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The Journal of Immunology, 2006, 176: 2448-2454.
Copyright © 2006 by The American Association of Immunologists

The Molecular Basis for Gal{alpha}(1,3)Gal Expression in Animals with a Deletion of the {alpha}1,3Galactosyltransferase Gene1,2

Julie Milland, Dale Christiansen, Brooke D. Lazarus3, Simon G. Taylor, Pei Xiang Xing and Mauro S. Sandrin4

The Austin Research Institute, Austin Health, Heidelberg, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The production of homozygous pigs with a disruption in the GGTA1 gene, which encodes {alpha}1,3galactosyltransferase ({alpha}1,3GT), represented a critical step toward the clinical reality of xenotransplantation. Unexpectedly, the predicted complete elimination of the immunogenic Gal{alpha}(1,3)Gal carbohydrate epitope was not observed as Gal{alpha}(1,3)Gal staining was still present in tissues from GGTA1–/– animals. This shows that, contrary to previous dogma, {alpha}1,3GT is not the only enzyme able to synthesize Gal{alpha}(1,3)Gal. As iGb3 synthase (iGb3S) is a candidate glycosyltransferase, we cloned iGb3S cDNA from GGTA1–/– mouse thymus and confirmed mRNA expression in both mouse and pig tissues. The mouse iGb3S gene exhibits alternative splicing of exons that results in a markedly different cytoplasmic tail compared with the rat gene. Transfection of iGb3S cDNA resulted in high levels of cell surface Gal{alpha}(1,3)Gal synthesized via the isoglobo series pathway, thus demonstrating that mouse iGb3S is an additional enzyme capable of synthesizing the xenoreactive Gal{alpha}(1,3)Gal epitope. Gal{alpha}(1,3)Gal synthesized by iGb3S, in contrast to {alpha}1,3GT, was resistant to down-regulation by competition with {alpha}1,2fucosyltransferase. Moreover, Gal{alpha}(1,3)Gal synthesized by iGb3S was immunogenic and elicited Abs in GGTA1 –/– mice. Gal{alpha}(1,3)Gal synthesized by iGb3S may affect survival of pig transplants in humans, and deletion of this gene, or modification of its product, warrants consideration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In pig-to-primate or human xenografts, Gal{alpha}(1,3)Gal has a central immunological role in hyperacute rejection (HAR)5 as it is bound by natural Abs (1, 2). If HAR can be prevented, the next rejection phenomenon unique to xenotransplantation is delayed xenograft rejection, also called acute vascular rejection (AVR) (3, 4). Although the mechanisms for AVR are unclear, it is most likely that anti-Gal{alpha} (1,3)Gal Abs play a central role (5), in part, by activating endothelial cells (6). Furthermore, recent studies show that anti-Gal{alpha}(1,3)Gal IgG is required for human NK cell migration across porcine aortic endothelial cells in vitro, a process that is independent of Ab-dependent cellular cytotoxicity (7). Gal{alpha}(1,3)Gal is also directly recognized by NK cells (8) although the nature of the receptor is not known.

Systemic coagulation disturbances that result in disseminated intravascular coagulation also occur during AVR (9). Disseminated intravascular coagulation is a consequence of xenoreactive Abs (natural or elicited) binding to and activating endothelial cells, or unidentified cross-species molecular incompatibilities.

Given the essential role of Gal{alpha}(1,3)Gal in the various phases of rejection, the production of homozygous pigs with a disruption in the GGTA1 gene (10, 11, 12), which encodes {alpha}1,3galactosyltransferase ({alpha}1,3GT), clearly represented a critical step toward the clinical reality of xenotransplantation. Analysis of GGTA1–/– pig fetal fibroblasts with Griffonia simplicifolia B4 (IB4) lectin, the standard lectin used to detect Gal{alpha}(1,3)Gal, showed that these cells were Gal{alpha}(1,3)Gal negative. These results were in accordance with those obtained for typing GGTA1–/– mice (13, 14). However, several years ago, we produced anti-Gal{alpha} (1,3)Gal mAbs that, unlike the IB4 lectin, stained a wide range of tissues in GGTA1–/– mice. Recently, Sharma et al. (15) showed that cells from GGTA1–/– pig lines also express low levels of Gal{alpha}(1,3)Gal when stained with anti-Gal{alpha}(1,3)Gal mAbs.

