Biosynthesis of glycosylated dolichol intermediates and asparagine-linked glycoproteins during the germination of soybean seed embryos

Abstract

Soybean embryos germinated in the presence of [14C]glucose or [14C]mannose incorporated considerable radioactivity into chloroform–methanol (2:1) (CM) soluble and chloroform–methanol–water (10:10:3) (CMW) soluble and insoluble components. The amount of [14C]mannosyl dolichyl phosphate in CM extracts increased over the 24 h of germination, while their [14C]glucosyl dolichyl phosphate content increased up to about 7 h. Incorporation of glucose exceeded incorporation of mannose by about fivefold. The amount of mannose- and glucose-labeled oligosaccharyl dolichyl pyrophosphates in CMW-soluble extracts increased during germination and, based on Concanavalin A Sepharose binding, never exceeded 2% of the total label incorporated into CM-soluble and CMW-soluble, and CMW-insoluble components combined. Nearly equal amounts of [14C]glucose and [14C]mannose appear in oligosaccharyl dolichyl pyrophosphates in the CMW-soluble fraction. Although the CMW-insoluble component contained the largest single amount of radioactivity from either sugar, much of it was not in protein. The protein content of embryos increased during germination, as did the incorporation of [14C]mannose into concanavalin A binding and nonbinding proteins. Microsomes isolated from nongerminated and germinated embryos contained (i) a dolichol kinase, (ii) glycosyl transferases required for the formation of oligosaccharyl dolichyl pyrophosphates, and (iii) a phosphatase which acts on dolichyl phosphate. Kinase activity doubled in 20 h, while phosphatase activity remained at the relatively high initial level. Activity of all three glycosyl transferases was highest after 2 h of germination, but decreased back to zero-time levels by 20 h. The N-acetylglucosaminyl, mannosyl, and glucosyl transferase activities were initially present in a ratio of about 1:10:40 and this ratio did not change appreciably over the period of observation.Key words: dolichol, dolichyl phosphate, glycosyl transferases, oligosaccharyl dolichyl pyrophosphate, soybean seed embryo germination.