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.
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.
Song
Kwon-Ho
KH
Molecular and Cellular Glycobiology Unit, Department of Biological Science, Sungkyunkwan University, Chunchun-Dong, Suwon 440-746, South Korea.
Kang
Yun-Jeong
YJ
Jin
Un-Ho
UH
Park
Yong-Il
YI
Kim
Sung-Min
SM
Seong
Hwan-Hoo
HH
Hwang
Seongsoo
S
Yang
Boh-Suk
BS
Im
Gi-Sun
GS
Min
Kwan-Sik
KS
Kim
Jin-Hoi
JH
Chang
Young-Chae
YC
Kim
Nam-Hyung
NH
Lee
Young-Choon
YC
Kim
Cheorl-Ho
CH
D000595:Amino Acid Sequence; D000818:Animals; D015478:Antigens, Heterophile / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007421:Intestine, Small / Q000201:enzymology; D007668:Kidney / Q000166:cytology / Q000201:enzymology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009438:Neuraminic Acids / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013552:Swine; D014183:Transplantation, Heterologous
D015478:Antigens, Heterophile; D009438:Neuraminic Acids; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C032592:N-glycolylneuraminic acid; D006899:Mixed Function Oxygenases; D007770:L-Lactate Dehydrogenase; C074383:CMPacetylneuraminate monooxygenase