Influenza A virus-binding activity of glycoglycerolipids of aquatic bacteria

As the aqueous sphere has been proposed to be an important source medium for the virus infection of land animals, the glycolipids of some aquatic organisms were examined for human influenza A virus-binding activity. Active compounds were not found among the eight echinoderm gangliosides, but two active non-sialylated glycoglycerolipids were isolated from an aquatic bacterium, Corynebacterium aquaticum. The structural formula of one of them, H632A, was elucidated to be 1-14-methyl-hexadecanoyl-3-alpha-D-galactopyranosyl-(1-->3)-6-(12-met hyl-tetradecanoyl)-1-alpha-D-mannopyranosyl]-sn-glycerol. The latter together with reported one elsewhere, S365A, 1-14-methyl-hexadecanoyl-3-[alpha-D-mannopyranosyl-(1-->3)-6-(12-meth yl-tetradecanoyl)-1-alpha-D-mannopyranosyl]-sn-glycerol, apparently bound to three human influenza viruses, A/PR/8/34 (H1N1), A/Aichi/2/68 (H3N2), and A/Memphis/1/71 (H3N2), exhibiting 7-12% (H632A) and 10-22% (S365A) of the activities of the control substances (Neu5Acalpha2-3-paragloboside and Neu5Acalpha2-6- paragloboside). Additionally, these glycolipids were assumed to have virus-neutralizing activities for the following two reasons: (i) The hemagglutination and hemolysis activities of the viruses were inhibited by the glycolipid. (ii) The leakage of a cytosolic enzyme (lactate dehydrogenase) from Madin-Darby canine kidney cells on virus infection was prevented by the glycolipids to nearly the same extent as by fetuin. This is the first evidence of the binding- and neutralizing-abilities of native glycoglycerolipids as to influenza viruses.     

Monoclonal antibody recognizing a determinant on type 2 chain blood group A and B oligosaccharides detects oncodevelopmental changes in azoxymethane-induced rat colon tumors and human colon cancer cell lines

Altered expression of ABH blood group substances is a common feature of human colorectal carcinoma, yet it remains unclear how these structural changes influence the biological properties of tumor cells. Azoxymethane-induced rat colon tumors display many features of the human disease, thereby providing a potentially useful model to study the role of blood group substances in colon cancer progression. We have prepared monoclonal antibodies to a microsomal fraction isolated from an azoxymethane-induced rat colon tumor and selected an antibody that detects cancer-associated changes. Monoclonal antibody (mAb) 3A7 recognizes a determinant on type 2 chain blood group A (GalNAcα1–3[Fucα1–2]Galβ1–4GlcNAc-R) and B (Galα1–3[Fucα1–2]Galβ1–4GlcNAc-R) oligosaccharides. Expression of the epitope detected by this antibody was developmentally regulated in rat colon, with maximal expression from day 4–21 after birth. Immunohistochemical staining and Western blotting analyses of azoxymethane-induced colon tumors revealed increased expression of the epitope in all of the 21 colonic tumors examined, including preneoplastic glands within transitional mucosa. Conventional and signet-ring adenocarcinomas that had invaded through the muscularis propria (Duke's B2) consistently showed the most intense staining with mAb 3A7, including regions depicting angioinvasion. Some of the lymph node metastases (Duke's C2) stained poorly with the antibody. The epitope was also expressed in blood group A positive human colon carcinoma cell lines, including HT29 and SW480 but not by SW620, a cell line derived from a lymph node metastasis isolated in vivo from the SW480 primary tumor, or in the blood group B cell line SW1417. The glycoproteins detected by mAb 3A7 in rat colon tumors and HT29 cells ranged in size between 50 and 200 kd, including a major species of 140 kd. Affinity chromatography of detergent lysates of normal rat colon on the blood group A specific lectin Dolichos biflorus (DBA)-agarose resulted in nearly quantitative binding of glycoprotein species detected by the antibody. By contrast, immunoreactive glycoproteins from rat colon tumors or HT29 cells bound poorly to DBA-agarose but were retained by another blood group A-binding lectin, Helix-pomatia (HPA)-agarose. These results indicate that colon carcinogenesis results in quantitative as well as qualitative changes in oligosaccharides detected by mAb 3A7 and suggest that the combined use of mAb 3A7 and blood group A-specific lectins may provide a useful tool for early detection of colon cancer.     

