In the present study, the pig CMP-N-acetylneuraminic acid hydroxylase gene (pcmah), a key enzyme for the synthesis of NeuGc (N-glycolylneuraminic acid), was cloned from pig small intestine and characterized. The ORF (open reading frame) of pcmah was 1734 bp, encoding 577 amino acids and consisting of 14 exons. Organ expression pattern analysis reveals that pcmah mRNA is mainly expressed in pig rectum, tongue, spleen and colon tissues, being the most highly expressed in small intestine. In the ectopic expression of pcmah, when pig kidney PK15 cells and human vascular endothelial ECV304 cells were transfected with the cloned pcmah, the NeuGc contents of these transfectants were greater in comparison with vector transfectants used as controls. In addition, in the functional analysis of NeuGc, HSMC (human-serum-mediated cytotoxicity) was elevated in the ectopic NeuGc-expressing pcmah-transfected cells compared with controls. Moreover, binding of human IgM to the pcmah-transfected cells was significantly increased, whereas binding of IgG was slightly increased, indicating that the human IgM type was a major anti-NeuGc antibody. Furthermore, pcmah silencing by shRNA (short hairpin RNA) resulted in a decrease in NeuGc content and xenoantigenicity in PK15. From the results, it was concluded that the pcmah gene was capable of synthesizing the NeuGc acting as a xenoantigen in humans, confirming the NeuGc-mediated rejection response in pig–human xenotransplantation.
- CMP-N-acetylneuraminic acid hydroxylase
- human-serum-mediated cytotoxicity
- hyperacute rejection
- N-glycolylneuraminic acid
Sialic acids are typically found at the non-reducing ends of oligosaccharide chains, which are involved in various biological processes, such as immune response, inflammation and tumour cell metastasis [1–3]. In general, ‘sialic acid’ is the term indicating N-acylneuraminic acids and their many derivatives [4,5]. Among sialic acids, NeuAc (N-acetylneuraminic acid) and NeuGc (N-glycolylneuraminic acid) are two of the most common sialic acid types. NeuAc is expressed ubiquitously and is the major sialic acid form found in humans. NeuGc is abundant in most mammals, but not in humans owing to a homozygous deletion mutation in the enzyme responsible for NeuGc biosynthesis . However, NeuGc is produced from NeuAc, through the reaction catalysed by a specific hydroxylase, CMAH (CMP-N-acetylneuraminic acid hydroxylase), that converts CMP-NeuAc into CMP-NeuGc [7–10].
In immune reactions, glycoconjugate-bound NeuGc is the target epitope recognized by HD (Hanganutziu–Deicher) antibodies, first described by Hanganutziu and Deicher independently in the 1920s [11,12]. These HD antibodies appeared in patients after the therapeutic injection of animal antisera and were able to agglutinate animal erythrocytes [13,14]. It was also discovered that HD antibodies were in the sera of unaffected humans, as well as some disease-related patients [15,16]. These findings indicate that NeuGc is a potential immunogen for humans.
In xenotransplantation, the pig has been identified as a suitable organ donor candidate for humans because of its compatible organ size and short breeding time. However, in pig–human xenotransplantation, exposure of pig organs to human blood results in HAR (hyperacute rejection) . The rejection is caused by the presence of Galα1,3Gal antigen on the pig vascular endothelium and natural anti-Galα1,3Gal antibodies in human serum [17,18]. When pig organs or tissues are transplanted into the human body, the human IgM isotype of anti-Galα1,3Gal binds to Galα1,3Gal antigens on the pig tissues, which causes the activation of the complement cascade resulting in cell lysis . Although the Galα1,3Gal antigen was eliminated by knocking out the α-1,3-galactosyltransferase , the remaining antigens, so called non-Galα1,3Gal antigens, are considered to be the next xenoantigen involved in the rejection phenomenon . It has been suggested that NeuGc has the potential to be one of the non-Galα1,3Gal xenoantigens in pig–human xenotransplantation after α-1,3-galactosyltransferase is knocked out [21,22].
Although CMAH has been cloned and characterized from various animals [7,23–25], the pig CMAH gene (pcmah) has only been partially cloned so far . The amino acid sequences of the mouse, pig and chimpanzee CMAH enzymes have been found to be highly homologous [6,7,24]. Therefore we have cloned the complete pcmah that encodes the CMAH from pig tissues, based on the sequences of various CMAH genes and 5′-RACE (rapid amplification of cDNA ends). The mRNA expression of the pcmah was analysed in various pig tissues by RT (reverse transcription)–PCR. When stably transfected into pig kidney PK15 cells and human endothelial ECV304 cells, the transfectant was increased in NeuGc content and has been capable of increasing HSMC (human-serum-mediated cytotoxicity) and HSMX (human-serum-mediated xenoreactivity).
