De-O-Acetylation of mucin-derived sialic acids by recombinant NanS-p esterases of Escherichia coli O157:H7 strain EDL933.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL933 encodes the single chromosomal 9-O-acetylesterase NanS, and several copies of prophage-encoded 9-O-acetylesterases (NanS-p). These enzymes have recently been shown to cleave 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac2) to yield de-O-acetylated Neu5Ac, the latter of which may serve as a carbon and/or nitrogen source. In the current study, we investigated the NanS- and NanS-p-mediated digestion of synthetic O-acetylated neuraminic acids and bovine submaxillary glands mucin (BSM)-derived O-acetylneuraminic acids by high-performance thin-layer chromatography (HPTLC) and nano electrospray ionization mass spectrometry (nanoESI MS). Initial HPTLC analyses showed the expected activity of NanS and NanS-p variants for Neu5,9Ac2. However, all tested enzymes were unable to de-O-acetylate 5-N-acetyl-4-O-acetylneuraminic acid (Neu5,4 Ac2) in our test system. The nanoESI MS analysis of neuraminic acids after treatment of BSM with NanS-p gave evidence that NanS-p variants of EHEC O157:H7 strain EDL933 cleave off O-acetyl groups from mono-, di-, and tri-O-acetylated Neu5Ac and N-glycolylneuraminic acid (Neu5Gc), regardless of the carbon positions C7, C8 or C9 of the acetate esters. This enzyme activity leads to neuraminidase-accessible Neu5Ac and Neu5Gc on mucin glycans. Moreover, we could demonstrate by HPTLC analyses that recombinant Bacteroides thetaiotaomicron sialidase (BTSA-His) was able to cleave Neu5Ac and Neu5,9Ac2 from BSM and that the combination of BTSA-His with both NanS-His and NanS-p-His derivatives enhanced the release of de-O-acetylated core Neu5Ac and Neu5Gc from mammalian mucin O-glycans. Growth experiments with EHEC wildtype strain EDL933, its nanS and nanS/nanS-p1a-p7 mutant and exogenous BTSA-His in BSM demonstrated that the presence of BTSA-His enhanced growth of EDL933 and the nanS deletion mutant but not the nanS/nanS-p1a-p7 mutant. Thus, we hypothesize that the expression of sialic acid O-acetylesterases with a broad specificity could be an advantage in competition with the gut microbiota for nutrients and facilitate EHEC colonization in the human large intestine.

Characterization of α-mannosidase from Dolichos lablab seeds: partial amino acid sequencing and N-glycan analysis.

α-Mannosidase is a key enzyme in processing and degradation of N-glycans in plants and animals. In the present study α-mannosidase from crude extracts of Dolichos lablab (Indian beans) has been purified by ammonium sulfate precipitation, anion exchange, galactose Sepharose, phenyl Sepharose, gel permeation and Con A Sepharose chromatography. The purified protein migrated as a single band corresponding to 116 kDa on SDS-PAGE under reducing conditions. The pH and temperature optima of α-mannosidase activity determined by use of p-nitrophenyl-α-D-mannopyranoside as substrate were found to be 5.0 and 60-65°C, respectively. The KM was 1.48 mM and swainsonine was a potent inhibitor of the enzyme with IC(50) value 50-80 nM. Additionally, the de novo amino acid sequencing showed active site regions highly conserved among other plant acidic α-mannosidases and yielded sequence coverage of approximately 32.5%. N-glycopeptide analysis revealed the presence of paucimannosidic type structure in a conserved N-glycosylation site as well as at least one oligo mannosidic glycan at an undetermined site after ZIC-HILIC enrichment of proteolytic glycopeptides. The partial biochemical and molecular characterization of this enzyme reveals that it is a class II α-mannosidase from the glycosyl hydrolase family 38.

Neoglycolipids derived from phosphatidylethanolamine serve as probes in cell culture studies on glycolipid metabolism.

