John Connell Laboratory for Glycobiology, Austin Research Institute, Austin and Repatriation Medical Centre, Heidelberg, VIC, 3084, Australia
Received on July 9, 2002; revised on November 12, 2002; accepted on November 12, 2002
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Abstract |
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Key words:
1,3galactosyltransferase
/
Gal
(1,3)Gal
/
glycolipid
/
xenotransplantation
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Introduction |
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The ABO blood group antigens are carbohydrate structures that, if mismatched during transplantation, can elicit a strong antibody mediated rejection process known as hyperacute rejection (HAR) (Nelson et al., 1992; Williams et al., 1968
). Likewise, the carbohydrate epitope Gal
(1,3)Gal, found on the cell surface of pig tissue, also causes HAR during pig-to-human xenotransplantation, mediated by naturally occurring human preformed anti-Gal
(1,3)Gal antibodies (Sandrin and McKenzie, 1999
; Sandrin et al., 1993
, 1994b
). For xenotransplantation to become a clinical reality, HAR must be overcome. Hence an active area of xenotransplantation is the characterization of Gal
(1,3)Gal glycoconjugates and their synthesis.
Gal(1,3)Gal is synthesized by the transferase
(1,3)galactosyltransferase (
(1,3)GT) which, like many other glycosylating enzymes, resides within the Golgi of the cell. It catalyzes the biochemical reaction on glycoproteins and glycolipids, transferring a UDP-galactose to N-acetyllactosamine (NAcLac) in an
(1,3) linkage to form Gal
(1,3)Gal (Blanken and Van den Eijnden, 1985
). The amino acid sequence of
(1,3)GT shares a high degree of homology with other cloned glycosyltransferases, including the A and B blood group transferases and Forssman synthase, all of which can be classified in the
(1,3)Gal/GalNAc transferase family. In many species Gal
(1,3)Gal was thought solely to be synthesized by one gene, the
(1,3)GT, but the recent cloning of rat iGb3 (isoglobotriaosylceramide) synthase (iGb3S) (Keusch et al., 2000b
), which also synthesizes Gal
(1,3)Gal, has suggested otherwise. The amino acid sequence of iGb3S has a much lower identity (
40%) compared with other mammalian
(1,3)GT transferases, such as pig, ox, and mouse, although still enough to classify it in the
(1,3)Gal/GalNAc transferase family. Functionally, iGb3S is unique in synthesizing the glycolipid structure iGb3, transferring an
-Gal from UDP-Gal to lactosyl-ceramide (Keusch et al., 2000b
). Synthesis of iGb3 is the initial committed step in the formation of the isoglobo-series glycolipid pathway and is the precursor to isogloboside (iGb4) and isoForssman (Keusch et al., 2000b
). To date, iGb3S is the only known transferase in rat to catalyze the formation of Gal
(1,3)Gal.
At least two Gal(1,3)Gal glycolipid structures have been isolated in the rat. The first is iGb3, the other is Gal
(1,3) Galß(1,4)GlcNAc
(1,3)Galß(1,4)Glcß1Cer (Gal
(1,3)nLc4) (Ogiso et al., 1995a
,b
). Gal
(1,3)nLc4 is a well-characterized Gal
(1,3)Gal glycolipid structure isolated from many species that is synthesized by the
(1,3)GT (Basu and Basu, 1973
; Blanken and Van den Eijnden, 1985
). This raises an important question of whether iGb3S is the only Gal
(1,3)Gal synthesizing transferase in rat or whether the rat expresses the
(1,3)GT homolog as found in other species. It was assumed by Keusch et al. (2000b)
that the rat iGb3S was a different enzyme from
(1,3)GT; to investigate this and formally show the presence of two
1,3galactosyltransferases in one species, clones encoding rat
(1,3)GT were isolated from spleen cell mRNA using reverse-transcription polymerase chain reaction (RT-PCR) and oligonucleotide primers to the mouse
(1,3)GT. These were characterized and their expression and function compared to that of rat iGb3S. The finding of two
1,3galactosyltransferases has significant implications for xenotransplantation, where major efforts have been directed to the production of cloned pigs with an inactivated
(1,3)GT with a view toward producing Gal
(1,3)Gal- pigs.