The pig kidney cell line PK15 was obtained from the Korean Cell Line Bank (Seoul, South Korea) and was cultured in DMEM (Dulbecco's modified Eagle's medium) (WelGENE). ECV304 cells, an immortalized human vascular endothelial cell line, was obtained from the A.T.C.C. (Manassas, VA, U.S.A.) and cultured in M199 medium (WelGENE). All culture media were supplemented with 10% heat-inactivated FBS (fetal bovine serum) (WelGENE) and 100 unit/ml penicillin and 100 μg/ml streptomycin. The cultures were maintained in a 5% CO2 atmosphere at 37 °C.
PCR for molecular cloning of pcmah
For cloning of full-length pcmah cDNA, degenerative forward sense primer [5′dgP: 5′-ATGR(A/G)GCAGCATCGAACAAACR(A/G)GCT-3′] was designed on the basis of conserved nucleotide sequences among the several kinds of CMAH genes. Reverse antisense primer (3′GSP: 5′-CTACCCAGAGCACATCAGGAA-3′) was designed on the basis of partial pig CMAH sequence published previously by Schlenzka et al. .
Total RNA was isolated from fresh pig small intestine tissues using TRIzol® reagent (Invitrogen), and the cDNAs were synthesized by RT with an oligo(dT)-adaptor primer using AccuPower® RT-PreMix (Bioneer), according to the manufacturer's recommended protocol. The pcmah fragment was amplified by the PCR system with the above primer set. The amplified DNA fragments were subcloned into the EcoRV site of pSTBlue-1 AccepTor™ vector (Novagen) by a TA-cloning method. The multiple independent clones were isolated and sequenced to confirm the sequence data.
To obtain the 5′-end of the ORF (open reading frame) and 5′-UTR (untranslated region), a 5′-RACE reaction was performed using a GeneRacer™ kit (Invitrogen) according to the manufacturer's recommended protocol. The total RNA from pig small intestine was used as a template for RACE. The first-strand cDNA was synthesized by RT with a 5′ gene-specific primer (5′GSP1: 5′-AACCACCATCCTCGCGCAAAAGC-3′) using a SuperScript™ III Reverse Transcriptase (Invitrogen). The first PCR was performed using two primers (GeneRacer™ 5′primer: 5′-CGACTGGAGCACGAGGACCATGA-3′ and 5′GSP2: 5′-GCGTGAGTAAG GTACGTGATCTGC-3′). The second PCR was performed using two primers (GeneRacer™ 5′nested primer: 5′-GGACACTGACATGGACTGAAGGAGTA-3′ and 5′GSP3: 5′-TCGTCTTGACAGAAGCTTCCAGGA-3′). The amplified 5′-RACE products were subcloned and sequenced.
Construction of the pcmah expression vector
The cDNAs of the full pcmah ORF was inserted at the 3′-end of the CMV (cytomegalovirus) promoter into the KpnI and XhoI sites of pcDNA™ 3.1/myc-His expression vector (Invitrogen). For the cloning of the full pcmah ORF, PCR was performed with pig cDNA as the template and the following primer set containing restriction enzyme sites (KpnI and XhoI respectively, underlined): pig CMAH full, 5′-ACGGTACCATGAGCAGCATCGA-3′ (sense) and 5′-AACTCGAGCCCAGAGCACATCA-3′ (antisense).
Establishment of stable transfectants
Transfection of pcmah into PK15 and ECV304 cells was performed using a WelFect-EX™ PLUS Transfection Reagent (WelGENE). The cells were selected for stable integration of transfected DNA by changing to a medium containing 0.6 mg/ml G418 (AG Scientific) for several days. To confirm the establishment of PK15 and ECV304 cell lines transfected with pcmah, Western blot analysis was performed.