{alpha}1,3GT is not the sole enzyme able to synthesize Gal{alpha}(1,3)Gal. The most likely candidate for the production of Gal{alpha}(1,3)Gal observed in GGTA1–/– animals is iGb3 synthase (iGb3S) (16), another member of the ABO blood group glycosyltransferase family. In the rat, synthesis of Gal{alpha}(1,3)Gal was initially ascribed to iGb3S alone (16). Subsequently, we demonstrated that the rat has two functional {alpha}1,3galactosyltransferases, the reported iGb3S (16) and the ortholog of {alpha}1,3GT (17). This raised the possibility that iGb3S may also be present in other species and therefore account for the remaining Gal{alpha}(1,3)Gal expression in GGTA1–/– animals. Recently, it was suggested that iGb3 lipid or a close structural analog may be recognized by NKT cells in mice and humans (18).

We show that iGb3S is an additional enzyme in the mouse that can synthesize the reactive xenoepitope Gal{alpha}(1,3)Gal. iGb3S cDNA was cloned from the thymus of GGTA1–/– mice. The predicted protein is a 370-aa type II integral membrane protein with a cytoplasmic domain that is atypically long for a glycosyltransferase. In contrast to {alpha}1,3GT, iGb3S synthesizes Gal{alpha}(1,3)Gal uniquely on the isogloboside subset of glycolipids, called iGb3. Importantly, iGb3S-synthesized Gal{alpha}(1,3)Gal elicits an Ab response in GGTA1–/– mice. It is evident that synthesis of the immunogenic Gal{alpha}(1,3)Gal carbohydrate is more complex than originally thought and current protocols have failed to completely eliminate all Gal{alpha}(1,3)Gal. At this point in time, it is unknown what the functional consequence(s) of this lipid-linked Gal{alpha}(1,3)Gal synthesized by iGb3S will be in a pig-to-primate xenograft. However, we propose that this enzyme may impact on the survival of pig tissues after transplantation into humans.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

C57BL/6 and GGTA1 –/– mice were maintained at The Austin Research Institute; mice aged 8–12 wk were used for preparation of splenocytes and for tissue sections. GGTA1–/– mice were originally obtained from the University of Michigan (13). Pig tissues were obtained from freshly slaughtered pigs at the abattoirs.

Antibodies

Human anti-Gal{alpha}(1,3)Gal IgG Abs were purified as previously described (19). mAbs were produced after fusing splenocytes from GGTA1–/– mice (immunized with rabbit RBC) with NS-1 hybridoma cells. Specificity was originally selected using Gal{alpha}(1,3)Gal-specific ELISAs. Two of these clones, 15.101 and 22.121, were used in this study. The Ig gene usage and binding specificity to Gal{alpha}(1,3)Gal-containing carbohydrates has been characterized for these Abs (20).

Cell culture and transfection

The pig endothelial cell line, PIEC, was a gift from K. Welsh (Transplant Immunology, Oxford Transplant Centre, Churchill Hospital, Oxford, U.K.). Primary splenic fibroblast cultures were prepared from GGTA1–/– mice or C57BL/6; single cells were prepared from spleens and cultured in DMEM (CSL) supplemented with 10% FCS overnight at 37°C. The medium was changed, removing nonadherent cells, and fibroblasts were grown until confluent when they were used for analysis. Human embryonic kidney cells (E-293) and CHOP cells (Chinese hamster ovary cells transformed with polyoma large T Ag) (21) were cultured in DMEM with 10% FCS. Transfections were with LipofectAMINE Plus (Invitrogen Life Technologies) as recommended by the manufacturer. Cells were examined after 48 h for cell surface expression of Gal{alpha}(1,3)Gal using FITC-labeled IB4 lectin (Sigma-Aldrich) and mAbs 15.101 and 22.121, iso-Forssman glycolipid using an anti-Forssman mAb, M1/87 (Sigma-Aldrich). Carbohydrate inhibition studies using lactose and galactose disaccharide were as described (1). All mAbs were detected with FITC-labeled sheep anti-mouse IgG (DakoCytomation) and either microscopy or flow cytometry (BD Biosciences FACSCalibur). Experiments were repeated three times.

RNA analysis

Northern blotting was performed (22) on a mouse Multiple Tissue Northern blot (Sigma-Aldrich) and a second blot containing poly(A)+ RNA (RNAeasy Maxi and Oligotex kits; Qiagen) from GGTA1–/– mouse thymus (13), PIEC, pig thymus, and lung. Both blots were probed with rat iGb3S cDNA (16, 17) radiolabeled using the Megaprime DNA labeling system (Amersham Biosciences) and exposed to a phosphor screen for 1 wk and scanned by the Storm840 PhosphorImager (both from Molecular Dynamics).

