Glycosylphosphatidylinositol membrane anchors in Saccharomyces cerevisiae: absence of ceramides from complete precursor glycolipids.

Glycosylphosphatidylinositol (GPI) anchoring of membrane proteins occurs through two distinct steps, namely the assembly of a precursor glycolipid and its subsequent transfer onto newly synthesized proteins. To analyze the structure of the yeast precursor glycolipid we made use of the pmi40 mutant that incorporates very high amounts of [3H]mannose. Two very polar [3H]mannose-labeled glycolipids named CP1 and CP2 qualified as GPI precursor lipids since their carbohydrate head group, Man alpha 1,2(X-->PO4-->6)Man alpha 1,2Man alpha 1,6Man alpha-GlcN-inositol (with X most likely being ethanolamine) comprises the core structure which is common to all GPI anchors described so far. CP1 predominates in cells grown at 24 degrees C whereas CP2 is induced by stress conditions. The apparent structural identity of the head groups suggests that CP1 and CP2 contain different lipid moieties. The lipid moieties of both CP1 and CP2 can be removed by mild alkaline hydrolysis although the protein-bound GPI anchors made by the pmi40 cells under identical labeling conditions contain mild base resistant ceramides. These findings imply that the ceramide moiety found on the majority of yeast GPI anchored proteins is added through a lipid remodeling step that occurs after the addition of the GPI precursor glycolipids to proteins.     

Characterization of glycophospholipid intermediate in the biosynthesis of glycophosphatidylinositol anchors accumulating in the Thy-1-negative lymphoma line SIA-b.

Several mammalian mutant cell lines are deficient in the biosynthesis of glycophosphatidylinositol anchors for membrane proteins. When metabolically labeled with [3H]myo-inositol or [3H]mannose, two out of five mutant lines (SIA-b and EL4-f) accumulated abnormal lipids which remained undetectable in the corresponding parental cell lines. The most abundant glycolipid of SIA-b cells (named lipid X) was isolated and partially characterized using hydrofluoric acid, nitrous acid deamination, acetolysis, and exoglycosidase treatments alone or in combination. The partial structure for the carbohydrate moiety of lipid X is Man alpha-(X----)Man alpha-GlcN-inositol, X being a charged, HF-sensitive substituent (possibly phosphoethanolamine). Lipid X is largely resistant to phosphatidylinositol-specific phospholipase C treatment but can be rendered sensitive to the enzyme by treatment with methanolic NH3, which suggests the presence of an acyl chain on the inositol moiety. The lipid moieties of lipid X are heterogenous in that about 50% of headgroups remain bound to a lipid moiety after mild alkaline hydrolysis. Similarly, about 50% of the lipid moieties of Thy-1, a glycophosphatidylinositol-anchored surface glycoprotein, isolated from SIA, the parent of SIA-b cells or from EL4 lymphoma cells, are resistant to mild alkaline hydrolysis. Altogether the data suggest that the SIA-b mutant line lacks an enzyme acting late in the anchor glycolipid biosynthesis pathway.     

Galactose-containing glycosylphosphatidylinositols in Trypanosoma brucei.

Many eukaryotic surface glycoproteins, including the variant surface glycoproteins (VSGs) of Trypanosoma brucei, are synthesized with a carboxyl-terminal hydrophobic peptide extension that is cleaved and replaced by a complex glycosylphosphatidylinositol (GPI) membrane anchor within 1-5 min of the completion of polypeptide synthesis. We have reported the purification and partial characterization of candidate precursor glycolipids (P2 and P3) from T. brucei. P2 and P3 contain ethanolamine-phosphate-Man alpha 1-2Man alpha 1-6Man alpha 1-GlcN linked glycosidically to an inositol residue, as do all the GPI anchors that have been structurally characterized. The anchors on mature VSGs contain a heterogenously branched galactose structure attached alpha 1-3 to the mannose residue adjacent to the glucosamine. We report the identification of free GPIs that appear to be similarly galactosylated. These glycolipids contain diacylglycerol and alpha-galactosidase-sensitive glycan structures which are indistinguishable from the glycans derived from galactosylated VSG GPI anchors. We discuss the relevance of these galactosylated GPIs to the biosynthesis of VSG GPI anchors.     