Establishment of stable pcmah silencing-PK15 cell lines by shRNA (short hairpin RNA)
Stable pcmah RNA-interference PK15 cell lines were generated using the pSilencer™ 3.1-H1 puro vector (Ambion). Four DNA oligonucleotides, containing a terminal BamHI or HindIII restriction site and linker sequence (TTCAAGAGA) that forms looped structures, were designed for knocking down pcmah following the manufacturer's protocol linking the 19-nucleotide sense and antisense sequences as follows: pcmah-sh1-sense, 5′-GATCCGCTGCCAATCTCAAGGAAGTTCAAGAGACTTCCTTGAGATGGCAGCTTTTTTGGAAA-3′; pcmah-sh1-antisense, 5′-AGCTTTTCCAAAAAAGCTGCCAATCTCAAGGAAGTCTCTTGAACTTCCTTGAGATTGGCAGCG-3′; pcmah-sh2-sense, 5′-GATCCGTTTACTGAGGAATGGAAAGTTCAAGAGACTTTCCATTCCTCAGTAAATTTTTTGGAAA-3′; and pcmah-sh2antisense, 5′-AGCTTTTCCAAAAAATTTACTGAGGAATGGAAAGTCTCTTGACTTTCCATTCCTCAGTAAACG-3′. The oligonucleotides were annealed, and the resulting insert was subcloned into the BamHI or HindIII site of pSilencer™ 3.1-H1 puro vector. The purified plasmid was transfected into PK15 cells by using a WelFect-EX™ PLUS Transfection Reagent. Stable clones were selected using 2 μg/ml puromycin and screened for their ability to knock down pcmah mRNA expression by RT–PCR.
RT–PCR in various pig tissues
The cDNAs from various pig tissues were amplified by PCR with the following primers using EF-Taq polymerase (SolGent): sense, 5′-ATGAGCAGCATCGAACAAACG-3′, and antisense, 5′-ACAACCAGTTCGTCTTGACAG-3′. The use of equal amounts of mRNA in the RT–PCR assay was confirmed by analysing the expression levels of β-actin.
Total cell lysates were treated with 0.1 M HCl at 80 °C for 1 h to release sialic acids from glycoconjugates. The sialic acid hydrolysates were labelled using a fluorescent labelling kit [DMB (1,2-diamino-4,5-methylenedioxybenzene) labelling kit, TaKaRa] according to the manufacturer's instructions. HPLC of the labelled sialic acids was performed using a Gemini 5 μC18 column (4.6 mm×250 mm) (Phenomenex). Elution was performed with a methanol/acetonitrile/water mixture (7:9:84, by vol.), and at a flow rate of 0.9 ml/min. The fluorescence (excitation at 373 nm, emission at 448 nm) was monitored using a fluoromonitor (RF-2000, Dionex). For all HPLC chromatograms, sialic acids were quantified by comparison with known quantities of DMB–NeuAc (TaKaRa) or DMB–NeuGc (Sigma) derivatives as standards.
HPAEC-PAD (high-performance anion-exchange chromatography with pulsed amperometric detection) analysis
The NeuGc content of pcmah-sh-PK15 cells was determined by HPAEC-PAD using the CarboPac PA-1 column-equipped chromatographic system DX 500 (Dionex) in combination with the Aminotrap columns, according to the modified method of Townsend et al. . The modification is as follows: eluent-1, 100 mM NaOH; eluent-2, 100 mM NaOH with 300 mM sodium acetate. The system was eluted at a flow rate of 1 ml/min through a Gradient Pump Module using the following gradients: linear gradient to 20% eluent-1 and 80% eluent-2 at 20 min; linear gradient to 100% eluent-1 at 25 min; and column equilibration: 20 min at 100% eluent-1; linear gradient to 90% eluent-1 and 10% eluent-2 with elution at 100 mM NaOH/100 mM sodium acetate and flow rate of 1 ml/min. Then, 100 μg of protein was hydrolysed in 0.1 M HCl at 80 °C, for 1 h to release sialic acids from glycoconjugates. The sialic acid hydrolysates were applied in the chromatography.
LDH (lactate dehydrogenase) release assay
The LDH assay was performed according to the manufacturer's recommended protocol, using a CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit (Promega). The PK15 or ECV304 transfectants with the construct were plated at 5×104 and 2×104 cells in 96-well trays respectively. After 15 h of incubation, the wells were washed twice with serum-free medium to remove the LDH and then incubated with 40% NHS (normal human serum) that had been diluted with medium. The plate was incubated for 2 h at 37 °C, and the released LDH was then measured.