Cloning mouse iGb3S

Mouse iGb3S cDNA was amplified using the TITANIUM One-Step RT-PCR kit (BD Clontech). Using a series of degenerate primers designed from the rat iGb3S cDNA sequence (16), we were unable to clone mouse iGb3S. A search of the National Center for Biotechnology Information (NCBI) mouse genome database showed a predicted cDNA sequence for mouse iGb3S cDNA and oligonucleotides were designed. The oligonucleotide encoding the N terminus was 5'-ATGGAAGTTGCCGAGAACAAGAAGGAC-3' and for the C terminus 5'-CTACTTTCGCACCAGCGTATATTCCTTGG-3'. Amplification and DNA sequencing (using Big Dye 3.1; Applied Biosystems) of the generated fragment showed a four-base deletion in the N-terminal sequence compared with the predicted sequence in the database, suggesting that the database initiation methionine was incorrect. We examined the intron sequence immediately 5' of the predicted first coding exon and found an in-frame initiation methionine existing 45 nucleotides (15 aa) upstream. An oligonucleotide was designed using this sequence (5'-ATGGCTCTGGGGACAGAGTTGGGAG-3'). Amplification from RNA using this oligonucleotide gave a product that enabled an in-frame translation of the open reading frame of mouse iGb3S.

The mouse and rat genomic iGb3S sequences were obtained by searching mouse and rat genomic databases (<www.ncbi.nlm.nih.gov/BLAST/and http://www.ensembl.org/>) using the cloned mouse and rat iGb3S cDNAs as reference. Two contig records were identified: NT 039265 (Mus musculus chromosome 4 genomic contig, strain C57BL/6J) and NW 047721 (Rattus norvegicus chromosome 5 WGS supercontig). From these genomic sequences the exon/intron boundaries were elucidated for both the mouse and rat iGb3S genes (data not shown).

Canine Forssman synthetase (FS) cDNA was a gift from Dr D. B. Haslam (Department of Pediatrics, Washington School of Medicine, St. Louis, MO) (23). The cDNA for pig {alpha}1,2fucosyltransferase ({alpha}1,2FT) was as previously reported (24).

Microscopy

Fixed mouse or human tissue sections were stained with anti-Gal{alpha}(1,3)Gal mAb, 15.101, and streptavidin-HRP and counterstained with hematoxylin and examined as described previously (25).

Serum Ab analysis

GGTA1–/– mice (in groups of five) were injected with 5 x 106 E-293 cells, either nontransfected or transfected with pig {alpha}1,3GT (26) or rat iGb3S. Three injections were given 1 wk apart, starting on day 0, and blood was collected on day 21 and serum was generated. Anti-CD4 Ab (GK1.5) was injected on days –1, 0, and +1. The amount of GK 1.5 injected was 1 mg/mouse over three injections. Levels of Abs to Gal{alpha}(1,3)Gal were measured using an ELISA as previously described (19). The results represent the mean of data for each individual mouse, i.e., serum was not pooled.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The Gal{alpha}(1,3)Gal epitope is expressed in GGTA1–/– mice

The unexpected presence of the Gal{alpha}(1,3)Gal epitope on GGTA1–/– animals was confirmed on splenic fibroblasts from GGTA1–/– mice (Fig. 1). The level of surface Gal{alpha}(1,3)Gal on GGTA1–/– fibroblasts was typically 10–20% of that observed for Gal{alpha}(1,3)Gal+ fibroblasts using a Gal anti-Gal{alpha}(1,3)Gal mAb (Fig. 1A). Similar results were obtained using affinity purified human anti-Gal{alpha}(1,3)Gal Ig (Fig. 1B). Carbohydrate inhibition studies confirmed mAb specificity as binding of the mAb to GGTA1–/– fibroblasts was almost completely inhibited with 2 mM Gal{alpha}(1,3)Gal disaccharide (Fig. 1C) but not with 2 mM lactose (Galbeta(1,4)Glu) (Fig. 1D).