GPI anchor biosynthesis in yeast: phosphoethanolamine is attached to the alpha1,4-linked mannose of the complete precursor glycophospholipid

Cells synthesize the GPI anchor carbohydrate core by successively adding N-acetylglucosamine, three mannoses, and phosphoethanolamine (EtN-P) onto phosphatidylinositol, thus forming the complete GPI precursor lipid which is then added to proteins. Previously, we isolated a GPI deficient yeast mutant accumulating a GPI intermediate containing only two mannoses, suggesting that it has difficulty in adding the third, alpha1,2-linked Man of GPI anchors. The mutant thus displays a similar phenotype as the mammalian mutant cell line S1A-b having a mutation in the PIG-B gene. The yeast mutant, herein named gpi10-1 , contains a mutation in YGL142C, a yeast homolog of the human PIG-B. YGL142C predicts a highly hydrophobic integral membrane protein which by sequence is related to ALG9, a yeast gene required for adding Man in alpha1,2 linkage to N-glycans. Whereas gpi10-1 cells grow at a normal rate and make normal amounts of GPI proteins, the microsomes of gpi10-1 are completely unable to add the third Man in an in vitro assay. Further analysis of the GPI intermediate accumulating in gpi10 shows it to have the structure Manalpha1-6(EtN-P-)Manalpha1-4GlcNalpha1- 6(acyl) Inositol-P-lipid. The presence of EtN-P on the alpha1,4-linked Man of GPI anchors is typical of mammalian and a few other organisms but had not been observed in yeast GPI proteins. This additional EtN-P is not only found in the abnormal GPI intermediate of gpi10-1 but is equally present on the complete GPI precursor lipid of wild type cells. Thus, GPI biosynthesis in yeast and mammals proceeds similarly and differs from the pathway described for Trypanosoma brucei in several aspects.      

Glycolipid precursors for the membrane anchor of Trypanosoma brucei variant surface glycoproteins. I. Can structure of the phosphatidylinositol-specific phospholipase C sensitive and resistant glycolipids

A number of eukaryotic surface glycoproteins, including the variant surface glycoproteins of Trypanosoma brucei, are synthesized with a carboxyl-terminal hydrophobic peptide extension that is cleaved and replaced by a complex glycosyl-phosphatidylinositol (GPI) membrane anchor within 1-5 min of the completion of polypeptide synthesis. The rapidity of this carboxyl-terminal modification suggests the existence of a prefabricated precursor glycolipid that can be transferred en bloc to the polypeptide. We have reported the purification and partial characterization of a candidate precursor glycolipid (P2) and of a compositionally similar glycolipid (P3) from T. brucei (Menon, A. K., Mayor, S., Ferguson, M. A. J., Duszenko, M., and Cross, G. A. M. (1988) J. Biol. Chem. 263, 1970-1977). The primary structure of the glycan portions of P2 and P3 have now been analyzed by a combination of selective chemical fragmentation and enzymatic glycan sequencing at the subnanomolar level. The glycans were generated by deamination, NaB3H4 reduction, and dephosphorylation of glycolipids purified from different trypanosome variants. Glycan fragments derived from biosynthetically labeled glycolipids were also analyzed. The cumulative data strongly suggest that P2 and P3 contain ethanolamine-phosphate-Man alpha 1-2Man alpha 1-6Man alpha 1-GlcN linked glycosidically to an inositol residue, as do all the GPI anchors that have been structurally characterized. The structural similarities suggest that GPI membrane anchors are derived from common precursor glycolipids that become variably modified during or after addition to newly synthesized proteins.     

Structural studies on the glycosylphosphatidylinositol membrane anchor of Trypanosoma cruzi 1G7-antigen. The structure of the glycan core.