The neoglycolipid (NeoGL) N-acetyl-1-deoxy-1-phosphatidylethanolamino lacto-N-tetraositol [Lc4Ose-PtdEtn(NAc)] and the radioactivly labeled analog [Lc4Ose-PtdEtn(N[14C]Ac)] were synthesized by coupling the corresponding oligosaccharide to phosphatidylethanolamine (dihexadecyl) via reductive amination and subsequent N-acetylation with unlabeled and [14C]acetic acid anhydride, respectively. Lc4Ose-PtdEtn(N[14C]Ac) was then incubated with homogenates of rat small intestine epithelial cells (IEC-6) at pH 4. The reaction products were shown to be the degradation products formed by glycosidases by fast atom bombardment mass spectrometry (FAB MS). On the other hand, incubation of Lc4Ose-PtdEtn(NAc) with IEC-6 cell homogenates in sialyltransferase assays yielded the corresponding sialylated product. When Lc4Ose-PtdEtn(N[14C]Ac) was fed to IEC-6 cells as BSA complex, up to 5% of the NeoGL administered were taken up by the cells. After extraction of the NeoGL and separation by thin layer chromatography (TLC) the catabolic products Lc3Ose-PtdEtn(N[14C]Ac), Lac-PtdEtn(N[14C]Ac), and Glc-PtdEtn(N[14C]Ac), as well as the main anabolic product NeuGc-Lc4Ose-PtdEtn(N[14C]Ac) could be identified by FAB MS. These results demonstrate that PtdEtn-derived NeoGL can be used as probes for studies on the metabolism of specific oligosaccharide structures in cell culture.

Cardiolipin, alpha-D-glucopyranosyl, and L-lysylcardiolipin from gram-positive bacteria: FAB MS, monofilm and X-ray powder diffraction studies.

Cardiolipin preparations from Streptococcus B, Listeria welshimeri, Staphylococcus aureus, and a glucosyl and lysyl derivative of cardiolipin were analysed for fatty acid composition and fatty acid combinations. Three different fatty acid patterns are described and up to 17 molecular species were identified in Streptococcus B lipids by high resolution FAB MS. The physicochemical properties of these lipids were characterised in the sodium salt form by monofilm experiments and X-ray powder diffraction. All lipids formed stable monofilms. The minimal space requirement of unsubstituted cardiolipin was dictated by the fatty acid pattern. Substitution with L-lysine led to a decrease of the molecular area, substitution with D-glucopyranosyl to an increase. On self assembly at 100% relative humidity, all preparations adopted lamellar structures. They showed a high degree of order, in spite of the heterogeneous fatty acid compositions and numerous fatty acid combinations. The repeat distances in lamellar fluid phase varied between 4.99 and 5. 52 nm, the bilayer thickness between 3.70 and 4.46 nm. Surprising were the low values of sorbed water per molecule of the glucosyl and lysyl derivatives which were 58 and 60%, compared with those of the respective cardiolipin. When Na(+) was replaced as counterion by Ba(2+), the bilayer structure was retained, but the lipids were in the lamellar gel phase and the fatty acids were tilted between 32 and 53 degrees away from the bilayer normal. Wide angle X-ray diffraction studies and electron density profiles are also reported. Particular properties of glucosyl cardiolipin are discussed.

Secretion of 13C-labelled oligosaccharides into human milk and infant's urine after an oral [13C]galactose load.

Human milk oligosaccharides seem to play an important role in the infant's defense against bacterial and viral infections of the gastrointestinal and the urogenital tract. In this study, we investigated the influence of dietary carbohydrates on the biosynthesis of lactose and oligosaccharides in the human mammary gland and their renal excretion by the human milk-fed infant. For this purpose, a lactating woman was given 27 g galactose (Gal) containing 2 g [13C] Gal (1-13C/99%) immediately after breakfast. In the following 36 h, milk (5-10 ml) was collected before each nursing. Infant's urine was collected over a period of 24 h. 13C-enrichment was measured in total milk, milk fat and protein, in the carbohydrate fraction as well as in urine by isotope ratio mass spectrometry (IRMS). Milk carbohydrates and deproteinized urine samples were fractionated by Sephadex G25 gel filtration and further analyzed by IRMS, high performance thin layer chromatography and and high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). IRMS revealed that in milk a maximal delta 13CPDB was reached within 8 h after Gal intake which then rapidly declined in the following 8 h. The cumulative 13C-elimination over this first peak was 6.9% of the oral 13C-dose. The highest 13C-enrichment was detectable in the carbohydrate fraction, mainly in lactose and neutral oligosaccharides. Compared to the enrichment of human milk, the delta 13CPDB of infant's urine was delayed. In urine, the highest amount of 13C was found in the Sephadex G25 fractions which mainly contained lactose, fucosyl-lactose, lacto-N-tetraose (LNT), fucosyl-LNT and difucosyl-LNT. For further characterization, individual components were separated by HPAEC-PAD and subsequently analyzed by fast atom bombardment mass spectrometry and IRMS. The data show, that orally applied Gal is incorporated in milk, especially in lactose and neutral oligosaccharides. Obviously, some of these components were absorbed by the infant and then excreted with urine. There, oligosaccharides may serve as analogous receptors for bacterial or viral adhesion molecules, and, hence, may prevent urogenital infections in breastfed infants.

