stained for surface expression of terminal carbohydrates on GSL: GalNAc (SMLDN1.1 mAb); Galalpha 1,3Gal (G. simplicifolia lectin I-B4-biotin); Gb5 (GalNAcalpha 1,3GalNAcbeta 1,3Galalpha 1,4Galbeta 1,4Glcbeta 1Cer) using anti-Gb5 (Forssman) glycolipid M1/22.21 mAb; or Gb3 (Galalpha 1,4Galbeta 1,4Glcbeta 1Cer) using anti-CD77 mAb clone 38-13 prior to analysis by FACS. Control primary antibodies are shown using a dotted line. Secondary reagents included anti-IgM-FITC and streptavidin-FITC.

Extensive homology seen between iGb3 synthase and a previously cloned murine alpha 1,3GalT prompted us to compare their ability to synthesize isoglobo-GSL. CHO cells transfected with either alpha 1,3GalT cDNA or iGb3 synthase cDNA express Galalpha 1,3Gal epitopes on their cell surface that are reactive with the lectin G. simplicifolia lectin I-B4 (Fig. 3). However, only 5% of CHO cells transfected with murine alpha 1,3-galactosyltransferase cDNA express GSL with terminal GalNAc as determined with the SMLDN1.1 mAb ±PPPP, whereas 70% of CHO cells transfected with iGb3 synthase cDNA are reactive with the mAb (Fig. 3). This suggests that CHO cells transfected with alpha 1,3GalT cDNA preferentially synthesize Galalpha 1,3Gal on glycoprotein substrates, most likely using the preferred acceptor Galbeta 1,4GlcNAcbeta 1-R (16). In contrast, CHO cells transfected with iGb3 synthase cDNA synthesize Galalpha 1,3Gal on GSL, primarily LacCer (Galbeta 1,4Glcbeta 1Cer) (see Fig. 5 below), producing iGb3 that is subsequently converted to iGb4 and iGb5 (reactive to SMLDN1.1 mAb).

Isolation of iGb5 from CHO Cells Transfected with iGb3 cDNA-- CHO cells were transfected with iGb3 synthase cDNA or Gb3 synthase cDNA and metabolically labeled with [3H]GlcNAc. The Gb3 synthase transfectants have been previously characterized as synthesizing the globo-series GSL, including Gb4 and Gb5 GSL (35) (Fig. 4, lane 2). Cells can incorporate [3H]GlcNAc into GlcNAc-, GalNAc-, and sialic acid-containing glycans. Glycolipids were extracted, isolated over a Sep-Pak C18 column, and separated by TLC. The resulting autoradiographs show a small amount of material migrating as a doublet at the position of Gb4 and a large amount of material migrating as a doublet at the position of Gb5 produced by CHO cells transfected with iGb3 synthase cDNA (Fig. 4, lane 1). This labeled GSL extract was purified on a Sep-Pak C18 cartridge and separated by HPLC using an Iatrobead column. Fractions containing each of the four major peaks were pooled and re-analyzed by TLC. The two fractions containing material that migrates with Gb5 (Fig. 4, lanes 3 and 6) were treated with exoglycosidases. Both bands migrating at the position of Gb5 were resistant to digestion with beta -N-acetylhexosaminidase (Fig. 4, lanes 5 and 8) but sensitive to alpha -N-acetylgalactosaminidase (Fig. 4, lanes 4 and 7). Following digestion with alpha -N-acetylgalactosaminidase, the two bands that had migrated at the position of Gb5 now migrate at the position of Gb4. Note that the loss in the intensity of the bands following digestion reflects the loss of one of the two [3H]GalNAc residues.


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  		Fig. 4.   Detection of isoglobo-GSL from CHO cells transfected with iGb3 synthase cDNA. TLC separation of glycolipid extracts from metabolically [3H]GlcNAc-labeled CHO cells transfected with iGb3 synthase cDNA (lanes 1, 3-8) or Gb3 synthase cDNA (lane 2). Material migrating with standard Gb4 and Gb5 is indicated by a the brackets. The labeled GSL from iGb3 synthase transfectants was separated by HPLC using an Iatrobead column (9), and pooled fractions of two peaks comigrating with Gb5 were re-run on TLC before (lanes 3 and 6) and after digestion with alpha -N-acetylgalactosaminidase (lanes 4 and 7) or beta -N-acetylhexosaminidase (lanes 5 and 8). The asterisk indicates GM3, which is also produced in parent CHO cells.