The 1G7-antigen is expressed by the infective metacyclic trypomastigote stage of the protozoan parasite Trypanosoma cruzi. The 1G7-antigen is a 90-kDa glycoprotein, present at about 40,000 copies/cell, which is anchored in the plasma membrane via a glycosylphosphatidylinositol (GPI) membrane anchor. The glycan of the GPI anchor has been isolated from immunopurified 1G7-antigen and its structure determined using a combination of methylation linkage analysis and exoglycosidase sequencing. The structure of the glycan is Man alpha 1-2Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH2. The glucosamine residue is in glycosidic linkage to a phosphatidylinositol moiety. The penultimate nonreducing alpha-Man residue is substituted with phosphate, which is most likely part of an ethanolamine phosphate bridge linking the GPI anchor to the 1G7-antigen polypeptide. The glycan sequence was obtained from 1.1 nmol of glycoprotein isolated from a detergent lysate of whole cells. The procedures reported here represent a high sensitivity protocol for determining GPI glycan structures from small quantities of biological material. The structure of the 1G7-antigen GPI anchor is consistent with the conserved core structure of all GPI anchors analyzed to date and is similar to that of the T. cruzi lipopeptidophosphoglycan. The biosynthesis of GPI anchors and lipopeptidophosphoglycan in T. cruzi is discussed in the light of this structural homology.     

Effect of growth temperature on lipid composition of Streptococcus faecium

The effect of growth temperature on the lipid and fatty acid composition of Streptococcus faecium has been studied. No differences in the qualitative composition of S. faecium lipids were observed. In all isolated fractions (neutral lipids, glycolipids, and phospholipids plus other polar lipids), the major fatty acids were palmitic (C-16:0), palmitoleic (C-16:1), octadecenoic (C-18:1), and cyclopropane (C-19:0). Changes in the fatty acid composition of the different fractions were observed which depended on growth temperature; the most significant one was the decrease of octadecenoic acid and the increase of palmitic acid in glycolipids and polar lipids as the temperature increased. The level of cyclopropane C-19:0 was approximately eightfold lower at 8 degrees C than at the other temperatures tested (20, 30, and 45 degrees C).     

Neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii

The neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii were characterized. The most abundant glycolipid was ceramide monosaccharide, followed by ceramide trisaccharide, ceramide tetrasaccharide, and ceramide disaccharide. More complex neutral glycolipids accounted for almost one-third of the total. The total amount of these glycolipids was 0.59 mg/g of dry weight of the ova preparation, a yield which was one-seventh of that of spermatozoa neutral glycolipids. Structural analyses were performed by enzymatic hydrolysis of the glycolipids with exoglycosidases, permethylation experiments, and also immuno-chemical assays. The proposed structures are as follows: ceramide monosaccharides, Gal-Cer and Glc-Cer; ceramide disacharides, Gal(beta 1-4)Gal-Cer, Gal(beta 1-4)Glc-Cer, and Man(beta 1-4)Glc-Cer; ceramide trisaccharide, Man(alpha 1-3)Man(beta 1-4)Glc-Cer; ceramide tetrasaccharides, Man(alpha 1-3)[Xyl(beta 1-2)]Man(beta 1-4)Glc-Cer, GlcNAc(beta 1-2)Man(alpha 1-3)Man(beta 1-4)Glc-Cer, Man(alpha 1-3)[Gal(beta 1-2)]Man(beta 1-4)Glc-Cer, and Man(alpha 1-2?)Man(alpha 1-3)Man(beta 1-4)Glc-Cer. The latter two ceramide tetrasaccharides were new types of glycosphingolipids. The spectrum of ova glycolipids appeared to be more complicated than that of the spermatozoa glycolipids. The ova glycolipids characterized here, with the exception of ceramide tetrasaccharides, contained considerable amounts of 2-hydroxy fatty acids, which were not observed in the spermatozoa glycolipids. The major sphingosine base was C18-sphingenine in all the ova glycolipids as well as in the spermatozoa glycolipids. However, the content of anteiso type of sphingosine base was 2- to 3-fold higher in the ova than in the spermatozoa.     

