Characterization of Mannolipids Formed in a Calf Pancreatic Microsomal Fraction from Guanosine Diphosphate [14C]Mannose and either Endogenous Lipid or Exogenous Polyisoprenyl Phosphates

In some glycoproteins, a glycosylamine bond between 2-acetamido-2-deoxy-~-glucose and the amide group of asparagine serves to link oligosaccharide with peptide. A common oligosaccharide unit containing 2-acetamido-2-deoxy-~-glucose and D-mannose residues has been suggested for the linkage region of these glycoproteins (Lee & Scocca, 1972). Although mannosyltransferase and N-acetylglucosaminyltransferase activities have been demonstrated in several tissues, little is known about the assembly of this unit. Further, the occurrence in some tissues of polyisoprenoid lipids capable of acting as D-mannose and 2-acetamido-2-deoxy-~-g~ucose acceptors has raised the possibility that lipid intermediates are involved in the transfer of these sugars to glycoproteins. Mannosyltransferase activity was investigated in calf pancreas. I t was shown that one of the products formed when a rough-microsomal fraction (microsomes) is incubated with GDP-[14C]mannose in the presence of Mn2+ is an alkali-stable acid-labile [14C]mannolipid (Herscovics et al., 1973). The acidic nature of this lipid was established by its chromatographic behaviour on silicic acid and DEAE-cellulose columns. When a silicic acid column on which the mannolipid was adsorbed was sequentially eluted with chloroform, acetone and chloroform-methanol (1 : 1, v/v), the lipid was recovered in the chloroform-methanol fraction. The radioactive lipid was not eluted from a DEAEcellulose column by chloroform or chloroform-methanol (4: 1, v/v), but could be eluted with chloroform-methanol (4: 1, v/v) containing ammonium acetate. Short-term acid hydrolysis (0.01 M-HCI, 100°C, 1 min) of the pancreatic lipid liberated [14C]mannose but no detectable ['4C]mannosyl phosphate. These properties are consistent with the view that the labelled pancreatic lipid is a polyisoprenyl mannosyl monoor pyro-phosphate (Richards & Hemming, 1972). Production of the mannolipid proceeded linearly with time for approx. lOmin under the incubation conditions employed. The addition of excess of GDP to the microsomes Smin after the beginning of the incubation resulted in a subsequent disappearance of label from the mannolipid. GMP at the same concentration was ineffectual. This evidence suggests that the mannose is linked to the lipid via a phosphodiester rather than a pyrophosphate linkage. Authentic dolichyl a-D-mannopyranosyl phosphate was prepared chemically for comparison with the pancreatic mannolipid. The procedure of MacDonald, as previously modified for the synthesis of 2,3,4,6-tetra-O-acety~-cc-~-galactopyranosyl phosphate (Warren & Jeanloz, 1972), was employed for the preparation of the corresponding a-Dmannopyranosyl derivative. This compound was coupled with pig liver dolichol in the presence of dicyclohexylcarbodi-imide or tri-isopropylbenzenesulphonyl chloride to give the acetylated phosphodiester, which was purified by either silica-gel column chromatography or t.1.c. After deacetylation with sodium methoxide in methanol, the product was purified by preparative t.1.c. (Warren & Jeanloz, 1973). The labelled mannolipid formed in pancreas was compared with the synthetic dolichyl