CLONED BETA-1,4N-ACETYLGALACTOSAMINYLTRANSFERASE SYNTHESIZES G(A2) ASWELL AS GANGLIOSIDES G(M2) AND G(D2) - G(M3) SYNTHESIS HAS PRIORITY OVER G(A2) SYNTHESIS FOR UTILIZATION OF LACTOSYLCERAMIDE SUBSTRATE IN-VIVO

Earlier studies reached conflicting conclusions as to the ability of t he beta 1,4 N-acetylgalactosaminyltransferase (GalNAc-T) that synthesi zes gangliosides G(M2), and G(D2) to also produce gangliotriosylcerami de (G(A2)). We constructed an experimental system in which to address this question. Wild type Chinese hamster ovary (CHO) cells contain gan glioside G(M3) as the most complex glycosphingolipid (GSL), whereas th e CHO glycosylation mutant Lec2, which is deficient in sialylation, ac cumulates lactosylceramide with little G(M3) being produced. We transf ected both cell types with a plasmid containing a cloned GalNAc-T. Whe reas transfected CHO cells produced G(M2), as the major complex GSL, t he major product in transfected Lec2 cells was G(A2). Both types of tr ansfected cells but not the untransfected cells expressed the transfec ted gene and contained high levels of enzyme activity for synthesizing both G(M2) and G(D2) in vitro. In summary, these results indicate tha t this enzyme can in fact synthesize G(A2) as well as G(M2) and G(D2). In addition, these findings suggest that in CHO cells the synthesis o f G(M3) in vivo has priority over G(A2) synthesis for utilization of t he substrate lactosylceramide, resulting in little G(A2) being produce d even though GalNAc-T is present and active. Thus, competition for su bstrate between glycosylation pathways may have profound effects on th e GSL pattern of cells.