The fatty acid and monosaccharide compositions of three neutral and three phosphorylated glycolipids isolated from Leishmania donovani promastigotes grown in a chemically defined medium

Several lipids and macromolecular lipoconjugates of Leishmania spp. have now been well characterized; however, the glycolipids of L. donovani have not been thoroughly examined. In the present study, 3 neutral and 3 phosphorylated glycolipids were detected in promastigote forms of the organism grown in a chemically defined medium. The fatty acid and sugar compositions of these glycolipids, isolated and purified by adsorption column chromatography and thin-layer chromatographic procedures, were identified and quantified by gas-liquid chromatography and mass spectrometry. Myristate (14:0), palmitate (16:0), palmitoleate (16:1), stearate (18:0), oleate (18:1), and linoleate (18:2) were the major fatty acids in all 6 glycolipids. Arabinose, mannose, glucose, and galactose were detected in the glycolipids. The biochemical nature of these lipids suggested that the major components in the isolated preparations of the 6 glycolipids are diacylglycerophospholipids, distinct from the major precursors of macromolecular lipoconjugates such as the lipid anchors of cell surface antigens that have been reported. These appear to be terminal products of lipid biosynthesis in this parasite.     

The structure of a neural specific carbohydrate epitope of horseradish peroxidase recognized by anti-horseradish peroxidase antiserum.

Antiserum raised against horseradish peroxidase (HRP) recognizes a neural specific carbohydrate antigen in Drosophila and other insects. The epitopic activity of the carbohydrate moiety of HRP recognized by anti-HRP antiserum was measured by a newly developed enzyme-linked immunosorbent assay, in which HRP glycopeptides conjugated with bovine serum albumin were coated onto the wells and then reacted with goat anti-HRP antiserum. HRP sugar moieties released by almond glycopeptidase A digestion of HRP pepsin digests were subjected to pyridylamination. Pyridylamino oligosaccharides were separated into seven fractions by reverse-phase high performance liquid chromatography. The major fraction, which comprised about 80% of the total sugars, reacted strongly with anti-HRP antiserum. The carbohydrate structure of this fraction was determined by sugar composition analysis and 600-MHz 1H NMR spectroscopy as follows: Man alpha 1----6(Man alpha 1----3)(Xyl beta 1----2)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----3)GlcNAc. Analyses of reactivity with anti-HRP antiserum of various oligosaccharide derivatives obtained from the major fraction by exoglycosidase digestion and partial acid hydrolysis indicated that alpha 1----6-linked mannose and alpha 1----3-linked fucose are predominantly involved in the epitopic structure.     

Novel fucolipids accumulating in human adenocarcinoma. I. Glycolipids with di- or trifucosylated type 2 chain

Among a series of fucolipids accumulating in human colonic and liver adenocarcinoma, two neutral fucolipids have been isolated and their carbohydrate structures have been characterized by methylation analysis, mass spectrometry, enzymatic degradation, and proton NMR spectrometry as shown in Structures 1 and 2 below. These glycolipids are either absent or present in very small quantity in normal colonic mucosa and normal liver tissue. (formular; see text) Difucosyl neolactonorhexaosylceramide (Structure 1) gives a doublet or a triplet on high performance thin layer chromatography and can be separated into four to five fractions on high pressure high performance liquid chromatography. These components have the same carbohydrate structure, but have different fatty acid composition. Fractions are characterized by a predominance of either alpha-hydroxy C16 fatty acid or alpha-hydroxy C24 fatty acid. Trifucosyl neolactonoroctaosylceramide (structure 2) gives two discrete bands, which are designated Z3 and Z4, on high performance thin layer chromatography. Methylation analysis and nuclear magnetic resonance spectra show that the Z3 and Z4 have identical carbohydrate structures. All the cases of cancer tissue that accumulate di- or trifucosyl structure (1 or 2 above) also accumulated lactofucopentaose(III)ceramide. A possible enhancement in a specific synthetic pathway including type 2 chain elongation coupled with alpha 1----3 fucosylation in human cancer is discussed.     