Integrin alpha3beta1 expressed by human colon cancer cells is a major carrier of oncodevelopmental carbohydrate epitopes

Oncodevelopmental carbohydrate epitopes are a common feature of human colorectal carcinoma, yet their biological significance remains unclear. We have shown previously that monoclonal antibody (MAb) 3A7, which recognizes a determinant on type 2 chain blood group A and B oligosaccharides, detects oncodevelopmental changes in azoxymethane-induced rat colon tumors and some human colon cancer cell lines. (Laferté S et al. [19951 J. Cell. Biochem. 57:101-119). In this study, we set out to purify gp140, the major glycoprotein carrier of the 3A7 epitope expressed by human colon cancer cells, as a first step towards elucidating the contribution of the 3A7 epitope and its major glycoprotein carrier to colon cancer progression. To this end, gp140 was purified from HT29 cells and used for the preparation of polypeptide-specific monoclonal antibodies. Five monoclonal antibodies (7A8, 7B11, 8C7, 8H7, and 11D4) immunoprecipitated a 3A7-immunoreactive glycoprotein complex of 140 kDa which was subsequently identified by partial protein sequencing as alpha3beta1 integrin. Flow cytometric analysis of Chinese hamster ovary (CHO) cells expressing different human integrin chains revealed that MAbs 7A8 and 7B11 detect the alpha3 integrin subunit whereas MAbs 8C7 and 8H7 detect the beta1 integrin subunit. MAb 11D4, which did not bind to any of the CHO transfectants, detected type 2 chain blood group A determinant. Flow cytometric analysis of a panel of human colon carcinoma cell lines obtained from blood group A, AB, or B individuals revealed a direct correlation between cell-surface expression of the 3A7 epitope and alpha3 integrin subunit, suggesting that alpha3beta1 integrin is a preferred target of the 3A7 epitope in colon cancer cells. Using lectins and glycosidases to examine further the carbohydrate structure of alpha3beta1 integrin, we demonstrated that it is a sialoglycoprotein containing both N- and O-linked oligosaccharides. In addition, both alpha3 and beta1 integrin subunits express beta1-6 branched Asn-linked oligosaccharides and short poly-N-acetyllactosamine units (Galbeta1-4GlcNAc-R; n < or = 3), glycans previously implicated in cancer metastasis.Thus, alpha3beta1 integrin expressed by human colon carcinoma cells is a major carrier of oncodevelopmental carbohydrate epitopes whose presence may modulate tumor cell adhesion, migration, and/or invasion.     

Increased BNip3 and decreased mutant p53 in cisplatin-sensitive PDT-resistant HT29 cells

We have reported previously the isolation of three photodynamic therapy (PDT)-resistant human colon carcinoma HT29 cell lines that show increased expression of the Hsp27 and BNip3 protein and a decreased expression of the mutant p53 protein compared to parental HT29 cells. Since mutant p53 and increased expression of Hsp27 have been associated with resistance to various chemotherapeutic agents, whereas BNip3 is a potent inducer of apoptosis, we were interested in determining whether these PDT-resistant cells were cross-resistant to other cytotoxic agents. We report here that the PDT-resistant HT29 cell lines showed a significant increase in cisplatin sensitivity and an increase in both spontaneous and cisplatin-induced apoptosis compared to parental HT29 cells. In addition, the cisplatin sensitivity of the PDT-resistant HT29 variants and several other clonal variants of HT29 cells correlated with increased BNip3 and decreased mutant p53 protein levels, but not Hsp27 protein levels.     

Intracellular inhibition of blood group A glycosyltransferase.

We report the intracellular inhibition of blood group A N-acetylgalactosaminyltransferase in the human colorectal carcinoma cell line HT29 by 3-amino-3-deoxy-[Fucalpha(1-2)]Galbeta-O(CH2)7CH3. Inhibition was demonstrated with a novel capillary electrophoresis assay that monitored decreased intracellular conversion of fluorescently labelled Fucalpha(1-2)Gal-R acceptor to the corresponding A epitope, GalNAcalpha(1-3)[Fucalpha(1-2)]Galbeta-R. Growth of HT29 cells with either the amino-inhibitor or a competitive substrate, Fucalpha(1-2)Galbeta-O(CH2)7CH3, also resulted in decreased expression of blood group A determinants on cell-associated glycoproteins, as detected by immunoprecipitation analysis using A-specific monoclonal antibodies. Furthermore, exposure of these cells to the amino-inhibitor or competitive substrate resulted in significant reduction of cell-surface expression of blood group A determinants. As integrin alpha3beta1, a cell-surface receptor mediating cell-cell and cell-extracellular matrix interactions, was shown previously to be a major carrier of blood group A determinants on HT29 cells, the studies described herein highlight the potential usefulness of these compounds for elucidating the role of blood group A determinants in biological phenomena.     

