Expression of alpha-gal epitopes on HeLa cells transduced with adenovirus containing alpha1,3galactosyltransferase cDNA.
Alpha1,3galactosyltransferase (alpha1,3GT) synthesizes alpha-gal epitopes (Gal(alpha)1-3Galbeta1-4GlcNAc-R) on glycoconjugates in nonprimate mammals but not in humans. Transduction of alpha1,3GT gene into human HeLa cells by an adenovirus vector allowed for accurate kinetics studies on the appearance of alpha1,3GT and of its product, the alpha-gal epitope, in the transduced cells. Mouse alpha1,3GT cDNA was inserted into a replication-defective adenovirus vector. This viral vector, designated Ad(alpha)GT, could be propagated in human 293 cells that have the viral E1 complementing gene. Transduction of HeLa cells resulted in immediate penetration of approximately 20 Ad(alpha)GT copies into each cell and the appearance of alpha1,3GT mRNA after 4h. Catalytic activity of alpha1,3GT was first detected in the cells after 6 h. The initial appearance of alpha-gal epitopes (approximately 6 x 10(4)/cell) on cell surface glycoconjugates was detected 10 h posttransduction, whereas 24 h posttransduction each cell expressed 2 x 10(6) epitopes. The activity of alpha1,3GT in cells transduced with approximately two copies of Ad(alpha)GT was eightfold lower than that in cells transduced with approximately 20 Ad(alpha)GT copies; however, the number of alpha-gal epitopes/cell remained closely similar. This implies that increased alpha1,3GT activity above a certain saturation level does not result in a corresponding increase in the carbohydrate product, possibly because of competing glycosyltransferases.
Deriy
Lucy
L
Department of Cardiovascular-Thoracic Surgery, Rush University, 1653 West Congress Parkway, Chicago, IL 60612, USA.
Chen
Zhao-chun
ZC
Gao
Guang-Ping
GP
Galili
Uri
U
D000256:Adenoviridae / Q000235:genetics*; D002455:Cell Division / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics*; D000939:Epitopes / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism*; D018628:Gene Dosage; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006367:HeLa Cells / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D037102:Lectins / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014161:Transduction, Genetic / Q000379:methods
D017931:DNA Primers; D018076:DNA, Complementary; D000939:Epitopes; D037102:Lectins; D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase