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Characterization of cDNA clones for porcine α(l,3)galactosyl transferase: The enzyme generating the Galα(l,3)Gal epitope
Author(s) -
Sandrin Mauro S.,
Dabkowski Paul L.,
Henning Margaret M.,
Mouhtouris Effie,
McKenzie Ian F.C.
Publication year - 1994
Publication title -
xenotransplantation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.052
H-Index - 61
eISSN - 1399-3089
pISSN - 0908-665X
DOI - 10.1111/j.1399-3089.1994.tb00053.x
Subject(s) - microbiology and biotechnology , complementary dna , biology , transferase , epitope , molecular cloning , cdna library , southern blot , northern blot , gene , antibody , biochemistry , enzyme , genetics
Galoα(l,3)Gal is a terminal carbohydrate found on many glycosylated cell surface molecules of species other than humans and Old World monkeys, and is produced by the α(l,3)galactosyl transferase enzyme's adding galactose to a substrate. We have previously shown, by the transfection of COS cells with the cloned mouse α(l,3)galactosyl transferase, that most human anti‐pig antibodies react with Galα(l,3)Gal. Using cross‐species hybridization with the mouse α(l,3)galactosyl transferase cDNA, bacteriophage λ, gt11 and λgt10 pig cDNA libraries were screened and overlapping clones isolated which encode the pig α(l,3)galactosyl transferase. Sequencing of the clones demonstrated a single open reading frame coding for a protein with high homology to murine (75% identity) and bovine (82% identity) α(l,3)galactosyl transferases. Southern blot analysis shows the porcine α(l,3)galactosyl transferase gene to be a single copy gene, and northern analysis demonstrated an mRNA of 3.9 kb. After splicing the clones to produce a single full length clone, transfection of Galα(l,3)Gal ‐ COS cells led to strong reactivity with human serum and with the IB4 lectin (which reacts only with Galα(l,3)Gal), indicating that the expression of the transferase led to the expression of Galα(l,3)Gal. The cloning of the cDNA gene for the pig α(l,3)galactosyl transferase is the first step in the production of a transgenic pig lacking the transferase and therefore the Galα(l,3)Gal epitope; such animals could serve as donors for human transplantation.