Glycolipids often migrate as doublets by TLC, reflecting a difference in the lipid tail rather than the carbohydrate structure (17). Digestion profiles similar to those described above were done using unlabeled glycolipids extracted from CHO cells transfected with iGb3 synthase cDNA. Following separation on TLC, the undigested glycolipids migrating at Gb5 and the alpha -N-acetylgalactosaminidase-digested glycolipids migrating at Gb4 both stained with SMLDN1.1 mAb, indicating the presence of terminal GalNAc (not shown). These results show that the major isoglobo-GSL isolated from CHO cells transfected with iGb3 synthase cDNA is iGb5. Note that, like parent CHO cells, CHO cells transfected with either murine alpha 1,3GalT cDNA (16) or the mutant 199AVA201 iGb3 synthase cDNA only synthesized GlcCer, LacCer, and GM3 in significant amounts, as determined by metabolically labeling with [3H]Gal or [3H]GlcNAc (not shown). A number of additional GSL products from CHO cells transfected with iGb3 synthase are observed when cells are metabolically labeled with [3H]Gal, but the identity of these products has not yet been established (data not shown).

In Vitro Activity of iGb3 Synthase-- Culture supernatant or detergent extracts from CHO cells transfected with iGb3 synthase cDNA were assayed for activity against several GSL acceptor substrates. An increase of approximately 100- to 200-fold in galactosyltransferase activity, using LacCer as acceptor, is seen when comparing extracts from CHO cells expressing iGb3 synthase to those expressing murine alpha 1,3-galactosyltransferase or mock-transfected with vector, respectively. A faint radioactive product migrating with Gb3 standard is seen in extracts assayed from the CHO cells expressing murine alpha 1,3GalT (Fig. 5, lane 3), but it is minor when compared with the product from CHO cells expressing iGb3 synthase (Fig. 5, lane 11). Parent CHO cells express sufficient levels of endogenous LacCer to allow the detection of iGb3 synthase activity in vitro. This accounts for the appearance of material migrating at the position of Gb3 in all cell extracts expressing iGb3 synthase, even in the absence of exogenous LacCer (Fig. 5, lane 9). Neither GlcCer nor Gb5 (not shown) are able to act as acceptor substrates. Significant, but lower activity was observed in the iGb3 synthase transfectants when using GalCer and Gb3 (Fig. 5, lanes 10 and 12), which contain terminal beta - and alpha -linked Gal, respectively. The predicted structure from the Gb3 reaction is Galalpha 1,3Galalpha 1,4Galbeta 1,4Glcbeta 1Cer, a GSL found in the rat small intestine (18). This is also the underlying structure of III3Galalpha Gb3Cer (Galalpha 1,3Galalpha 1,3Galalpha 1,4Galbeta 1,4Glcbeta 1Cer), which is present in the cryptic cells and circular muscle of the rat small intestine (19). The proposed structure for the product from the Galbeta 1Cer reaction, Galalpha 1,3Galbeta 1Cer, represents a novel GSL, to the best of our knowledge. CHO cells expressing iGb3 synthase secrete an active form of the enzyme into the culture supernatant that acts on LacCer but not on GalCer (Fig. 5, lanes 13 and 14). Radioactive material near the origin of the TLC plates is seen in all cell extracts and is nonspecific.


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  		Fig. 5.   TLC of [3H]Gal GSL products from the in vitro galactosyltransferase assay. Cell extracts from parent CHO cells, or cells transfected with either murine alpha 1,3GalT cDNA or iGb3 synthase cDNA, are used as enzyme source. Lanes 13 and 14 show products using supernatant from iGb3 synthase transfectants as the enzyme source. Products obtained using the exogenous acceptor substrates indicated along the bottom of the TLC were isolated following the enzyme reaction by Sep-Pak C18 prior to separation by TLC in a chloroform:methanol:water solvent (65:25:4, v/v). Arrows indicate material comigrating with a, LacCer; b, Gb3; and c, Gb4 standards.