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Results |
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From the genomic sequence of rat (1,3)GT the gene's exon/intron boundaries were elucidated and showed a preservation and arrangement of exons identical to that of mouse
(1,3)GT (Table I). The rat
(1,3)GT coding region is encoded by six exons: exons 4, 5, 6, 7, 8, and 9 (based on the numbering of mouse
(1,3)GT). Compared to the mouse, exons 5 and 7 have minor base changes at their exon splice boundaries (Table I). The isolated cDNA sequence represents an mRNA splice variant that lacks exons 5 and 6; thus, the nucleotide changes in exon 5 and 7 splice boundaries do not seem to affect splicing at these sites. This is consistent with observations in the mouse, in which a number of mRNA splice isoforms are found, with the splicing out of either exon 5, exon 6, or both (Joziasse et al., 1992
).
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One interesting finding was the large array of bands formed by iGb3S expression migrating at and below Gal(1,3)nLc4 (Figure 8, lane 2). These glycolipids are either due to the direct glycosylation by iGb3S using its own synthesized product as an acceptor or the action of another endogenous transferase using iGb3 as an acceptor. To investigate this further, Gb3 synthase (Gb3S) glycolipid expression was compared with iGb3S. Apart from the isoglobo-series pathway in which iGb3S synthesizes an
(1,3)Gal linkage, an analogous pathway exists, the globo-series pathway, in which Gb3S synthesizes an
(1,4)Gal linkage to Lac-Cer (Keusch et al., 2000a
). Expression of Gb3S enables the downstream expression of Forssman. The glycolipids isolated from Gb3S expressing CHOP cells show similarities and differences in the banding pattern compared to iGb3S expressing cells (Figure 9, lanes 1 and 3). Similar to iGb3S, there is the presence of a doublet migrating with CTH, corresponding to Gb3 and a second doublet migrating below Gb3 corresponding to Gb4, although in both cases much more intense. In contrast, there are no bands migrating at or below Gal
(1,3)nLc4 as is observed for iGb3S. The greater intensity of the Gb3 and Gb4 band can be explained by the inability of Gb3S to synthesize the large array of glycolipid structures migrating at or below Gal
(1,3)nLc4 seen for iGb3S. Therefore Gb3 can only be converted toGb4, whereas iGb3 is converted to multiple glycolipid products migrating below Gal
(1,3) nLc4, hence depleting the amount of steady-state iGb3 present in the cell. This does, however, assume that these glycolipid structures are caused by the direct utilization of the iGb3 structure. When iGb3S and Gb3S glycolipids are treated with coffee bean
-galactosidase (Figure 9, lanes 2 and 4), which can cleave
-Gal, the iGb3S bands migrating below Gal
(1,3)nLc4 were cleaved (Figure 9, lane 2) and converted back to Lac-Cer, shown by a significant intensity increase in this doublet. This shows that these structures are in fact a direct utilization of iGb3. In contrast, in Gb3S-expressing cells Gb3 was sensitive due to the cleavage of
(1,4)Gal (Figure 9, lane 4). This shows the existence of large
-Gal linked glycolipids derived from iGb3, which are either synthesized by iGb3S or by the action of another transferase endogenous in CHOP cells, which can only utilize iGb3 and not Gb3.
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Discussion |
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Rat (1,3)GT has a more similar genomic sequence and genetic structure to mouse
(1,3)GT than iGb3S, as shown by Southern blot analysis using restriction mapping with PvuII (Figure 2). This becomes more apparent when comparing the conservation of exon boundaries and number of exons present for rat
(1,3)GT and mouse
(1,3)GT (Figure 3). Interestingly, the rat
(1,3)GT clone isolated here (Figure 1) lacks exons 5 and 6, a feature not uncommon to
(1,3)GT cDNA clones isolated from mouse and pig (Joziasse et al., 1992
; Vanhove et al., 1997
). This provides evidence that, like mouse and pig,
(1,3)GT in the rat may express other mRNA splice variants of exons 5, 6, or 7. The amino acid alignments (Figure 3) between rat
(1,3)GT and other
(1,3)GT of other species, such as mouse, ox, and pig, typically shows >70% identity. This clearly defines this transferase to be the
(1,3)GT homolog in rats. Rat iGb3S, on the other hand, has a much lower identity,
40%, indicating that the two transferases,
(1,3)GT and iGb3S, probably diverged in the rat from a single ancestral gene to take up different functions within the rat. This is further supported by the differential expression of the two transferases shown by northern blot analysis (Figure 4).
The rat (1,3)GT is functionally active and capable of transferring
-Gal to glycoconjugates, forming Gal
(1,3)Gal on the cell surface of transfected CHOP cells, as is found for mouse
(1,3)GT and to a lesser extent, iGb3S (Figure 5). What makes rat
(1,3)GT distinct from iGb3S is its inability to cause the downstream synthesis of isoForssman (Figure 6), or to transfer an
-Gal to Lac-Cer to form the isoglobo-series glycolipid structure iGb3 (Figure 8). Furthermore, rat
(1,3)GT is capable of synthesizing Gal
(1,3)Gal on glycoproteins, a function not detected for iGb3S (Figure 7). This evidence unequivocally leads us to conclude that rat
(1,3)GT and iGb3S are functionally distinct transferases synthesizing Gal
(1,3)Gal by very different biochemical pathways.