Choline-linked phosphoglycerides. A source of phosphatidic acid and diglycerides in stimulated neutrophils

Stimulation of human polymorphonuclear leukocytes (PMN) may result in the metabolism of phospholipids other than phosphoinositides to generate second-messenger intermediary metabolites. We investigated agonist-induced breakdown of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acyl-GPC), which constitutes almost half the diradyl-GPC fraction in human PMN (Mueller, H. W., O'Flaherty, J. T., Green, D. G., Samuel, M. P., and Wykle, R. L. (1984) J. Lipid Res. 25: 383-388), in cells prelabeled with 1-O-[3H] alkyl-2-acyl-GPC. We also utilized normal-phase high pressure liquid chromatography to quantitate the accumulation of diradylglycerols (1-O-alkyl-2-acylglycerols and diacylglycerols) in stimulated PMN. Phorbol-12-myristate-13-acetate (PMA), 1-oleoyl-2-acetyl-sn-glycerol-, calcium ionophore A23187-, and f-methionyl-leucyl-phenylalanine (fMLP) stimulation of PMN resulted in a time- and concentration-dependent hydrolysis of 1-O-[3H]alkyl-2-acyl-GPC and the formation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid (PA) and 1-O-[3H]alkyl-2-acylglycerol. In all cases formation of 1-O-[3H]alkyl-2-acyl-PA preceded that of 1-O-[3H]alkyl-2-acylglycerol. The times between addition of stimulus and appearance of 1-O-[3H] alkyl-2-acylglycerol varied for PMA (40 s at 1.6 microM), A23187 (5 min at 5 microM), and fMLP (30 sec at 1 microM). Preincubation of cells with 1 microgram/ml pertussis toxin (PT) inhibited the breakdown of 1-O-[3H]alkyl-2-acyl-GPC in cells stimulated with 1 microM fMLP, indicating a role for a PT-sensitive G protein with this stimulus. Quantitation of diglycerides as diradylglycerobenzoates in PMN stimulated with PMA (10 min), A23187 (10 min), or fMLP demonstrated marked accumulation of both 1-O-alkyl-2-acylglycerols and diacylglycerols. The highest increases over controls were observed for fMLP (33-fold for 1-O-alkyl-2-acylglycerols and 17-fold for diacylglycerols). In stimulated PMN prelabeled with 1-O-[3H]hexadecyl-2-acyl-GPC and 1-O-alkyl-2-acyl-sn-glycero-3-[32P]phosphocholine, the ratio of 3H to 32P in 1-O-alkyl-2-acyl-PA compared to 1-O-alkyl-2-acyl-GPC suggested the involvement of a phospholipase D in the hydrolysis of 1-O-[3H]-alkyl-2-acyl-GPC. Thus, stimulation of human PMN results in the hydrolysis of 1-O-[3H]alkyl-2-acyl-GPC to yield 1-O-[3H] alkyl-2-acyl-PA and 1-O-[3H]alkyl-2-acylglycerol possibly initiated by activation of a phospholipase D.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structures of minor ether lipids isolated from the aceticlastic methanogen, Methanothrix concilii GP6

Structures were determined for two phospholipids and three glycolipids purified from chloroform-methanol extracts of Methanothrix concilii GP6. Together they accounted for 14% of the total lipid and were based on a C20,20-diether core structure consisting of either 2,3-di-O-phytanyl-sn-glycerol or its 3'-hydroxy analog, namely, 2-O-[3,7,11,15-tetramethylhexadecyl]-3-O-[3'- hydroxy-3',7',11',15'-tetramethylhexadecyl]-sn-glycerol. These two core lipids formed phosphodiester bonds to ethanolamine and glycosidic bonds to beta-D-galactopyranose. A third glycolipid consisted of the triglycosyl head group beta-D-galactopyranosyl-(1----6)-[beta-D-glucopyranosyl-(1----3)]-beta-D - galactopyranose in glycosidic linkage to the 3'-hydroxydiether core lipid.     

