Cloning and functional characterization of pig CMP-N-acetylneuraminic acid hydroxylase for the synthesis of N-glycolylneuraminic acid as the xenoantigenic determinant in pig–human xenotransplantation

Abstract

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.

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups

“…Although this tissue contains more total Sias than any other (34), Neu5Gc is reported to be present at only trace levels, if at all. Studies of mouse and pig tissues have accordingly found Cmah mRNA to be undetectable in brain (35,36). As we confirm and expand in this study, no other tissue displays this unusual suppression of Neu5Gc expression across vertebrate taxa.…”

Housekeeping promoter 5’pcmah-2 of pig CMP-N-acetylneuraminic acid hydroxylase gene for NeuGc expression

“…1b). This result entirely corresponded with the previously described tissue expression patterns of pcmah, indicating that 5'pcmah-2 is responsible for housekeeping expression [16]. The different lengths of pcmah promoter regions were PCR-amplified from the pig BAC clone containing the pcmah gene (Fig.…”

“…Our previous study [16] isolated two 5'alternative splicing variants of pcmah and demonstrated their tissue-specific expression pattern, as illustrated in Fig. 1a.…”

“…Previously, we have cloned the complete pig cmah (pcmah) and isolated two 5' alternative transcripts (5'untranslated region (UTR)-1 and −2), suggesting the existence of alternative promoters for the pcmah [16]. Among two transcripts, 5'pcmah-2 containing exon 0, exon 1b and common ORF region, is expressed in most pig tissues expressing pcmah suggesting its housekeeping expression.…”

Effect of gangliosides on LPS stimulation and nitric oxide release in porcine kidney cell line PK15

“…It is important that the nature of immune suppression in gangliosides is determined because of their potential for reducing xenotransplantation immune rejection. Moreover, the mechanism of cell-mediated xenotransplantation rejection is only partially understood (Mejia-Laguna et al 1971;Ozawa et al 1992;Lin et al 1997;Magnusson et al 2005;Jung et al 2009;Song et al 2010;Liu & Yang 2012). In particular, it is likely that the innate immunity effects that are mediated by gangliosides will influence cell functions and contribute to the strength and complexity of xenotransplantation rejection (Platt et al 1991).…”