CHO Cells Expressing iGb3 Synthase Produce iGb3 in Vitro-- Cell extracts from CHO cells expressing iGb3 synthase show high levels of galactosyltransferase activity toward LacCer, yielding a product that migrates at the position of Gb3 standard. Digestion of this isolated [3H]Gal in vitro product with specific glycosidases revealed the terminal galactose to be in an alpha 1,3-linkage (Fig. 6, lane 2). Hence, CHO cells expressing the iGb3 synthase cDNA produce iGb3 (Galalpha 1,3Galbeta 1,4Glcbeta 1Cer). This confirms the FACS analysis, where CHO cells transfected with iGb3 synthase are reactive to G. simplicifolia lectin I-B4 (a lectin that recognizes alpha 1, 3-galactosyl residues) but not to anti-CD77 mAb (a monoclonal antibody that binds to Gb3, Galalpha 1,4Galbeta Glcbeta 1Cer) (Fig. 3). Similarly, total GSL extracts from CHO cells expressing iGb3 synthase do not react with the B-subunit of Shiga toxin (35), which specifically binds Gb3 (20). The globoside and Forssman structures produced in CHO transfected with iGb3 synthase cDNA must, therefore, be synthesized on the iGb3 glycolipid, Galalpha 1,3Galbeta 1,4Glcbeta 1Cer rather than the more common Gb3, Galalpha 1,4Galbeta 1,4Glcbeta 1Cer (see Fig. 8).


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  		Fig. 6.   Exoglycosidase digestion of the iGb3 synthase in vitro product. The isolated [3H]Gal product from an in vitro enzyme assay using LacCer as acceptor substrate and cell extract from CHO cells transfected with iGb3 synthase cDNA was treated with exoglycosidases. After digestion the products were purified on Sep-Pak C18 and analyzed by autoradiography following TLC. Digests are: lane 1, mock-digested; lane 2, alpha 1,3-galactosidase; and lane 3, beta 1,3,4,6-galactosidase.

Expression of iGb3 Synthase in Rat Tissues-- RT-PCR using iGb3 synthase-specific primers shows a widespread tissue expression of the iGb3 synthase enzyme (Fig. 7). Highest levels of expression are seen in the spleen, thymus, and skeletal muscle. The lung, uterus, pituitary, and heart have intermediate iGb3 synthase expression, whereas other tissues indicate low or undetectable amounts. Similar amounts of control glyceraldehyde-3-phosphate dehydrogenase gene expression is seen in all tissues (35).


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  		Fig. 7.   Expression of iGb3 synthase RNA in rat tissues. Total RNA was isolated from rat tissues and assayed for iGb3 synthase RNA expression using RT-PCR and gene-specific primers. Gene-specific primers for glyceraldehyde-3-phosphate dehydrogenase RNA, a housekeeping gene, produced similar levels of product in each tissue (35). The expected iGb3 synthase PCR product size of 780 base pairs is indicate by the arrow. pop. LN, popliteal lymph node; mes. LN, mesenteric lymph node; and skel. muscle, skeletal muscle.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have described the cloning of a new member of the ABO gene family, iGb3 synthase, an alpha 1,3-galactosyltransferase that initiates the synthesis of isoglobo-GSL. LacCer (Galbeta 1,4Glcbeta 1Cer) is the preferred acceptor substrate for iGb3 synthase, however, alternative substrates include Galbeta 1Cer and, surprisingly, Gb3 (Galalpha 1,4Galbeta 1,4Glcbeta 1Cer). This suggests that iGb3 synthase is able to utilize multiple substrates containing a terminal Gal residue, irrespective of its anomeric configuration (Fig. 8). Hence, iGb3 synthase may be regarded as an exception to the one-linkage-one-enzyme rule. The ability to add alpha 1,3-linked Gal to these other structures may result in the appearance of novel structures in tissues depending on the available substrates. Even though iGb3 synthase acts principally on GSL, treatment of cells expressing iGb3 synthase with PPPP did not completely abolish the expression of Galalpha 1,3Gal seen using the lectin, G. simplicifolia lectin I-B4 (data not shown). It remains to be determined if this PPPP-resistant expression of the Galalpha 1,3Gal epitope is occurring on GalCer or glycoprotein acceptor substrates.


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  		Fig. 8.   The major mammalian GSL pathways. Major GSL series are noted in boxes. Structures with Galalpha 1,3Gal represent the product catalyzed by iGb3 synthase (thick arrows). Isoglobo-series are synthesized in CHO cells following transfection with iGb3 synthase cDNA. Black arrows indicate reactions in the biosynthetic pathways where the pertinent glycosyltransferases are cloned. Digalacto GSL represents a novel glycolipid structure.