CD4 molecules are restricted to the basolateral membrane domain of in vitro differentiated human colon cancer cells (HT29-D4)

The CD4 glycoprotein serves as a receptor for the human immunodeficiency virus HIV, the etiologic agent of acquired immunodeficiency syndrome (AIDS). We have examined the expression of CD4 molecules in a clone (HT29-D4) derived from a human colon adenocarcinoma cell line. HT29-D4 cells synthesized a 60 kDa polypeptide immunoprecipitated with two anti-CD4 monoclonal antibodies after metabolic or cell surface labeling. This 60 kDa polypeptide was also immunodetected using the same antibodies in human acute lymphoblastic leukemia cells CEM which are known to express CD4. HT29-D4 cells can be induced to differentiate into enterocyte-like cells by removing glucose from the culture medium. Under these conditions, HT29-D4 cells form a polarized epithelial monolayer in which tight junctions separate the plasma membrane in an apical and a basolateral domain. The localization of CD4 molecules in differentiated HT29-D4 cells was exclusively restricted to the basolateral membrane domain as demonstrated by radioimmunoassay and indirect immunofluorescence studies. Therefore the HT29-D4 clonal cell line represents a unique model for polarized HIV infection of colonic epithelial cells and may be useful to understand some of the gastrointestinal disorders occurring in AIDS patients.     

Conversion of 1-O-[3H]alkyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine to lyso platelet-activating factor by the CoA-independent transacylase in membrane fractions of human neutrophils

The first step in the synthesis of platelet-activating factor (PAF) in stimulated neutrophils is generally accepted to be hydrolysis of 1-O-alkyl-2-acyl-sn-glycero-3-phosphorylcholine (1-O-alkyl-2-acyl-GPC), with 1-O-alkyl-2-arachidonoyl-GPC being the preferred precursor. Characterization of the enzymatic activity responsible for the hydrolysis of 1-O-alkyl-2-arachidonoyl-GPC has been hampered by lack of an active and reliable cell-free system for study. In the present studies, membrane preparations containing 1-O-[3H]alkyl-2-arachidonoyl-GPC were prepared from intact human neutrophils that had been labeled using 1-O-[3H]hexadecyl-2-lyso-GPC. When the labeled membrane preparations were incubated in the presence of unlabeled 1-O-alkyl-2-lyso-GPC (5 microM), rapid deacylation (up to 25% of the label in 10 min) of the 1-O-[3H]alkyl-2-arachidonoyl-GPC to 1-O-[3H]alkyl-2-lyso-GPC (lyso-PAF) was observed. The deacylation activity appeared to be the same in preparations from resting or stimulated cells. No requirement for Ca2+, various nucleotides, or protein kinase activation could be demonstrated. A number of observations indicated that [3H]lyso-PAF is formed in the system by the action of the CoA-independent transacylase present in the cells rather than by phospholipase A2. Both 1-O-alkyl-2-lyso-GPC and 1-acyl-2-lyso-GPC elicited deacylation of 1-O-[3H]alkyl-2-arachidonoyl-GPC, whereas neither 3-O-alkyl-2-lyso-GPC nor 1-O-alkyl-2-O-methyl-rac-glycero-3-phosphorylcholine, which should act as detergents but are not transacylase substrates, effected deacylation. The deacylation activity and CoA-independent transacylase activities were blocked in parallel by a number of inhibitors and by heat inactivation. In preparations containing 1-O-alkyl-2-[3H]arachidonoyl-GPC, no release of free [3H]arachidonic acid was observed. However, a shift of the [3H]arachidonate into exogenous 1-O-tetradecyl-2-lyso-GPC was observed in the system. These findings are consistent with the generation of [3H]lyso-PAF by the CoA-independent transacylase activity.     

Structural characterization of the lipids of Methanococcus voltae, including a novel N-acetylglucosamine 1-phosphate diether

The lipids of a moderately halophilic methanogen, Methanococcus voltae, accounted for 5.1% of the cell dry weight and consisted of 91% polar lipids and 9% neutral lipids. Twelve polar lipids were detected, three of which, all derivatives of 2,3-di-O-phytanyl-sn-glycerol, were identified as: 2,3-di-O-phytanyl-1-O-[beta-D-glucopyranosyl-(1-6)-beta-D- glucopyranosyl]-sn-glycerol, 2,3-di-O-phytanyl-1-O-[beta-D-glucopyranosyl]-sn-glycerol and a novel NAc-glucosamine 1-phosphate diether, and 2,3-di-O-phytanyl-1-[phosphoryl-2-acetamido-2-deoxy-beta-D- glucopyranosyl]-sn-glycerol. The neutral lipids consisted mainly of squalenes: squalene, dihydrosqualene, tetrahydrosqualene, hexahydrosqualene, and unidentified squalenes.     