The transfected cells were seeded 1 day before the assay. The cells were washed twice with PBS and then harvested by scraping. For the human IgG- or IgM-binding assay, the gathered cells were allowed to react with NHS. After 30 min of incubation on ice, the cells were washed twice and then incubated on ice for 30 min with FITC-conjugated goat anti-(human IgG) and anti-(human IgM) (Zymed Laboratories). To analyse the amount of Galα1,3Gal antigen, the gathered cells were reacted with FITC-conjugated GSIB4 lectin (Sigma) on ice for 30 min. In amelioration by flow cytometry, the cells were also stained using 1 μg/ml PI (propidium iodide) (BD Pharmingen) for 10 min in the dark at room temperature (25 °C). Then stained cells were washed twice and analysed using a FACSCalibur (Becton Dickinson).
Western blot analysis
Anti-c-Myc and horseradish-peroxidase-conjugated goat anti-(mouse IgG) were purchased from Santa Cruz Biotechnology. The cells were harvested by scraping and washed twice with PBS and resuspended in RIPA lysis buffer (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 10% glycerol, 0.1% SDS and 0.5% sodium deoxycholate) containing protease inhibitor cocktail (1 mM Na3VO4, 20 μg/ml PMSF, 10 μg/μl leupeptin and 50 mM NaF). Whole lysates from transfectants were separated by SDS/PAGE (12% gels) and transferred on to a nitrocellulose membrane and immunoblotted with antibodies. Bands were visualized by ECL (enhanced chemiluminescence).
Isolation of pcmah
The nucleotide sequence of the partial pcmah gene (GenBank® accession number Y15010) was initially used to generate the reverse antisense primer and perform a BLAST analysis. As a result of BLASTn analysis, high sequence identities were observed among the CMAH genes of various species, such as cattle (95%, GenBank® accession number XP_617171) and chimpanzee (94%, GenBank® accession number NP_001009041). Because the initial sequences of cattle and chimpanzee CMAH genes were highly conserved, the sequences were used to generate a forward degenerative primer. Then, the pcmah-1 fragment of 1734 bp was isolated by PCR from pig small intestine RNAs using degenerative forward primer (5′dgP) and gene-specific reverse primer (3′GSP) (Figure 1). To isolate the complete coding sequence and 5′-UTR, we performed 5′-RACE and finally obtained two specific products of 527 bp (5′pcmah-1) and 626 bp (5′pcmah-2) (Figure 1).
From DNA sequence analysis using these three fragments, pcmah-1, 5′pcmah-1 and -2, the complete sequence of the ORF containing 1734 bp encoding 577 amino acids and two 5′-UTR sequences, 5′UTR-1 (228 bp) and 5′UTR-2 (327 bp), of pcmah was established (Figure 2A). These cDNA sequences have been deposited in GenBank® (accession numbers EU204974, FJ907456 and FJ907457). To understand the genomic structural organization of the pcmah, BLASTn analysis of GenBank® was performed. As shown in Figure 3 and Figure 2(B), pcmah had two alternatively spliced forms within the 5′-UTR. The longer form (5′UTR-2) has a 5′-UTR of 327 bp containing exons 0–1b and a length of 76863 bp with 15 exons separated by 14 introns. The shorter form (5′UTR-1) has a 5′-UTR of 228 bp containing exon 1a and a length of 36241 bp with 14 exons. DNA sequence analysis revealed that 5′UTR-2 was overlapped for 103 bp containing the translation start site with the 3′-end of 5′UTR-1 (Figure 2C). These results indicated that alternatively spliced forms of the 5′-UTR of pcmah do not affect the ORF sequence of pcmah (Figure 2D).
A multiple alignment analysis among the CMAH amino acid sequences of various species such as cattle, chimpanzee, mouse and human was performed. The deduced amino acid sequence of pcmah has high identities with that of various species (Figure 2E). Moreover, it was demonstrated that the deduced amino acid sequence of pcmah has typical motifs, such as a binding site of the Rieske iron–sulfur centre, postulated binding sites of a mononuclear iron centre, a possible CMP-NeuAc-binding site and a possible site of interaction with cytochrome b5, which are highly conserved with the known sequences with the exception of humans  (Figure 2E).
