Cell-free labeling in thyroid rough microsomes of lipid-linked and protein-linked oligosaccharides. I. Mannosylated units.

In order to evaluate the possibility in a pig thyroid rough microsomal system of a transfer of pre-assembled sugar cores from sugar-lipids to protein, we have examined after incubation with GDP-[14C]Man the compounds bearing labeled saccharides and have determined some properties of their released saccharide moieties. The [14C]Man material specifically soluble in CHCl3/CH3OH/H2O, 10:10:3, behaved on DEAE-cellulose and when treated with hot alkali and alkaline phosphatase as a lipid pyrophosphate (sometimes accompanied by some dolichol-P-[14C]Man). Its saccharide moiety, released by mild acid, exhibited properties (molecular size, sensitivity to alpha-mannosidase, affinity for concanavalin A and charge modification introduced by a strong reductive alkaline treatment) pointing to a polymannosylated N,N'-diacetylchitobiose containing an average of nine monosaccharide units (from six to twelve). The [14C]mannosylated glycoproteins have represented all the polymeric label remaining after lipid extraction. From the susceptibility of their pronase glycopeptides to a differential reductive alkaline hydrolysis, it was concluded that their label belonged mainly to N-glycosidically linked units. Released saccharides exhibited the same properties as those from lipids, a result substantiating the possibility raised from previous studies of a transfer of pre-assembled moieties.