Figure 1
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  		FIGURE 1. Anti-Gal{alpha}(1,3)Gal Ab binds to fibroblasts from GGTA1–/– mice and binding is specifically inhibited. A, Anti-Gal(1,3)Gal mAb, 22.121, or B, human anti-Gal IgG staining of splenic fibroblasts from GGTA1–/– mice (bold lines) or C57BL/6 mice (solid lines). Background secondary Ab staining on both types of cells is shown (broken lines). C, Splenic fibroblasts from GGTA1–/– mice are stained with anti-Gal{alpha}(1,3)Gal mAb, 22.121, (bold line), with anti-Gal{alpha}(1,3)Gal mAb preincubated with 2 mM Gal{alpha}(1,3)Gal (solid line) or with secondary Ab (broken line). D, Splenic fibroblasts from GGTA1–/– mice are stained with anti-Gal{alpha}(1,3)Gal mAb, 22.121, (bold line), with anti-Gal{alpha}(1,3)Gal mAb preincubated with 2 mM lactose (solid line and superimposed by the bold line) or with secondary Ab (broken line).

 
The presence of Gal{alpha}(1,3)Gal was also examined in fixed tissue sections from GGTA1–/– mice using the anti-Gal{alpha}(1,3)Gal mAb, 22.121. In the pancreas of GGTA1–/– mice, Gal{alpha}(1,3)Gal staining was observed in the cytoplasm of cells of the islets of Langerhans and the acinar cells, the cytoplasm and cell surface of endothelial cells lining the small blood vessels and cells in the basement membrane of the small ducts (Fig. 2A). In contrast, staining was not observed in human pancreatic cells (Fig. 2B, note: the weak staining observed in some human erythrocytes is due to incomplete inhibition of endogenous peroxidase). In GGTA1–/– mice, lymphocytes in the red pulp area of spleen were strongly stained for Gal{alpha} (1,3)Gal, as were some lymphocytes in the white pulp (Fig. 2C). In contrast, lymphocytes infiltrating a human breast carcinoma did not stain for Gal{alpha}(1,3)Gal, nor did the carcinoma cells themselves (Fig. 2D). Staining was also observed in kidney (including endothelial cells) and liver cells of GGTA1–/– mice, but not in sections of human prostate or normal breast tissue (data not shown).


Figure 2
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  		FIGURE 2. The Gal{alpha}(1,3)Gal epitope is present on tissues from GGTA1–/– mice but not humans. Fixed tissue sections were stained by immunoperoxidase with anti-Gal{alpha}(1,3)Gal mAb, 15.101, followed by H&E. A, Pancreas from GGTA1–/– mice contained Gal{alpha}(1,3)Gal on the endothelium of blood vessels (upper white arrow) and on islets of Langerhans (lower white arrow) and acinar cells whereas, B, pancreas from humans did not stain for anti-Gal{alpha}(1,3)Gal (weak staining of human erythrocytes is due to endogenous peroxidase, white arrow). C, Lymphocytes in spleen from GGTA1–/– mice were positive for Gal{alpha}(1,3)Gal (white arrow) whereas, D, human lymphocytes infiltrating breast carcinoma cells were negative.

 
The presence of Gal{alpha}(1,3)Gal in the tissues of GGTA1–/– mice (i.e., after the removal of the {alpha}1,3GT gene) suggested that another transferase is able to synthesize the Gal{alpha}(1,3)Gal xenoepitope.

iGb3 synthase mRNA is expressed in mouse and pig tissues

The most likely candidate for the production of Gal{alpha}(1,3)Gal observed in GGTA1–/– animals is iGb3S. The expression of iGb3S mRNA was examined by Northern blot. Poly(A)+ RNA from thymus and lung of Gal{alpha}(1,3)Gal+ mice (Fig. 3A) contained iGb3S mRNA. Poly(A)+ RNA from thymi of GGTA1–/– mice was used to isolate iGb3S cDNA (see below) and a signal for iGb3S mRNA was observed in a Northern blot (data not shown). Similarly, poly(A)+ RNA from pig thymus, lung, and an endothelial cell line (PIEC) also contained iGb3S mRNA (Fig. 3B). Two species of mRNA of ~2.0 and 4.0 kb were observed (Fig. 3) similar to those reported for rat (17). Two mRNA species usually suggest alternate 3' untranslated regions and this is currently under investigation. The level of iGb3S mRNA in both mouse and pig was very low, as has been observed with other glycosyltransferases such as the GGTA1 in the rat (17).