Structure analysis of an allene-containing estolide tetraester triglyceride in the seed oil of Sebastiana commersoniana (Euphorbiaceae).

The seed oil of Sebastiana commersoniana (Euphorbiaceae) was separated into triglyceride and an estolide fraction by preparative thin-layer chromatography. The triglyceride band was characterized by spectroscopic methods, and its fatty acids have been analyzed by gas chromatography (GC) and GC-mass spectrometry (MS) as their methyl esters. Linolenic acid was the main fatty acid (65%). The estolide band was examined by a combination of spectroscopic and chromatographic methods (ultraviolet, infrared, nuclear magnetic resonance, fast atom bombardment-MS, GC-MS of the fatty acids before and after silylation) and was identified as a tetraglyceride, where one alpha-carbon of the triglyceride backbone was esterfied with 8-hydroxy-5,6-octadienoic acid, which itself was esterfied with trans-2,cis-4-decadienoic acid. The remaining positions of the triglyceride backbone were occupied by common fatty acids.

Heart muscle cells share common neutral glycosphingolipids with Trypanosoma cruzi.

Neutral glycosphingolipids were isolated from mouse heart muscle cells and their structures were analyzed. The molecular compositions of these glycosphingolipids were examined using column chromatography, HPTLC, GC-MS and fast atom bombardment-mass spectrometry (FAB-MS). Monohexosylceramides are a mixture of glucosyl- and galactosylceramides in a ratio of 1:1, sphingosine as the long chain base and as fatty acyl groups mainly C16, C18 saturated and C22 and C24 hydroxy fatty acids. Dihexosylceramide, identified as lactosylceramide contains C18 sphingosine and C18, C20 and C22 were the major fatty acids. No evidence for the occurrence of hydroxylated fatty acids in this glycolipid could be obtained from the GC-MS data. Our results clearly demonstrated that Trypanosoma cruzi and heart muscle cells have similar glycosphingolipid structures. In addition, heart muscle cells neutral glycosphingolipids have been shown to be immunoreactive. Antibodies reactive with each of the immunogenic glycolipids from heart cells or T. cruzi epimastigotes were present in the sera of human patients with Chagas disease as detected by ELISA. These cross-reactive antigens could be involved in the Chagasic autoimmunity.

Synthesis and mass spectrometric characterization of digoxigenin and biotin labeled ganglioside GM1 and their uptake by and metabolism in cultured cells.

Selective acylation of mono-deacetyl lyso-GM1, i.e. beta-D-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta-D-galactopyr ano syl -(1-->4)-(alpha-D-neuraminyl-(2-->3))-beta-D-galactopyranosyl- (1-->4)-beta-D-glucopyranosyl-(1-->1)-(2S,3R,4E)-2-amino-4-octa decen-1,3-diol, with N-succinimidyl-[1-14C]stearate afforded labeled mono-deacetyl GM1, i.e. beta-D-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta-D-galactopyr ano syl- (1-->4)-(alpha-D-neuraminyl-(2-->3)-beta-D-galactopyranosyl-(1-->4)-beta -D- glucopyranosyl-(1-->1)-(2S,3R,4E)-2-[1-14C]octadecanamido-4- octadecen-1, 3-diol, in good yield. Its condensation with either N-succinimidyl-digoxigenyl-3-O-methyl carbonyl-epsilon-amino caproate or N-succinimidyl-D-biotinyl-epsilon-aminocaproate led to radioactive GM1 derivatives carrying a tag for immuno-electron microscopy at the sialic acid residue. These GM1 derivatives could be hydrolyzed to the corresponding GM3 derivatives by treatment with GM1-beta-galactosidase and beta-hexosaminidases. There was no further degradation by sialidases due to the bulky tag in the sialic acid residue. The uptake of biotin labeled GM1 by human skin fibroblasts, rat neuroblastoma cells B104 and human neuroblastoma cells SHSY5Y was 0.85, 0.58 and 1.62 nmol lipid/mg cellular protein, respectively, after an incubation for 66 h at 37 degrees C and was similar to that of untagged GM1. The uptake of digoxigenin labeled GM1 by these cell types was, however, significantly higher (3.1, 6.8, and 20.0 nmol lipid/mg cellular protein, respectively). Both the biotin and digoxigenin labeled GM1 analogs were catabolized to the corresponding GM2 and GM3 derivatives in lysosomes of cultured cells. This demonstrates that these synthetic analogues are suitable for studying, by immuno-electron microscopy, their endocytosis and distribution in intralysosomal membranes.