The inability of iGb3S to glycosylate glycoproteins was based on IB4 binding to Gal(1,3)Gal structures in western blots (Figure 7). Unfortunately IB4 was less effective at detecting iGb3S derived cell surface Gal
(1,3)Gal compared to rat
(1,3)GT (Figure 5) and may therefore not be as sensitive at detecting such structures. To improve the detection, a mouse monoclonal antibody specific for Gal
(1,3)Gal that detects iGb3S synthesized structures on transfected cells more effectively than for rat
(1,3)GT derived structures was also used in parallel for the immunoprecipiation studies. The result obtained (not shown) confirmed the IB4 data shown here (Figure 7). Previously, Keusch et al. (2000b)
, used an inhibitor of glucosylceramide synthase, phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol to block glycolipid synthesis on iGb3S expressing CHO cells. Surprisingly, levels of Gal
(1,3)Gal expression were still detected despite the use of this inhibitor. The results were attributed to either Gal
(1,3)Gal-synthesized glycoproteins (which, in light of our results, seems unlikely) or, a more probable explanation, the synthesis of Gal
(1,3)Gal on galactosylceramide forming the novel structure Gal
(1,3)Galß1Cer. In theory, a distinction in protein/lipid utilization between rat
(1,3)GT and iGb3S does seem plausible with rat
(1,3)GT like other
(1,3)GT, utilizing terminal NAcLac, expressed on both glycoproteins and glycolipids, whereas iGb3S utilizes a terminal lactose, a structure in mammalian cells only found on glycolipids. These findings open up an important biological question of why two Gal
(1,3)Gal synthesizing transferases, producing Gal
(1,3)Gal structures by different biochemical pathways, are required in the same species.
A glycolipid analysis of rat small intestine shows that a number of unique poly--Gal structures have been synthesized: Gal
(1,3)Gal
(1,4)LacCer, Gal
(1,3)Gal
(1,3) Gal
(1,3)Gal
(1,4)LacCer, and larger repeats (Breimer et al., 1981
). All these structures are on a core backbone Gb3 glycolipid sturcture. Our glycolipid analysis shows iGb3S transfected cells are capable of forming such large (equal to or greater than Gal
(1,3)nLc4) poly-
-Gal structures as determined by their sensitivity to coffee bean
-galactosidase (Figure 9). Surprisingly, especially given the glycolipid structures isolated from the rat small intestine (Breimer et al., 1981
), Gb3S was incapable of forming these poly-
-Gal structures. The evidence discounts the possibility that another transferase galactosylates iGb3 in CHOP cells as it should also be capable of utilizing Gb3. In vitro enzyme assays from cell lysates with iGb3S activity (Keusch et al., 2000b
) have shown the synthesis of Gal
(1,3) Gal
(1,4)Lac using Gb3 as the acceptor substrate. Thus we propose a model of glycolipid synthesis for rat iGb3S in which it is capable of glycosylating Lac-Cer to produce iGb3, which in turn can be further glycosylated to form larger poly-
-Gal structures (Figure 10). In addition iGb3S can also utilize Gb3 if present, again forming large poly-
-Gal structures (Figure 10). It is therefore apparent that the acceptor specificity of iGb3S is unusual compared to other transferases with a common requirement for a terminal galactose irrespective of anomer or linkage. Another feature of iGb3S is its ability, in in vitro enzyme assays, to transfer
-Gal to Galß1Cer, forming Gal
(1,3) Galß1Cer (Keusch et al., 2000b
), although our in vivo glycolipid analysis of coffee bean
-galactosidase treatment was unable to show any increase in Galß1Cer (Figure 9). However, we cannot eliminate the possibility that Gal
(1,3)Galß1Cer exists as minor product of iGb3S expression in vivo and was not detected by our analysis.