Figure 3
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  		FIGURE 3. Expression of iGb3S mRNA. A, iGb3S mRNA expression in mouse tissues as labeled using Northern blot of 2 mg of poly(A)+ RNA. B, iGb3S mRNA expression in pig tissues as labeled using Northern blot of 2 mg of poly(A)+ RNA. Arrows indicate mRNA species of ~2 and 4 kb.

 
Cloning and exon arrangement of mouse iGb3S cDNA

Mouse iGb3S cDNA was isolated from GGTA1–/– thymus mRNA. The cDNA coding sequence and predicted amino acid sequences (Fig. 4) are shown. The predicted protein is a 370-aa type II integral membrane protein with several unique features. The cytoplasmic domain is atypically long for a glycosyltransferase, being 42 aa in length. Comparison with the rat iGb3S cytoplasmic domain (11 aa) shows an insertion of 31 aa after E6, before the conserved 38RAKKR42 flanking the cytoplasmic domain (data not shown). The additional amino acids do not contain any obvious protein motifs. The 18-aa transmembrane domain (predicted by the TMHMM program, <www.cbs.dtu.dk/services/TMHMM/>) is one residue smaller than the rat iGb3S, and also contains a charged R46 residue. The 274-aa C-terminal catalytic domain containing the essential 229DVD231) sequence is preceded by a 36-aa stalk region. The catalytic domain has 91% aa identity with the rat, with a conserved pattern of cysteine residues.


Figure 4
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  		FIGURE 4. iGb3S cDNA and predicted protein sequence. The deduced amino acid sequence of mouse iGb3S is shown in single letter code and is shown above the nucleotide sequence. The putative transmembrane domain is underlined.

 
This difference in the cytoplasmic domains between rat and mouse iGb3S is due to the use of an alternative first coding exon in the mouse as shown. The rat uses exon 1 (7457 bp upstream of coding exon 2) whereas the mouse uses the alternative exon 1a (1278 bp upstream of coding exon 2). The mouse homolog of rat exon 1, located 7430 bp upstream of exon 2, encodes a similar amino acid sequence to the rat (MALIEFR) but was not used in the sequence we isolated. The rat homolog of mouse exon 1a contains an in-frame stop codon 15 bp downstream of the initiation Met and therefore would encode a truncated protein. In both mouse and rat, the iGb3S coding regions are encoded by five exons and, apart from the first exons, consensus sequences at exon-intron boundaries and overall exon and intron lengths are conserved.

Mouse iGb3S generates Gal{alpha}(1,3)Gal on lipid in the iosglobo-series pathway

To demonstrate that mouse iGb3S cDNA is functional and synthesizes Gal{alpha}(1,3)Gal, we transfected CHOP cells that are Gal{alpha}(1,3)Gal negative (Fig. 5). A high level of surface Gal{alpha}(1,3)Gal (mean fluorescence units (mfu) of 540) was detected by the binding of the anti-Gal{alpha}(1,3)Gal mAb, 15.101 (Fig. 5A).


Figure 5
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  		FIGURE 5. Transfection of mouse iGb3S into CHOP cells results in Gal{alpha}(1,3)Gal expression on cell surface glycolipid (mfu are shown). A, CHOP cells were transfected with mouse iGb3S cDNA and then stained for Gal{alpha}(1,3)Gal with anti-Gal{alpha}(1,3)Gal mAb, 15.101. B, Coexpression of {alpha}1,2fucosyltransferase with mouse iGb3S cDNA did not result in a decrease in level of staining with anti-Gal{alpha}(1,3)Gal Ab, 15.101, (solid line) compared with mouse iGb3S transfected alone, A. C, Cells were transfected with FS alone (solid line) or D, mouse iGb3S was cotransfected with FS (solid line). Cells were then stained for isoForssman epitope with anti-Forssman Ab. Only coexpression of iGb3S and FS cDNA enabled expression of the cell surface isoForssman epitope, D. Background staining with secondary Ab is shown in all panels by a broken line and the markers represent the population of positive stained cells.

 
Rat iGb3S synthesizes Gal{alpha}(1,3)Gal exclusively on glycolipid (16, 17) whereas {alpha}1,3GT can synthesize Gal{alpha}(1,3)Gal on either protein or glycolipid. iGb3S expression is required to initiate the isoglobo-series pathway to enable the subsequent production of isoForssman lipid. Using a complementation assay in CHOP cells, which lack both iGb3S and FS, we examined whether coexpression of mouse iGb3S with FS resulted in expression of isoForssman glycolipid. As expected, cells transfected with FS alone did not stain for isoForssman (Fig. 5C), whereas cells transfected with both iGb3S and FS were positive for isoForssman (mfu of 475, Fig. 5D), demonstrating that iGb3S synthesizes Gal{alpha}(1,3)Gal on glycolipid in the isoglobo-series pathway.

