Regulation of alpha1,3galactosyltransferase expression in pig endothelial cells. Implications for xenotransplantation.
The disaccharide galactose(alpha)1,3 galactose (the alphaGal epitope) is the major xenoantigen responsible for the hyperacute vascular rejection occurring in pig-to-primates organ transplantation. The synthesis of the alphaGal epitope is catalyzed by the enzyme alpha1,3-galactosyltransferase (alpha1,3GalT). To be able to control porcine alpha1,3GalT gene expression specifically, we have analyzed the upstream portion of the alpha1,3GalT gene, and identified the regulatory sequences. Porcine alpha1,3GalT transcripts were detected by 5' RACE analysis, and the corresponding genomic sequences were isolated from a phage library. The porcine alpha1,3GalT gene consists of at least 10 different exons, four of which contain 5' untranslated sequence. Four distinct promoters, termed A-D, drive alpha1,3GalT gene transcription in porcine cells. A combination of alternative promoter usage and alternative splicing produces a series of transcripts that differ in their 5' portion, but encode the same protein. Promoters A-C have been isolated, and functionally characterized using luciferase reporter gene assays in transfected porcine endothelial cells (PEC-A). Promoter preference in porcine endothelial cells was estimated on the basis of relative transcript levels as determined by real-time quantitative PCR. More than 90% of the alpha1,3GalT transcripts in PEC-A cells originate from promoter B, which has characteristics of a housekeeping gene promoter. While promoter preference remains unchanged, alpha1,3GalT mRNA levels increase by 50% in 12 h upon tumour necrosis factor alpha-activation of PEC-A cells. However, the magnitude of this change induced by inflammatory conditions could be insufficient to affect cell surface alpha1,3-galactosylation.
Mercier
Dominique
D
Department of Molecular Cell Biology, Research Institute of Immunology and Inflammatory Diseases, Amsterdam, The Netherlands. d.mercier.pharm@med.vu.nl
Charreau
Beatrice
B
Wierinckx
Anne
A
Keijser
Remco
R
Adriaensens
Lize
L
van den Berg
Renate
R
Joziasse
David H
DH
D000818:Animals; D019556:COS Cells; D003001:Cloning, Molecular; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D005700:Galactosyltransferases / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D013552:Swine; D014183:Transplantation, Heterologous; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D005700:Galactosyltransferases