Structural analysis of a glycosylphosphatidylinositol glycolipid ofLeishmania donovani

A glycosylphosphatidylinositol (GPI) glycolipid antigen recognized by sera from patients with visceral leishmaniasis was isolated from Leishmania donovani promastigotes. The carbohydrate moiety was cleaved from the lipid part by digestion with specific phosphatidylinositol phospholipase C. After separation, structural analysis was carried out on the phosphorylated inositol oligosaccharide and the alkylacyl glycerol. The following major structures were found: [formula: see text] The presence of the conserved sequence Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN-PI of glycosyl phosphatidylinositol protein anchors in this antigen may be consistent with a precursor role of Leishmania glycosyl phosphatidylinositol anchored proteins for this glycolipid.     

Developmental variation of glycosylphosphatidylinositol membrane anchors in Trypanosoma brucei. Identification of a candidate biosynthetic precursor of the glycosylphosphatidylinositol anchor of the major procyclic stage surface glycoprotein.

The major surface antigen of the mammalian bloodstream form of Trypanosoma brucei, the variant surface glycoprotein (VSG), is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. The VSG anchor is susceptible to phosphatidylinositol-specific phospholipase C (PI-PLC). Candidate precursor glycolipids, P2 and P3, which are PI-PLC-sensitive and -resistant respectively, have been characterized in the bloodstream stage. In the insect midgut stage, the major surface glycoprotein, procyclic acidic repetitive glycoprotein, is also GPI-anchored but is resistant to PI-PLC. To determine how the structure of the GPI anchor is altered at different life stages, we characterized candidate GPI molecules in procyclic T. brucei. The structure of a major procyclic GPI, PP1, is ethanolamine-PO4-Man alpha 1-2Man alpha 1-6 Man alpha 1-GlcN-acylinositol, linked to lysophosphatidic acid. The inositol can be labeled with [3H]palmitic acid, and the glyceride with [3H]stearic acid. We have also found that all detectable ethanolamine-containing GPIs from procyclic cells contain acylinositol and are resistant to cleavage by PI-PLC. This suggests that the procyclic acidic repetitive glycoprotein GPI anchor structure differs from that of the VSG by virtue of the structures of the GPIs available for transfer.     

Mycolipenates and mycolipanolates of trehalose from mycobacterium tuberculosis

Analysis of the lipids of Mycobacterium tuberculosis, by thin-layer chromatography, revealed the presence of two families of novel glycolipids each having two closely-related members but differing widely in polarity. The least and most polar families of lipids were characterized from M. tuberculosis strains C and H37Rv, respectively; all were based on trehalose, the least polar pair of glycolipids having more long-chain substituents than the more polar pair. The acyl substituents of the least polar of the four glycolipids were mainly straight-chain C16 and C18 acids and 2,4,6-trimethyltetracos-2-enoic (C27-mycolipenic) acid, and the second least polar glycolipid contained major amounts of 3-hydroxy-2,4,6-trimethyltetracosanoic (C27-mycolipanolic) acid in addition to these non-hydroxylated acids. The relatively polar pair of glycolipids were analysed together and released mainly straight-chain C16 and C18 acids, C27-mycolipanolic acid, minor amounts of C25- and C27-mycolipenic acids and major proportions of an acid having the chromatographic properties of 2,4-dimethyldocosanoic acid. The most polar pair of glycolipids co-chromatographed with glycolipid antigens previously detected in Mycobacterium bovis BCG.     