The dependence of glyceroglycolipid orientation and dynamics on head-group structure

The head-group orientations and molecular dynamics of three glyceroglycolipids in aqueous dispersions, as determined by 2H-NMR, are compared. 1,2-Di-O-tetradecyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerol (alpha-DTGL) and 1,2-di-O-tetradecyl-3-O-(alpha-D-mannopyranosyl)-sn-glycerol (alpha-DTML), selectively 2H-labelled on the pyranose ring, at the exocyclic hydroxymethyl group, and at C3 of glycerol, have been studied by 2H-NMR and the results compared with those reported earlier for 1,2-di-O-tetradecyl-3-O-(beta-D-glucopyranosyl)-sn-glycerol (beta-DTGL). The alpha-glucolipid exhibits a gel-to-liquid crystal phase transition and a lamellar to hexagonal mesophase transition at temperatures which are similar to those of the beta-anomer, beta-DTGL. However, alpha-DTGL exhibits head group orientations and molecular ordering in the lamellar and hexagonal phases which differ strikingly with those reported for the corresponding beta-glucolipid. Whereas the head group of beta-DTGL is extended away from the bilayer surface into the aqueous phase, that of alpha-DTGL is almost parallel to the bilayer surface. alpha-DTGL exhibits a molecular order parameter of 0.56 which is substantially greater than that of its anomer, beta-DTGL, 0.45. The latter indicates that the head group region of the alpha-glyceroglucolipid is characterized by smaller angular fluctuations than that of beta-DTGL. On entering the hexagonal mesophase the pyranose ring of the beta-glucolipid undergoes a large reorientation relative to the motional axis of the head group, whereas the alpha-anomer exhibits only a small orientational change. 1,2-Di-O-tetradecyl-3-O-(alpha-D-mannopyranosyl)-sn-glycerol (alpha-DTML) undergoes a phase transition at 47 degrees C, attributed to the unusual lamellar gel to hexagonal phase transition. The pyranose ring of alpha-DTML, in a mixture with dimyristoylphosphatidylcholine (1:9 mol ratio) to give a lamellar liquid crystalline phase, is oriented away from the bilayer surface into the aqueous environment and has an Smol of 0.75. The results for alpha-DTML, 2H-labelled at the C3 position of glycerol, suggest that this segment also has high molecular ordering. alpha-DTML in a lamellar environment has the least flexible membrane surface of the glyceroglycolipids investigated to date. 2H-NMR spin lattice relaxation times have been used to probe the head group motions of the glycolipids. The results indicate that the rate of head group motion increases in the order alpha-DTML less than alpha-DTGL less than beta-DTGL.(ABSTRACT TRUNCATED AT 400 WORDS)     

Production of MUC1 and MUC2 mucins by human tumor cell lines.

A mucus secreting, clonal derivative (HT29-SB) of the human colonic adenocarcinoma cell line HT29, and the LS174T colon cancer cell line, secrete mucin into the culture medium as a viscoelastic gel. Mab BC2, which defines a peptide epitope present in the variable number of tandem repeats (VNTR) of the MUC1 core protein, reacted with this material after deglycosylation. Two high molecular weight bands were detected in TFMSA treated gel-formed mucin from HT29-SB and LS174T by western blotting (Mr 580 kDa and 420 kDa). A similar pattern of reactivity was seen with the culture supernatants from HT29-SB, the ovarian tumor cell line COLO-316, and the breast cancer cell line MCF-7. Mab CCP58 (anti-MUC2 VNTR) reacted with a 580 kDa band in gel-formed mucin produced by LS174T, but was not reactive with mucin produced by the other cell lines. The findings indicate that human colonic cell lines, in addition to breast and ovarian cell lines, may both express and secrete the MUC1 protein core, and that the LS174T cell line expresses and secretes both the MUC1 and MUC2 core proteins.     

Effects of olive oil polyphenols on fatty acid synthase gene expression and activity in human colorectal cancer cells