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It seems likely that other species could also express iGb3S and therefore have a second Gal(1,3)Gal synthesizing pathway of critical importance to xenotransplantation. Our unpublished data show that similar to the rat, both mice and pigs have genes for both
(1,3)GT and iGb3S as shown by genomic Southern blots. Northern blot studies show that both glycosyltransferases are expressed in different tissues in mice and pigs (unpublished data). Analysis of the mouse genomic structure shows that, like
(1,3)GT, the iGb3S coding region is also encoded by five exons. The human homolog is located on chromosome 1, but the results of mRNA analysis suggest that this is a nonprocessed pseudogene (unpublished data). Furthermore, using monoclonal anti-Gal antibodies residual staining was found in tissues, such as thymus, liver, and heart, of Gal o/o mice. We have presented this preliminary data on mouse and pig iGb3S at the recent Xenotransplantation Meeting (Sandrin et al., 2001
) and the International Transplantation meeting (Sandrin et al., 2002
). Furthermore an iGb3 structure has been identified in hog stomach mucosa in a fucosylated form (Fuc
(1,2)Gal
(1,3)LacCer) (Slomiany et al., 1974
), providing further evidence that iGb3S is expressed in pig. This could present problems for xenotransplantation, especially if iGb3S is expressed in other tissues of the pig, although it needs to be established whether the Gal
(1,3)Gal glycolipids synthesized by iGb3S are involved in HAR. In attempts to produce pigs devoid of Gal
(1,3)Gal strategies have assumed the presence of a single transferase,
(1,3)GT and thus focused on removal of
(1,3)GT or its product. This will require revision focusing on the removal of iGb3S and its product.
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Materials and methods |
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Cloning of the Rat Gb3 and iGb3 synthase
Rat Gb3S and iGb3S were PCR-amplified from the rat first-strand cDNA using the same conditions as for rat (1,3)GT. The oligonucleotide primers were designed from their relevant cDNA sequences (Keusch et al., 2000a
,b
) and are as outlined follows: iGb3S, MO509 5'CCCAAGCTTATGGCTCTGGAGGGACTCAGG3' (HindIII site underscored) and MO510 5' CGGGATCCTAGGTTCGCACCAGTGCGTA3' (BamHI site underscored); Gb3S, MO591 5'CCCAAGCTTATGTCCAAGCCCCCCGACTG3' (HindIII site underscored); and MO592 5'CGGGATCCTCACAAGTACATCGTCATGGCT3' (BamHI site underscored). The resultant amplified products were subcloned into pCDNA1 using the restriction enzyme sites (HindIII and BamHI).
Genomic Southern analysis
DNA extracted from rat spleen tissue or mouse tail tissue was prepared using a Puregene genomic DNA extraction kit (Gentra Systems Minneapolis, MN). Genomic DNA, 10 µg, was digested with the restriction enzyme PvuII and electrophoresised on a 1% agarose gel. Southern blotting was done (Sambrook and Maniatis, 1989) onto a nylon membrane, Highbond N+ (Amersham Pharmacia Biotech, Little Chalfton, United Kingdom). The membrane was hybridized with either the rat
(1,3)GT or rat iGb3S cDNA labeled with 32P. The membrane was washed in 0.1x Sodium chloride/sodium citrate buffer/1% SDS at 65°C and the remaining hybridized radioactivity cDNA detected by a phosphoimage screen (Molecular Dynamics, Sunnyvale, CA).
Rat northern analysis
A preblotted rat multitissue northern blot (Clontech, Palo Alto, CA) containing 2 µg poly A+ RNA of eight tissues (heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis) was hybridized with either the rat (1,3)GT cDNA or iGb3S labeled with 32P. The membrane was washed in 0.1xSodium chloride/sodium citrate buffer/1% SDS at 60°C and detected by a phosphoimage screen (Molecular Dynamics).
Transfections
CHOP cells (Heffernan and Dennis, 1991), were maintained in Dulbecco's modified Eagle's medium (DMEM) (Cytosystems, Sydney, Australia) with 10% fetal bovine serum. The cells were seeded in 6-well plates (Linbro, Aurora, OH) at 3x105 cells and 24 h later transfected using Lipofectamine Plus Reagent (Gibco BRL Life Technologies, Gaithersburg, MD). The following cDNAs were transfected: mouse
(1,3)GT, rat
(1, 3)GT, iGb3S, Gb3S, Forssman synthase (Haslam and Baenziger, 1996
) using 1 µg of each per well. After 48 h the cells were analyzed for expression.