Lipid analysis of Methanopyrus kandleri

Abstract Non-polar and polar lipids were isolated from Methanopyrus kandleri . Non-polar lipids accounted for 50% w/w of total lipids, with a high proportion of 2,3-di- O -geranylgeranyl-sn-glycerol, 2,3-di- O -phytanyl-sn-glycerol and geranylgeraniol. The core lipids prepared by mild acid methanolysis consisted exclusively of 2,3-di- O -phytanyl-sn-glycerol. Two-dimensional TLC showed mostly glycolipids, and minor amounts of aminophospholipids, phosphoglycolipids and phospholipids. The purification yielded three diglycosyl-lipids (50% of total polar lipids), one triglycosyl-lipid (5%) and six glycosyl-lipids with five glycosyl-groups (36%), which consisted of glucose, galactose and mannose. The lipid analysis supports the unique position of Methanopyrus kandleri within the 16S rRNA-based phylogenetic tree.     

Primary structure and carbohydrate binding specificity of a potent anti-HIV lectin isolated from the filamentous cyanobacterium Oscillatoria agardhii

The primary structure of a lectin, designated Oscillatoria agardhii agglutinin (OAA), isolated from the freshwater cyanobacterium O. agardhii NIES-204 was determined by the combination of Edman degradation and electron spray ionization-mass spectrometry. OAA is a polypeptide (Mr 13,925) consisting of two tandem repeats. Interestingly, each repeat sequence of OAA showed a high degree of similarity to those of a myxobacterium, Myxococcus xanthus hemagglutinin, and a marine red alga Eucheuma serra lectin. A systematic binding assay with pyridylaminated oligosaccharides revealed that OAA exclusively binds to high mannose (HM)-type N-glycans but not to other N-glycans, including complex types, hybrid types, and the pentasaccharide core or oligosaccharides from glycolipids. OAA did not interact with any of free mono- and oligomannoses that are constituents of the branched oligomannosides. These results suggest that the core disaccharide, GlcNAc-GlcNAc, is also essential for binding to OAA. The binding activity of OAA to HM type N-glycans was dramatically decreased when alpha1-2 Man was attached to alpha1-3 Man branched from the alpha1-6 Man of the pentasaccharide core. This specificity of OAA for HM-type oligosaccharides is distinct from other HM-binding lectins. Kinetic analysis with an HM heptasaccharide revealed that OAA possesses two carbohydrate binding sites per molecule, with an association constant of 2.41x10(8) m-1. Furthermore, OAA potently inhibits human immunodeficiency virus replication in MT-4 cells (EC50=44.5 nm). Thus, we have found a novel lectin family sharing similar structure and carbohydrate binding specificity among bacteria, cyanobacteria, and marine algae.     

Studies on glycosphingolipids in larvae of the green-bottle fly, Lucilia caesar: two neutral glycosphingolipids having large straight oligosaccharide chains with eight and nine sugars

Two neutral glycosphingolipids having large straight oligosaccharide chains with eight and nine sugars, provisionally named COS and CNS, were isolated and purified from larvae of the green-bottle fly, Lucilia caesar, as the only two remaining unidentified significant neutral glycolipids in this organism. From the results of sugar analysis, permethylation, negative-ion fast atom bombardment mass spectroscopy (FAB-MS), and 1H-NMR studies, the structures of the two glycolipids are proposed to be: COS, GalNAc beta 1-3GlcNAc beta 1-3Gal beta 1-3GalNAc alpha 1-4GalNAc beta 1-4GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer; and CNS, Gal beta 1-3GalNAc beta 1-3GlcNAc beta 1-3Gal beta 1-3GalNAc alpha 1-4GalNAc beta 1-4GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer. The fatty acid and long-chain base compositions of the above glycolipids were very similar, and were dominated by arachidic acid, and tetradeca- and hexadeca-4-sphingenines. The great similarity between the compositions of their ceramide moieties suggests that COS may be a precursor in the glycosylation reaction yielding CNS.     