Previously cloned alpha 1,3-glycosyltransferases (alpha 1,3GalT and Forssman or i/Gb5 synthase) are members of the ABO gene family. They have been described in numerous mammals but are nonfunctional in Old World primates, including humans (21, 22). These enzymes differ in their substrate specificity and may therefore have different functional significance. GSL containing the iGb3 structure have been described in fish, rat, dog, pig, and horse (23-27). At present it is not known if homologues of the rat iGb3 synthase exist in other species. The phenotypic cloning procedure used to clone iGb3 synthase has revealed that iGb3 may be efficiently capped with GalNAc residues to form iGb4 and iGb5. The presence of structures arising from iGb3 may not be apparent in other species, because iGb4 and iGb5 react with many of the same immunological reagents as Gb4 and Gb5, respectively. The fixative procedures used in the detection of Galalpha 1,3Gal epitopes on glycoproteins may eliminate or reduce the levels of iGb3.

Humans do not normally express the Galalpha 1,3Gal epitope and as a result have high circulatory levels of the natural anti-Galalpha 1,3Gal antibody (28). These antibodies mediate acute rejection of xenotransplants from species that express the Galalpha 1,3Gal epitope. The anomalous expression of Galalpha 1,3Gal epitope in humans has been described in human cancer cells and in the thyroid from patients with the autoimmune disorder, Graves' disease (29, 30). Furthermore, small amounts of Galalpha 1,3Gal have been reported on red cells (31). Low levels of Galalpha 1,3Gal expression in humans may reflect the activity of an alternative alpha 1,3-galactosyltransferase, possibly iGb3 synthase. The accumulation of GSL containing iGb3 structures has been noted in rat hepatomas, mammary adenocarcinomas, and tumors of colorectal origin (32-34). Furthermore, the presence of multiple alpha 1,3-galactosyltransferases may have an important clinical consequence in animals currently being assessed for xenotransplantation. Should they express the Galalpha 1,3Gal epitope on both glycoproteins and glycolipids, it may be essential to eliminate these epitopes from both proteins and lipids to prevent xenotransplant rejection.

In summary, we have cloned iGb3 synthase, an alpha 1,3-galactosyltransferase, that initiates the synthesis of the isoglobo-series GSL. This enzyme also has the ability to act on two further substrates found in different GSL series, the globo-series and the galactosylceramides. With the recent cloning of the Gb3 synthase (35), gene ablation studies will be possible to determine the functional significance of different GSL series.

    ACKNOWLEDGEMENTS

We thank Dr. D. Haslam for his helpful suggestions and generous gifts of the anti-Forssman (Gb5) and anti-SSEA-3 MC631 (GalGb4) mAb. We are also indebted to Dr. P. Smith for providing the RPL18 cDNA library and to Dr. J. Lowe for supplying the alpha 1,3GalT cDNA.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant R01-DK 41738 (to J. U. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF248543.

§ To whom correspondence should be addressed: Tel.: 314-362-8733; Fax: 314-362-8888; E-mail: jkeusch@pathbox.wustl.edu.

Current address: Dade-Behring Inc., Newark, DE.

Published, JBC Papers in Press, June 14, 2000, DOI 10.1074/jbc.M002629200

1 The abbreviations for the glycosphingolipids are in accordance with the 1997 recommendations of the IUPAC-IUB Joint Commission of Biochemical Nomenclature. The abbreviations used are: GSL, glycosphingolipid; LacCer, lactosylceramide (Galbeta 1,4Glcbeta 1Cer); Cer, ceramide; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; Gb3, Pk, CD77 or globotriaosylceramide (Galalpha 1,4Galbeta 1,4Glcbeta 1Cer); iGb3, isoglobotriaosylceramide (Galalpha 1,3Galbeta 1,4Glcbeta 1Cer); Gb4, globoside (GalNAcbeta 1,3Galalpha 1,4Galbeta 1,4Glcbeta 1Cer); iGb4, isogloboside (GalNAcbeta 1,3Galalpha 1,3Galbeta 1,4Glcbeta 1Cer); Gb5, Forssman (GalNAcalpha 1,3GalNAcbeta 1,3Galalpha 1,4Galbeta 1,4Glcbeta 1Cer); iGb5, isoForssman (GalNAcalpha 1,3GalNAcbeta 1,3Galalpha 1,3Galbeta 1,4Glcbeta 1Cer); GM3, Neu5Acalpha 2,3Galbeta 1,4Alcbeta 1Cer; mAb, monoclonal antibody; PPPP, 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol, hydrochloride; TLC, thin layer chromatography; FACS, fluorescence-activated cell sorting; RT-PCR, reverse transcriptase-polymerase chain reaction; PBS, phosphate-buffered saline; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; MES, 2-(N-morpholino)ethane sulfonic acid; HPLC, high performance liquid chromatography; Neu5Ac, sialic acid.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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