Expression pattern of the pcmah mRNAs in pig tissues
To investigate the tissue-specific expression pattern of pcmah, total RNAs were isolated from various pig tissues and then RT–PCR analysis was performed with a primer set amplifying exons 1–3. pcmah was found to exhibit distinct tissue-specific expression patterns. The gene was most highly expressed in small intestine, and moderately expressed in rectum, tongue, spleen, testicle, liver and colon. However, pcmah was minimally expressed in brain, bladder, stomach, muscle, kidney, spinal cord and heart (Figure 4). To investigate the tissue-specific expression of alternatively spliced forms of the 5′-UTR, RT–PCR analysis was performed with a specific primer set for 5′UTR-1 or 5′UTR-2. These alternatively spliced forms have different expression patterns in various pig tissues, except for rectum. 5′UTR-1 was mainly expressed in small intestine and colon. However, 5′UTR-2 was the major form in the spleen, tongue, testicle, kidney and liver (Figure 4).
Induced constitutive expression of NeuGc in pcmah-transfected PK15 cells
To investigate whether the expression of pcmah is active to synthesize the NeuGc, we have established pcmah-transfected PK15 cells. As shown in Figure 5, the pcmah-transfected PK15 cells significantly expressed the recombinant pig CMAH protein. Then, the sialic acid contents were measured in pcmah-transfected PK15 cells by using HPLC analysis. From the HPLC analysis, it was demonstrated that the pcmah-transfected PK15 cells contained a larger amount of NeuGc than the vector-transfected cells. These results indicate clearly that NeuGc biosynthesis of pcmah-transfected PK15 cells was enhanced by de novo synthesis, which would reflect the activity of the ectopically expressed pcmah within the PK15 cells.
Previous reports have suggested that NeuGc synthesized by CMAH shows xenoantigenicity, leading to increased HSMC [27–29]. To determine HSMC, the LDH assay was performed using 20% NHS. As shown in Figure 5(C), HSMC was greater in the pcmah-transfected PK15 cells than in the control. The amelioration of xenoantigenicity of CMAH was also analysed by flow cytometric analysis using PI. As expected, PI-positive populations were greater in the pcmah-transfected cells than in the vector-transfected cells (Figure 5D). These results demonstrated that NeuGc generated by pcmah in PK15 cells has potential xenoantigenicity.
Xenoreactivity was evaluated further by flow cytometric analysis using the pcmah-transfected PK15 cells. Binding of human IgM to the transfected cells was largely increased in the pcmah-transfected cells, whereas binding of human IgG was slightly increased in the pcmah-transfected cells, compared with controls (Figures 5E and 5F). These results suggest that xenoantigenicity of the pcmah-transfected cells is mainly caused by IgM rather than IgG in human sera. A binding analysis was used to investigate whether the increase of xenoantigenicity was affected by quantitative change of Galα1,3Gal antigen, using GS-IB4, Galα1,3Gal-specific lectin, with pcmah-transfected PK15 cells. Binding of GS-IB4 to pcmah-transfected cells was not altered in comparison with control (Figure 5G). It is suggested that increased xenoantigenicity of the pcmah-transfected PK15 cells is Galα1,3Gal antigen-independent.
Silencing of pcmah in PK15 cells
The functional activity of the pcmah in PK15 cells was characterized further using shRNAs to silence the pcmah expression. PK15 cells were stably transfected with pcmah-sh1, pcmah-sh2 and empty vector. As shown in Figure 6(A), pcmah mRNA expression was successfully down-regulated in the pcmah-sh1 and pcmah-sh2 cells compared with pSilencer™ 3.1 vector transfectants. Among the two pcmah-sh cells, pcmah-sh2 was more effectively silencing than pcmah-sh1, so the pcmah-sh2 cells were analysed further. When the NeuGc contents of pcmah-sh2 and pSilencer™ 3.1-transfectant were analysed using HPAEC-PAD and compared, the NeuGc content of the pcmah-sh2 cells was less than that of the pSilencer™ 3.1-transfected cells (Figure 6B). In addition, when HSMC was also determined by LDH assay using 20% NHS, as expected, HSMC was decreased in the pcmah-sh2 cells, compared with the control (Figure 6C). Therefore the results strongly support the hypothesis that NeuGc generated by pcmah in PK15 cells has a potential xenoantigenicity.