FACS analysis
Transfected CHOPs were harvested from the six-well plates using 0.5 mM ethylenediamine tetra-acetic acid (EDTA)/phosphate buffered saline (PBS), washed with 0.5% (w/v) bovine serum albumin (BSA) (Commonwealth Serum Laboratories, Parkville, Victoria, Australia)/PBS. Cells (5x105) were stained, incubated on ice for 30 min with either IB4 (Vector, Burlingame, CA) conjugated to fluorescein isothiocyanate (FITC) at 10 µg/ml or anti-mouse Forssman antibody, M1/87 (Pharmingen, San Diego, CA, and Becton Dickinson, Franklin Lakes, NJ) at 5 µg/ml. The anti-Forssman antibody stained cells were washed and incubated for a further 30 min on ice in the presence of a sheep anti-mouse Ig conjugated to FITC (Silenus, Hawthorne, Victoria, Australia). The cells were washed after FITC staining and then analyzed cytoflurometericly using a fluorescence activated cell sorter (FACS), Facscalibur (Becton Dickinson).
Cell surface biotinylation
Transfected CHOP cells were harvested from plates using 0.5 mM EDTA/PBS, washed in biotinylation buffer (PBS [pH 8.0] containing 1 mM MgCl and 0.1 mM CaCl). The cell surface proteins of the cells were biotinylated by incubating 1x107 cells in 1 ml 0.5 mg sulfo-NHS-LC-biotin (Pierce)/biotinylation buffer for 40 min on ice. The cells were pelleted by centrifugation and the biotinylation reaction stopped by incubating the cells a further 10 min in DMEM/10% fetal calf serum and then washing a number of times in PBS. The labeled cells were lysed in 1 ml lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 2.5 mM EDTA [Sigma, St. Louis, MO], 1% NP40, 1 mM phenylmethylsulfonylfluoride [Sigma], and 1 mM aprotinin [Sigma]), for 20 min and the nuclei and membrane fractions removed by centrifugation at 12,000xg.
Immunoprecipitation
The lysate was precleared with 50 µl of packed BSA-conjugated Sepharose beads (Pharmacia, Uppsala, Sweden) per 100 µl lysate and incubated at 4°C for 2 h. A second preclearing step was carried out overnight at 4°C. The precleared lystate was then immunoprecipitated using 25 µl of packed IB4 conjugated Sepharose beads per 100 µl precleared lysate and incubated at 4°C for a further 24 h. The beads were washed in lysis buffer and the proteins separated by electorophoresis on a 7.5% SDSPAGE gel as per standard protocals (Coligan, 1995). The electrophoresised proteins were transferred to a nitrocellulose membrane, Protran (Schleicher & Schuell, Einbeck, Germany), blocked in 2.5% casein (BDH Laboratory Supplies, Poole, England)/Tris buffered saline (0.5 mM Tris, pH 7.5, 0.15 NaCl). The membrane was incubated in Tris buffered saline containing streptavidin-conjugated horseradish peroxidase (Amersham) for 1 h. The detection of bound horseradish peroxidase was by chemiluminescence using the reagent Renaissance (NEN Life Science, Boston, MA) and imaged using X-OMAT AR X-ray film (Kodak, Rochester, NY).
Metabolic labeling and glycolipid purification
CHOP cells 24 h posttransfection were metabolically labeled with 3H-galactose (NEN) at 35 µCi/ml. At 48 h posttransfection cells were harvested using 0.5 mM EDTA/PBS, washed in PBS buffer, and the glycolipids extracted as described by Haslam and Baenziger (1996). Briefly, cells were pelleted by centrifugation and resuspended in a volume of methanol equal to the cell pellet. One volume of chloroform was added and then vortexed; an additional volume of methanol was added drop-wise, until the organic and aqueous phases resolved to a single phase. The cell debris was removed by centrifugation, and the extracted glycolipids were dried under N2. For analysis the glycolipids were resuspended in 60 µl chloroform/methanol/water (65:35:8), and 10 µl was spotted onto high performance thin layer chromatography (HPTLC) plates (Merck, Darmstadt, Germany). The glycolipids were resolved on the plates using chloroform/methanol/water 65:35:8 then dried and sprayed with En3Hance reagent (NEN) before exposure to X-OMAT AR X-ray film (Kodak) at 80°C for 25 days.
Glycolipid exoglycosidase digestions
The extracted radiolabeled glycolipids (10 µl, approximately 30,000 cpm) were dried under N2 and resuspended in 50 mM sodium citrate (pH 6.5) containing 0.05% sodium taurodeoxycholate (Sigma) and 0.1 U -galactosidase (from green coffee beans, Sigma). The digestions were incubated at 37°C overnight, dried under vacuum, resuspended in 10 µl of chloroform/methanol/water (65:35:8), and separated by HPTLC as described.
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Acknowledgements |
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Footnotes |
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1 To whom correspondence should be addressed; e-mail: m.sandrin{at}ari.unimelb.edu.au
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Abbreviations |
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References |
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