Switching amino-terminal cytoplasmic domains of alpha(1,2)fucosyltransferase and alpha(1,3)galactosyltransferase alters the expression of H substance and Galalpha(1,3)Gal.
When alpha(1,2)fucosyltransferase cDNA is expressed in cells that normally express large amounts of the terminal carbohydrate Galalpha(1,3)Gal, and therefore the alpha(1,3)galactosyltransferase (GT), the Galalpha(1,3)Gal almost disappears, indicating that the presence of the alpha(1,2)fucosyltransferase (HT) gene/enzyme alters the synthesis of Galalpha(1,3)Gal. A possible mechanism to account for these findings is enzyme location within the Golgi apparatus. We examined the effect of Golgi localization by exchanging the cytoplasmic tails of HT and GT; if Golgi targeting signals are contained within the cytoplasmic tail sequences of these enzymes then a "tail switch" would permit GT first access to the substrate and thereby reverse the observed dominance of HT. Two chimeric glycosyltransferase proteins were constructed and compared with the normal glycosyltransferases after transfection into COS cells. The chimeric enzymes showed Km values and cell surface carbohydrate expression comparable with normal glycosyltransferases. Co-expression of the two chimeric glycosyltransferases resulted in cell surface expression of Galalpha(1,3)Gal, and virtually no HT product was expressed. Thus the cytoplasmic tail of HT determines the temporal order of action, and therefore dominance, of these two enzymes.
Osman
N
N
Molecular Immunogenetics Laboratory, Austin Research Institute, Austin Hospital, Heidelberg Victoria 3084, Australia.
McKenzie
I F
IF
Mouhtouris
E
E
Sandrin
M S
MS
D000818:Animals; D019556:COS Cells; D003593:Cytoplasm / Q000378:metabolism; D005647:Fucosyltransferases / Q000737:chemistry*; D005697:Galactosides / Q000378:metabolism*; D005700:Galactosyltransferases / Q000378:metabolism*; D007700:Kinetics; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D013552:Swine; D000097631:Galactoside 2-alpha-L-fucosyltransferase
D005647:Fucosyltransferases; D005697:Galactosides; D005700:Galactosyltransferases; D011993:Recombinant Fusion Proteins; D000097631:Galactoside 2-alpha-L-fucosyltransferase; C037959:beta-D-galactosyl-N-acetylglucosaminylglycopeptide alpha-1,3-galactosyltransferase