Overexpression of pcmah on human endothelial ECV304 cells
Next, it was investigated whether pcmah acts on human cells which do not express NeuGc. The pcmah-transfected ECV304 cells significantly expressed pcmah mRNA and recombinant pig CMAH protein (Figure 7A). As shown in Figure 7(B), NeuAc/NeuGc contents of these transfectants were analysed using HPLC and compared. Vector-transfected ECV304 cells did not express NeuGc, but pcmah-transfected ECV304 cells expressed a large amount of NeuGc. To determine HSMC, the LDH assay was performed using 50% NHS. As shown in Figure 7(C), HSMC was significantly greater in the pcmah-transfected ECV304 cells than in the control. These results demonstrated that that immunogenic NeuGc of pcmah-transfected ECV304 cells was supplied by de novo synthesis, which would reflect the activity of pcmah within the ECV304 cells as also observed in PK15 cells.
NeuGc is produced from NeuAc through enzymatic hydroxylation of the N-acetyl residue of free NeuAc, CMP-NeuAc or glycoconjugate-linked NeuAc [30,31]. Even though several CMAH genes of animal species have been cloned successfully [7,23–25], only the pcmah gene has not been cloned completely . In the present study, a full ORF of the pcmah gene was cloned by using RT–PCR and 5′-RACE based on previously partially cloned pcmah gene and the known CMAH genes. The cloned pcmah was found to share approx. 93% sequence homology with CMAH of cattle, chimpanzee and human, and 89% sequence homology with Cmah of mouse. The expression of NeuGc is widespread in animals, and also shows tissue-specific and developmentally regulated expression [3,32]. It was demonstrated that mouse Cmah mRNA is expressed in various tissues including the liver, thymus, spleen and kidney, but not in the brain . The mRNA expression of pcmah was investigated in several pig tissues. RT–PCR analysis revealed that pcmah mRNA is highly expressed in the small intestine and spleen, and moderately expressed in the rectum, tongue, testes, liver and colon. However, it was not detected in the brain, bladder, stomach, muscle, pancreas, kidney, spinal cord or heart (Figure 2). The high hydroxylase activity, a large amount of the hydroxylase protein and the NeuGc/NeuAc ratio in small intestine and spleen  were evident by high levels of the pcmah mRNA (Figure 2). It was demonstrated that NeuGc-containing glycoconjugates in the pig small intestine play an important role in mediating infections by pathogens, such as Escherichia coli K99 .
Also, unexpectedly, 5′-RACE analysis revealed that pcmah has two alternatively spliced forms, having two novel initiation sites located in exons 0 and 1a of the gene expressed by pig small intestine. In pig small intestine, the shorter transcript, 5′UTR-1, for which transcription is initiated in exon 1a of pcmah is dominant over the longer transcript, 5′UTR-2, for which transcription is initiated in exon 0. Because of these results, we named the shorter transcript as 5′UTR-1. The expression of 5′UTR-1 and 5′UTR-2 of pcmah in various pig tissues were investigated using RT–PCR. 5′UTR-1 is mainly expressed in small intestine and colon, whereas 5′UTR-2 is highly expressed in spleen, and moderately expressed in most tissues expressing pcmah, including rectum, tongue, small intestine, testicle, kidney and colon.
Unexpectedly, multiple RT–PCR products were observed in the RNAs obtained from spleen tissue. However, from the sequencing analysis, it was confirmed that the longer or shorter forms than the original size were artefacts. Although the biological role of alternative splicing of pcmah is not yet clear, it will be useful to clarify the difference of NeuGc expression or CMAH activity in various pig tissues. Therefore we are now in the process of investigating further the biological role of alternative splicing of pcmah.
To examine the functional pcmah activity, we have analysed the change of NeuAc/NeuGc contents in both pcmah-transfected PK15 and ECV304 cells. When human ECV304 cells negative for NeuGc expression were transfected with pcmah cDNA, the NeuGc contents were significantly increased in the transfectants, compared with mock control cells (Figure 7B). However, NeuGc contents of the pcmah-transfected PK15 cells were slightly increased compared with mock transfectants (Figure 5B), possibly due to the constitutive level of the endogenous NeuGc contents of PK15 cells. With regard to NeuGc production, the expression of CMAH is known to be a dominant factor in the production of glycoconjugate-bound NeuGc , and expression of NeuGc glycoconjugates is regulated by various factors including post-translational modification of the enzyme, regulation of the accessibility of cytochrome b5 and cytochrome b5 reductase, etc. [7,35]. In the present study, the increased contents of NeuGc in the transfected PK15 and ECV304 cells are attributed to the increase in CMAH activity. Therefore, from these results, it was indicated that the cloned cDNA encodes for the pig CMAH.