We have previously shown that coexpression of {alpha}1,2FT with {alpha}1,3GT results in a reduction of cell surface Gal{alpha}(1,3)Gal (27). In contrast, coexpression of {alpha}1,2FT with mouse iGb3S in CHOP cells did not result in a reduction of cell surface Gal{alpha}(1,3)Gal (mfu 580 Fig. 5B, compared with mfu 540, Fig. 5A). Similar results were obtained with the rat iGb3S (data not shown). Thus, {alpha}1,2FT does not compete for the substrate of iGb3S as it does for {alpha}1,3GT.

Gal{alpha}1,3Gal glycolipid generated by iGb3S is immunogenic

The immunological significance of Gal{alpha}(1,3)Gal expressed by iGb3S was examined. We produced 293 cell lines expressing comparable levels of either {alpha}1,3GT or iGb3S which were used to examine the anti-Gal{alpha}(1,3)Gal Ab responses of GGTA1–/– mice (Fig. 6). As expected, a strong anti-Gal{alpha}(1,3)Gal response was observed in GGTA1–/– mice immunized with {alpha}1,3GT+ 293 cells (Fig. 6). A similar anti-Gal{alpha}(1,3)Gal response was observed in mice immunized with iGb3S+ 293 cells (Fig. 6). Nonimmunized GGTA1–/– mice contained very low levels of anti-Gal{alpha}(1,3)Gal Abs (data not shown) and this level was not increased by immunization with mock-transfected 293 cells (Fig. 6). Only IgM and IgG3 Abs were produced (data not shown), which is similar to previous reports (28). The anti-Gal{alpha}(1,3)Gal response in GGTA1–/– mice has previously been shown to be dependent on CD4+ T cells (28). When the immunizations were repeated in anti-CD4-treated GGTA1–/– mice, the Ab responses were almost completely suppressed for all the three cell lines used (Fig. 6). Therefore, the Ab response to Gal{alpha}(1,3)Gal synthesized by iGb3S, like that of {alpha}1,3GT, is dependent on CD4+ T cells.


Figure 6
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  		FIGURE 6. GGTA1–/– mice mount an Ab response to Gal{alpha}(1,3)Gal synthesized by iGb3S. Serum was produced from immunized mice and tested for anti-Gal{alpha}(1,3)Gal Abs in an ELISA. The y-axis shows OD at 450 nm and the x-axis shows doubling dilutions with the first point starting at 1/10. The results are the mean for serum from five mice per group, immunized with {alpha}1,3GT ({blacksquare}), iGb3S ({blacktriangleup}) expressing 293 cells, and those immunized with untransfected 293 cells (•). The results after anti-CD4 (GK1.5) treatment are for serum from five mice per group, immunized with {alpha}1,3GT ({square}), iGb3S ({triangleup}) expressing 293 cells, and those immunized with untransfected 293 cells ({circ}).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previous studies of GGTA1–/– animals could not detect Gal{alpha}(1,3)Gal expression on vascular endothelium using the IB4 lectin (10, 12, 13). Our data demonstrates low but significant levels of Gal{alpha}(1,3)Gal on several tissues, but in particular on vascular endothelium of GGTA1–/– mice. We attribute this Gal{alpha}(1,3)Gal expression to an additional transferase, iGb3S. Recently, Gal{alpha}(1,3)Gal expression was similarly reported on GGTA1–/– pig cell lines (15) and our mAbs and human serum bind tissues from GGTA1–/– pigs (M. S. Sandrin and A. J. d’Apice, unpublished data). We have shown that mRNA for iGb3S is expressed in pig tissues and a fucosylated form of iGb3 has been described in hog mucosa (29), suggesting that iGb3S may also be responsible for Gal{alpha}(1,3)Gal expression in GGTA1–/– pigs as it is in mice.

The key question is will Gal{alpha}(1,3)Gal synthesized by iGb3S have a role in xenotransplantation? Organs from GGTA1–/– pigs are not subject to HAR after transplantation into primates (30, 31). On face value, this suggests that the Gal{alpha}(1,3)Gal synthesized by iGb3S does not play a major role in this initial mode of rejection; this may need to be re-examined. First, humans have higher levels of anti-Gal{alpha}(1,3)Gal Ab than most primates (32). Second, transplanted primates are often subjected to potent immunosuppression that would best be avoided in humans. To test whether the presence of iGb3 will lead to rejection, GGTA1–/– pig organs need to be transplanted into nonimmunosuppressed primates to give a true indication of the ability of the natural preformed baboon Abs to react with the iGb3 lipid containing Gal{alpha}(1,3)Gal. We will be performing these experiments.

The Gal{alpha}(1,3)Gal on lipid, if recognized by Abs, may also play a role in AVR. Indeed, it has been demonstrated that pig GGTA1–/– cell lines (presumably expressing Gal{alpha}(1,3)Gal on iGb3) did not bind anti-Gal{alpha}(1,3)Gal Ab from preimmune human serum, but did bind anti-Gal{alpha}(1,3)Gal Ab from human serum that had been previously exposed to pig liver (extracorporeal circuit) and contained elicited Abs (15). This suggests that elicited Abs in humans can bind Gal{alpha}(1,3)Gal not synthesized by {alpha}1,3GT. However, our data on mouse GGTA1–/– cell lines (Fig. 1B) and preliminary histology on GGTA1–/– pig tissues (data not shown) show binding. This discrepancy requires further investigation. Nonetheless, the Ab in human serum, whether natural preformed or elicited, that can bind iGb3, may have consequences for Ab-mediated rejection processes such as AVR.

A role for anti-Gal{alpha}(1,3)Gal Abs in AVR has been demonstrated (5). A recent report showed that all anti-Gal{alpha}(1,3)Gal Abs, irrespective of isotype or affinity, were capable of promoting endothelial cell activation and apoptosis in vitro. Some of these Ab isotypes promoted HAR and AVR in vivo (6).

Pathological features similar to AVR can be induced by cross-linking of lipid. In humans, Gb3 (or CD77 or the P1 blood group) is the receptor for Shiga toxins produced by Escherichia coli (33). Binding of the bacteria (i.e., cross-linking) is frequently associated with systemic complications such as hemolytic-uremic syndrome, bloody diarrhea, acute renal failure, and neurological abnormalities (34). The tissue damage has the pathologic hallmark of the development of vascular lesions in which endothelial cells are swollen and detached from underlying basement membranes, a feature also observed in AVR. It is interesting to speculate that cross-linking of the glycolipid form of Gal{alpha}(1,3)Gal may lead to similar cell activation and/or apoptosis, and thus contribute to AVR. We have a group of aging mice that have been immunized with untransfected, iGb3-, or {alpha}1,3GT-expressing 293 cells and will be examined for pathological features.

Given that GGTA1–/– mice still express Gal{alpha}(1,3)Gal, the natural and elicited anti-Gal{alpha} (1,3)Gal Abs must be considered as autoantibodies. These natural anti-Gal{alpha}(1,3)Gal Abs are several orders of magnitude lower than those in humans (28, 35, 36, 37). Sykes and colleagues (38) demonstrated that in vivo T cell depletion facilitates Gal anti-Gal{alpha}(1,3)Gal IgM production in GGTA1–/– mice, and suggested that inhibitory T cells are responsible for the low Ab titers in these mice. It is interesting that the thymus (site of T cell education) is one of the organs where Gal{alpha}(1,3)Gal is still expressed in GGTA1–/– mice (see above). When GGTA1–/– mice are immunized with Gal{alpha}(1,3)Gal-positive cells or tissues there is an ~100-fold increase in titer and affinity of anti-Gal{alpha}(1,3)Gal Abs, with mostly IgM and IgG3, but not IgG1, being produced (28). Furthermore, this Ab response is T cell dependent (Fig. 6) (28).

Similar to Gal{alpha}(1,3)Gal-specific Abs, natural autoantibodies reacting with various self-Ags (39) (including ABO blood group Ags (40), heat shock protein 90, and glycolipids (41)) have been reported. Anti-glycolipid Abs are involved in autoimmune diseases of the nervous system, in peripheral neuropathies like Guillain Barre syndrome, multifocal motor neuropathy as well as the promotion of proteinuria in passive Heymann nephritis (42, 43, 44). Most natural IgM, IgG, and IgA autoantibodies have a relatively low affinity, are highly polyreactive, and are encoded by germline V genes (39). Their role in immune regulation and homeostasis or connection with pathological autoantibodies is not clear.

Our studies of iGb3S mRNA expression demonstrate that both GGTA1–/– mice and pigs transcribe the gene. In contrast, iGb3S mRNA was not detected by Northern blot or by RT-PCR across exon boundaries on RNA from a range of human tissues including thymus (data not shown). Furthermore, Gal{alpha}(1,3)Gal was not expressed in any of the human tissues examined (Fig. 2). Unlike the human GGTA1 gene, which has multiple mutations in the coding region, analysis of the human genome shows that the iGb3S gene has no such mutations except for changes at the intron/exon boundaries (data not shown). The reasons why humans do not transcribe the iGb3S gene are not clear, but may be due to either inappropriate splicing or mutation in the promoter region that does not permit initiation of transcription.

Analysis of the mouse iGb3S gene structure shows that the mature protein is encoded by five exons, with conservation of intron/exon boundaries between mouse and rat (data not shown) and, like {alpha}1,3GT, the catalytic domain is encoded in one exon. The mouse, however, has two potential exons containing the initiation Met: exon 1a identified by comparison with the isolated cDNA clones and exon 1 identified by homology analysis of the mouse genome database with rat exon 1. Mouse exon 1a encodes a longer cytoplasmic tail than exon 1, but the effect of this longer cytoplasmic domain is unknown. We have characterized an isoform containing mouse exon 1a, but it is unclear whether the mouse can use exon 1 to encode a second iGb3S isoform. There is, however, precedence for different isoforms of glycosyltransferases to be encoded by alternative first exons (45). The beta1,4galactosyltransferase also has isoforms with different cytoplasmic domains; the larger one localizes both to the Golgi and cell surface whereas the shorter one is exclusively in the Golgi (for review, see Ref.46). We are investigating whether this is also the case with iGb3S.

We have also previously shown that the cytoplasmic tails of {alpha}1,3GT and {alpha}1,2fucosyltransferase ({alpha}1,2FT) affect their subcellular localization (47, 48). The {alpha}1,2FT is localized in an earlier subcompartment of the Golgi and is able to reduce Gal{alpha}(1,3)Gal expression by competing for substrate (27). Reduction of Gal{alpha}(1,3)Gal by expression of other glycosyltransferases that compete with {alpha}1,3GT for the precursor acceptor have also been described (reviewed in Ref.4).

The {alpha}1,2FT, which produces H substance or O blood group Ag from N-acetyllactosamine, was very effective at reducing Gal{alpha}(1,3)Gal expression in cell lines and transgenic mice (49, 50). However, this strategy was not as effective in the several transgenic pig lines that were generated (51, 52, 53). This may reflect higher levels of Gal{alpha}(1,3)Gal in pigs, but we propose that the residual Gal{alpha}(1,3)Gal observed in {alpha}1,2FT transgenic pigs is partly due to iGb3S. The expression of Gal{alpha}(1,3)Gal in these pigs may be due to the inability of {alpha}1,2FT to use the lactosylceramide precursor of iGb3S (Fig. 5) because they may be in distinct compartments of the Golgi.

The removal of Gal{alpha}(1,3)Gal was a major aim in the field of xenotransplantation for a decade. We and others (15) have shown Gal{alpha}(1,3)Gal is still present in knockout animals. Consequently, the deletion of iGb3S, or modification of its product, warrants further investigation.


   Acknowledgments
 
We thank I. Dinatale and H. Dodson for technical assistance, and S. Russell and F. Ierino for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by funds obtained from the National Health and Medical Research Council of Australia and the Roche Organ Transplantation Research Foundation. Back

2 The mouse iGb3 cDNA sequence presented in this article has been deposited in GenBank with accession number AY91982. Back

3 Current address: National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 8, Bethesda, MD 20892-0851. Back

4 Address correspondence and reprint requests to Dr. Mauro S. Sandrin, The Austin Research Institute, Austin Health, Studley Road Heidelberg, 3084, Australia. E-mail address: m.sandrin{at}ari.unimelb.edu.au Back

5 Abbreviations used in this paper: HAR, hyperacute rejection; AVR, acute vascular rejection; {alpha}1,3GT, {alpha}1,3galactosyltransferase; iGb3S, iGb3 synthase; FS, Forssman synthetase; {alpha}1,2FT, {alpha}1,2fucosyltransferase; IB4, Griffonia Simplicifolia B4 lectin; mfu, mean fluorescence unit. Back

Received for publication October 13, 2004. Accepted for publication November 29, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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