Oligosaccharide structure of a functional unit RvH1-b of Rapana venosa hemocyanin using HPLC/electrospray ionization mass spectrometry

In the present study the structures of two glycopeptides (G1 and G1'), isolated from FU RvH(1)-b and two glycopeptides (G2 and G3), isolated from the structural subunit RvH(1) of Rapana venosa hemocyanin, were determined. To structurally characterize the site-specific carbohydrate heterogeneity and binding site of the N-linked glycopeptide(s), a combination of capillary reversed-phase chromatography and ion trap mass spectrometry was used. The amino acid sequences of glycopeptides G1 and G1' determined by Edman degradation and MS/MS sequencing demonstrated that the oligosaccharides are linked to N-glycosylation sites. Two peptides (a glycosylated (G1) and non-glycosylated one) were identified in this fraction and no linkage sites were observed in the latter one. Based on the sequencing of the glycosylated fractions G1, G1', G2 and G3, the carbohydrate structure Man(alpha1-6)Man(alpha1-3)Man(beta1-4)GlcNAc(beta1-4)[Fuc(alpha1-6)]GlcNAc-R could be identified for glycopeptides G1 and G3, and only the typical core structure Man(alpha1-6)Man(alpha1-3)Man(beta1-4)GlcNAc(beta1-4)GlcNAc-R was found for G1' and G2. The Fuc residue found in glycopeptides G1 and G3 is attached to N-acetyl-glucosamine of the carbohydrate core, as often found in other glycoproteins.     

The isolation and partial characterization of glycolipids of normal human leucocytes

1. The lipids of purified human leucocytes were extracted with chloroform-methanol and the extract was washed with water. Glycolipids, isolated by Florisil chromatography, were subjected to mild alkaline hydrolysis and the alkali-resistant fraction was fractionated on a silicic acid column. 2. Three classes of glycolipid were separated. The less polar, containing 3.6% of the total glycolipid hexose as galactose, was tentatively identified as ceramide monohexoside. The major glycolipid fraction was characterized as ceramide dihexosides. The more polar glycolipids comprised 1.6% of the total glycolipid hexose as galactose and glucose (in the molar ratio 2:1) and were non-acidic. This class was separated as a mixture containing ninhydrin-positive glycolipids. 3. The ceramide dihexosides taken from two leucocyte preparations accounted for 15.2% and 16.4% by weight of the total lipids. 4. The carbohydrate moiety of the ceramide dihexosides contained galactose and glucose in the molar ratio 2:1. Partial acid hydrolysis and paper chromatography indicated that the hexoses are present as disaccharides, lactose being identified as one of them. 5. Palmitic acid (C(16:0)) and nervonic acid (C(24:1)) were the major fatty acids of this glycolipid. Hydroxy fatty acids were not detected.     

Biosynthesis of lipid-linked oligosaccharides. I. Preparation of lipid-linked oligosaccharide substrates.

In order to purify the glycosyltransferases involved in the assembly of lipid-linked oligosaccharides and to be able to study the acceptor substrate specificity of these enzymes, methods were developed to prepare and purify a variety of lipid-linked oligosaccharides, differing in the structure of the oligosaccharide moiety. Thus, Man9 (GlcNAc)2-pyrophosphoryl-dolichol was prepared by isolation and enzymatic synthesis using porcine pancreatic microsomes, while Glc3Man9(GlcNAc)2-PP-dolichol was isolated from Madin-Darby canine kidney cells. Treatment of these oligosaccharide lipids with a series of selected glycosidases led to the preparation of Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6(Man alpha 1,3)Man alpha 1,6]Man beta 1,4GlcNAc beta 1,4GlcNAc-PP-dolichol; Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6]Man beta 1,4GlcNAc beta 1, 4GlcNac-PP-dolichol; and Man alpha 1,6(Man alpha 1,3)Man alpha 1, 6[Man alpha 1,3]Man beta 1,4GlcNAc-beta 1,4GlcNAc-PP-dolichol. The preparation, isolation, and characterization of each of these lipid-linked oligosaccharide substrates are described.