Previously, Cooper et al.  reported the importance of Galα1,3Gal antigen as the major antigen in pig–human xenotransplantation, and genetic approaches to modify this carbohydrate antigen have also progressed [36,37]. However, after the removal of the Galα1,3Gal antigen by knocking out the α-1,3 galactosyltransferase, non-Galα1,3Gal antigens including the HD antigen, Thomsen–Fridenreich (T or TF) or Forssman antigen were still xenoreactivity-causative [21,38]. NeuGc, also called the HD antigen, are widely expressed on endothelial cells of all mammals, except for humans, and are targets for non-Galα1,3Gal antibodies . It was also demonstrated that the human immune cells transfected with mouse CMAH increases HSMC . When treated with 20% NHS, HSMC was slightly elevated in pcmah-overexpressed PK15 cells, compared with control (Figure 5C). Moreover, pcmah silencing by shRNA resulted in reduction of the NeuGc content and xenoantigenicity in PK15 cells (Figure 6). To investigate whether the increase of xenoantigenicity was affected by the quantitative change of Galα1,3Gal antigen, a binding analysis of GS-IB4 to pcmah-transfected cells was performed. As expected, the binding of GS-IB4 to pcmah-transfected cells was not changed compared with control, suggesting that the increased xenoantigenicity of pcmah-transfected PK15 cells was Galα1,3Gal antigen-independent (Figure 5G). In general, the PK15 cells expressed NeuGc as well as Galα1,3Gal antigen, whereas human ECV304 cells do not express the NeuGc and Galα1,3Gal antigen. Therefore the 20% NHS treatment had little effect on pcmah-overexpressed ECV304 cells (results not shown). However, when treated with 50% NHS, HSMC was elevated in pcmah-overexpressed ECV304 cells, compared with control (Figure 7C). These results indicate that the cloned pcmah is capable of NeuGc expression, having xenoantigenicity.
It was reported that anti-Galα1,3Gal IgM is the predominant immunoglobulin involved in HAR, whereas anti-Galα1,3Gal IgG plays a central role in AVR (acute vascular rejection) [39–41]. Therefore the binding capacities of human immunoglobulins such as IgM and IgG to the pcmah transfectants were examined by flow cytometry. The binding of IgM to the transfectants was significantly greater and the binding of IgG was slightly elevated than in the control (Figures 5E and 5F). These results suggest that xenoantigenicity of pcmah-transfected cells was affected by the binding of IgM rather than IgG. In addition, it is suggested that NeuGcs were involved in HAR and AVR in pig–human xenotransplantation.
In summary, we have cloned the complete pcmah gene that encodes CMAH. The pig full-length cDNA directed the expression of an active enzyme capable of increasing the HSMC. Therefore, together with the pig Galα1,3Gal-knockout strain, knocking out pcmah, which synthesizes the NeuGc acting as another xenoantigen, will undoubtedly decrease the rejection response in pig–human xenotransplantation.
Kwon-Ho Song was responsible for all of the experimental work, with the exception of the establishment of the pcmah-transfected PK15 cell lines and HSMC of pcmah-transfected PK15 cells which were carried out by Yun-Jeong Kang and Un-Ho Jin, and HPLC and HPAEC-PAD analysis of the NeuGc contents which were carried out by Sung-Min Kim and Yong-Il Park. Seongsoo Hwang, Boh-Suk Yang, Gi-Sun Im, Hwan-Hoo Seong, and Jin-Hoi Kim provided various pig tissues. Kwan-Sik Min, Young-Chae Chang, Nam-Hyung Kim and Young-Choon Lee carried out FACS analysis for the xenoantigenic determination. Cheorl-Ho Kim, as principal investigator, was responsible for the planning of the work and writing the manuscript.
This study has been in part supported by Biogreen 21 Project, Rural Development Administration [grant number 20070401-034-030-008-02-00].
Abbreviations: AVR, acute vascular rejection; CMAH, CMP-N-acetylneuraminic acid hydroxylase; DMB, 1,2-diamino-4,5-methylenedioxybenzene; HAR, hyperacute rejection; HD, Hanganutziu–Deicher; HPAEC-PAD, high-performance anion-exchange chromatography with pulsed amperometric detection; HSMC, human-serum-mediated cytotoxicity; LDH, lactate dehydrogenase; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid; NHS, normal human serum; ORF, open reading frame; pcmah, pig CMAH gene; PI, propidium iodide; RACE, rapid amplification of cDNA ends; RT, reverse transcription; shRNA, short hairpin RNA; UTR, untranslated region
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