Cloned beta 1,4 N-acetylgalactosaminyltransferase synthesizes GA2 as well as gangliosides GM2 and GD2. GM3 synthesis has priority over GA2 synthesis for utilization of lactosylceramide substrate in vivo.
Earlier studies reached conflicting conclusions as to the ability of the beta 1,4 N-acetylgalactosaminyltransferase (GalNAc-T) that synthesizes gangliosides GM2 and GD2 to also produce gangliotriosylceramide (GA2). We constructed an experimental system in which to address this question. Wild type Chinese hamster ovary (CHO) cells contain ganglioside GM3 as the most complex glycosphingolipid (GSL), whereas the CHO glycosylation mutant Lec2, which is deficient in sialylation, accumulates lactosylceramide with little GM3 being produced. We transfected both cell types with a plasmid containing a cloned GalNAc-T. Whereas transfected CHO cells produced GM2 as the major complex GSL, the major product in transfected Lec2 cells was GA2. Both types of transfected cells but not the untransfected cells expressed the transfected gene and contained high levels of enzyme activity for synthesizing both GM2 and GA2 in vitro. In summary, these results indicate that this enzyme can in fact synthesize GA2 as well as GM2 and GD2. In addition, these findings suggest that in CHO cells the synthesis of GM3 in vivo has priority over GA2 synthesis for utilization of the substrate lactosylceramide, resulting in little GA2 being produced even though GalNAc-T is present and active. Thus, competition for substrate between glycosylation pathways may have profound effects on the GSL pattern of cells.
Lutz
M S
MS
Department of Biological, University of Louisville, Kentucky 40292.
Jaskiewicz
E
E
Darling
D S
DS
Furukawa
K
K
Young
W W
WW
D000818:Animals; D015703:Antigens, CD; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D005678:G(M2) Ganglioside / Q000096:biosynthesis*; D005679:G(M3) Ganglioside / Q000096:biosynthesis*; D005732:Gangliosides / Q000096:biosynthesis*; D006028:Glycosphingolipids / Q000096:biosynthesis* / Q000378:metabolism*; D007790:Lactosylceramides; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics* / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D013379:Substrate Specificity; D014162:Transfection
D015703:Antigens, CD; D005679:G(M3) Ganglioside; D005732:Gangliosides; D006028:Glycosphingolipids; D007790:Lactosylceramides; D009838:Oligodeoxyribonucleotides; D005678:G(M2) Ganglioside; C038244:ganglio-N-triaosylceramide; C009744:CDw17 antigen; C019403:ganglioside, GD2; D017350:N-Acetylgalactosaminyltransferases