Oleuropein (OL) and hydroxytyrosol (HT), the main olive oil polyphenols, possess anti-proliferative effects in vitro. Fatty acid synthase, a key anabolic enzyme of biosynthesis of fatty acids, plays an important role in colon carcinoma development. Our aim was to investigate whether gene expression of FAS, as well as its enzymatic activity, is regulated by HT and OL in two human colon cancer cell lines, as HT-29 and SW620. In addition, we investigated the effects of these polyphenols on growth and apoptosis in these cells. FAS gene expression and activity in treated HT-29 and SW620 cells were evaluated by real-time PCR and radiochemical assay, respectively. Cell growth and apoptosis, after polyphenols treatment, were measured by MTT test and flow cytometry, respectively. The inhibition of proliferation, detected after HT treatment, was mediated by an inhibition of FAS expression and its enzymatic activity in SW620 cells, while the anti-proliferative effect in HT-29 cells seems to be independent from FAS. OL exerted an anti-proliferative effect only on SW620 cells with a mechanism which excluded FAS. Olive oil polyphenols used were able to induce apoptosis in both cell lines studied. The increase of apoptosis in these cells was accompanied by the block of cell cycle in the S phase. This study demonstrates that HT and OL may induce anti-proliferative and pro-apoptotic effects only in certain human colorectal cancer cell types. These effects are FAS mediated only in SW620 cells after treatment with HT.     

Role of caspase activation in butyrate-induced terminal differentiation of HT29 colon carcinoma cells

Colon epithelial cells have a defined life span and undergo terminal differentiation as they mature and migrate to the luminal surface. The differentiation process can be induced in cultured colon cancer cells by sodium butyrate, which induces expression of various differentiation markers followed subsequently by cell death. In the present study, HT29 colorectal carcinoma cells were shown to undergo butyrate-induced caspase activation that was mainly produced through a mitochondrial pathway. Inhibition of caspase activation, either by peptide pan caspase inhibitor Z-VAD-FMK, by caspase 9 inhibitor Z-LEHD-FMK, or by overexpression of Bcl-XL, also inhibited the expression of differentiation markers. These findings suggest (a) that terminal differentiation of HT29 colon carcinoma cells is tightly linked to caspase activation and (b) that increased expression of anti-apoptotic members of the Bcl-2 family of proteins, as well as other inhibitors of caspase activation, has the potential to inhibit terminal differentiation and thereby may contribute to the progression of colon cancer.     

Novel processive and nonprocessive glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana synthesize glycoglycerolipids, glycophospholipids, glycosphingolipids and glycosylsterols.

A processive diacylglycerol glucosyltransferase has recently been identified from Bacillus subtilis [Jorasch, P., Wolter, F.P., Z?hringer, U., and Heinz, E. (1998) Mol. Microbiol. 29, 419-430]. Now we report the cloning and characterization of two other genes coding for diacylglycerol glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana; only the S. aureus enzyme shows processivity similar to the B. subtilis enzyme. Both glycosyltransferases characterized in this work show unexpected acceptor specificities. We describe the isolation of the ugt106B1 gene (GenBank accession number Y14370) from the genomic DNA of S. aureus and the ugt81A1 cDNA (GenBank accession number AL031004) from A. thaliana by PCR. After cloning and expression of S. aureus Ugt106B1 in Escherichia coli, SDS/PAGE of total cell extracts showed strong expression of a protein having the predicted size of 44 kDa. Thin-layer chromatographic analysis of the lipids extracted from the transformed E. coli cells revealed several new glycolipids and phosphoglycolipids not present in the controls. These lipids were purified from lipid extracts of E. coli cells expressing the S. aureus gene and identified by NMR and mass spectrometry as 1, 2-diacyl-3-[O-beta-D-glucopyranosyl]-sn-glycerol, 1, 2-diacyl-3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyrano-+ ++syl] -sn-glycerol, 1, 2-diacyl-3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-( 1-->6)-O-beta-D-glucopyranosyl]-sn-glycerol, sn-3'-[O-beta-D-glucopyranosyl]-phosphatidylglycerol and sn-3'-[O-(6"'-O-acyl)-beta-D-glucopyranosyl-(1"'-->6")-O-beta-D-gluco pyranosyl]-sn-2'-acyl-phospha-tidylglycerol. A 1, 2-diacyl-3-[O-beta-D-galactopyranosyl]-sn-glycerol was isolated from extracts of E. coli cells expressing the ugt81A1 cDNA from A. thaliana. The enzymatic activities expected to catalyze the synthesis of these compounds were confirmed by in vitro assays with radioactive substrates. Experiments with several of the above described glycolipids as 14C-labeled sugar acceptors and unlabeled UDP-glucose as glucose donor, suggest that the ugt106B1 gene codes for a processive UDP-glucose:1, 2-diacylglycerol-3-beta-D-glucosyltransferase, whereas ugt81A1 codes for a nonprocessive diacylglycerol galactosyltransferase. As shown in additional assays with different lipophilic acceptors, both enzymes use diacylglycerol and ceramide, but Ugt106B1 also accepts glucosyl ceramide as well as cholesterol and cholesterol glucoside as sugar acceptors.