Biosynthesis in vitro of mono- and di-sialosylgangliosides from gangliotetraosylceramide by cultured cell lines and young rat brain. Structure of the products, and activity and specificity of sialosyltransferase

Abstract

Incubations in vitro of G A1, labeled with 3H in the terminal d-galactopyranosyl group, with nonradioactive CMP-NeuNAc in the presence of homogenates of C2 1 rat brain glial cells, NIE mouse neuroblastoma cells, 3T3 mouse fibroblasts, SV 40-transformed 3T3 cells, chick embryo fibroblasts, Rous sarcoma virus-transformed chick embryo fibroblasts, and 9-day old rat brain resulted in all cases in the formation in high yield of G M1b, in which the neuraminidase-labile NeuNAc group is linked at O-3 of the terminal d-galactosyl residue, as shown by permethylation studies. No trace of the naturally occurring neuraminidase-stable G M1a was detected in any case. In addition, with NIE cells, and normal and RSV-transformed chick embryo fibroblasts, a disialosylganglioside (G D1) differing from G D1a and G D1b, and bearing only one substituent at O-3 of the terminal d-galactopyranosyl residue was formed. It was also biosynthesized from G M1b and CMP-NeuNAc by NIE and chick embryo cells but not by C2 1 cells, or rat brain. However, C2 1 cells and rat brain were capable of synthesizing G D1a from G M1a. Periodate oxidation degraded both NeuNAc groups in G D1 to a 7-carbon fragment, indicating lack of substitution at O-8. G M1b could not be detected as a natural product in rat brain.