The formation of lipid-linked sugars by cell-free preparations of lactating rabbit mammary gland

Abstract

1. A lactating rabbit mammary-gland microsomal system catalysed the incorporation of mannose from GDP-[U-14C]mannose into three endogenous acceptors, (i) polyprenyl phosphate mannose, (ii) lipid-linked oligosaccharide and (iii) protein. 2. Synthesis of polyprenyl phosphate mannose was stimulated by addition of dolichol phosphate to the incubation medium and was reversed by addition of GDP. The product had properties identical with those of authentic dolichol phosphate mannose. 3. The oligosaccharides derived from acid hydrolysis of the lipid-linked oligosaccharide fraction were of six, eight and nine to ten monosaccharide units, the octasaccharide being the major species formed. The oligosaccharide appeared to be attached to the lipid via a pyrophosphate bridge, since strong alkaline hydrolysis liberated an oligosaccharide phosphate. 4. Polyprenyl phosphate mannose served as a mannose donor to lipid-linked oligosaccharides and protein. When added as exogenous substrate it gave rise to a lipid-linked oligosaccharide of about six units. 5. Incorporation of radioactivity in protein was low, but polyacrylamide-gel electrophoresis of the protein fractions indicated that polypeptides of mol.wts. 115000, 75000 and 33000 were labelled.

The effect of tunicamycin on the glycosylation of lactating-rabbit mammary glycoproteins

“…The involvement of lipid-linked sugars as intermediates in glycoprotein biosynthesis in lactating rabbit mammary gland has been demonstrated in cell-free preparations (White, 1978), and a lipidlinked oligosaccharide containing mannose, Nacetylglucosamine (Speake & White, 1978a) and glucose (Speake & White, 1978b) has been isolated from explants of lactating mammary tissue. activity from D-[1-14C]mannose was largely incorporated by mammary explants into glycopolypeptides of mol.wts.…”

Lipid-linked oligosaccharides containing glucose in lactating rabbit mammary gland

“…Previous work in this laboratory (Speake & White, 1978;White, 1978) has shown that the oligosaccharide lipid synthesized from microsomal fractions (7 units) is shorter than that synthesized in mammary explants (10-12 units). Addition of UDPglucose to a mammary-gland microsomal system synthesizing mannose-containing oligosaccharide lipid increased the size of the oligosaccharide by approximately two monosaccharide units, as measured by paper chromatography (Fig.…”

“…Microsomal fractions were prepared from tissue homogenized in 250mM-potassium phosphate buffer, pH7.0, containing SmM-2-mercaptoethanol and 1 mM-EDTA as described by White (1978).…”

“…The explants were then washed with Medium 199 (3 x 1 ml) and homogenized in chloroform/methanol (2:1, v/v; 3 ml), and lipid-linked sugar and protein fractions were obtained as described above. The radioactivity in each fraction was determined as described by Speake & White (1978).…”

“…The involvement of polyprenol-linked sugars as intermediates in glycoprotein synthesis in lactating rabbit mammary gland has been demonstrated in cell-free preparations (White, 1978) and explants (Speake & White, 1978). Estimation of the size of the oligosaccharide moiety released from the oligosaccharide lipids by treatment with mild acid suggested that the major microsomal oligosaccharide was approximately seven glycose units, whereas that ofthe explants was 10-12 units.…”