Monohexosylceramides of Trypanosoma dionisii.

A detailed knowledge of the primary structure of neutral glycosphingolipids isolated and purified from Trypanosoma dionisii has been elucidated using a combination of techniques--as column chromatography, HPTLC and GC-MS together with fast atom bombardment spectrometry. The ceramide monohexoside fraction (CMH) contained both glucosyl- and galactosylceramides in a ratio of 1:1, sphingosine and as fatty acyl groups mainly C-24 saturated and 2-hydroxy fatty acids. A close similarity between Trypanosoma cruzi and T. dionisii monohexosylceramides was reported.

High-pH anion-exchange chromatography with pulsed amperometric detection and molar response factors of human milk oligosaccharides.

A method is described to separate and characterize neutral and acidic lactose-derived oligosaccharides without prior derivatization or reduction by high-pH anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD). This method has been applied to human milk oligosaccharides from donors with different blood group specificity (A, Le(a) and A, Le(b). Neutral and acidic components were separated from each other by anion-exchange chromatography. A distinct separation of individual components was obtained by size-exclusion chromatography on Fractogel TSK HW 50S (acidic oligosaccharides) or Fractogel TSK HW 40S (neutral oligosaccharides containing up to 6 monomers) and Bio-Gel P-4 size exclusion (neutral oligosaccharides containing more than 6 monomers). Furthermore the moral response factors after HPAEC-PAD have been determined for 28 components.

Urinary excretion of lactose and oligosaccharides in preterm infants fed human milk or infant formula.

At present, not much is known about the absorption and metabolism of human milk (HM) oligosaccharides in term and preterm infants. We investigated the renal excretion of lactose and complex oligosaccharides in preterm infants fed HM (n = 9, mean actual body weight 2290 g) or a cow's milk-based infant formula (n = 9, mean actual body weight 2470 g). We found that the renal excretion of lactose in HM-fed infants was slightly lower than in formula-fed infants (14.0 +/- 7.4 versus 20.4 +/- 8.7 mg kg-1 day-1, mean +/- SD). The excretion of neutral sugars deriving from oligosaccharides was similar in HM-fed and formula-fed infants (3.8 +/- 2.1 versus 2.9 +/- 0.9 mg kg-1 day-1); the difference between means was not statistically significant. The separation and characterization of oligosaccharides by high-pH anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) and subsequent analysis by fast atom bombardment-mass spectrometry (FAB-MS) revealed a more complex pattern in HM-fed infants compared to the formula-fed group. Lactose-derived oligosaccharides characteristic for HM (e.g. lacto-N-tetraose, and lacto-N-fucopentaoses I and II) were excreted in HM-fed but not in formula-fed infants. These results indicate that nutrition has a significant impact on the oligosaccharide composition in urine of preterm infants.

Cholesterol-containing lactose derived neoglycolipids serve as acceptors for sialyltransferases from rat liver Golgi vesicles.

The cholesterol-containing lactose derived neoglycolipids beta-Lactosylcholesterol, Cholesteryl-beta-lactosylpropane-1,3-diol, 3-Cholesteryl-1-beta-lactosylglycerol, 2-Cholesteryl-1-beta-lactosylglycerol, 2,3-Dicholesteryl-1-beta-lactosylglycerol, 1-Deoxy-1-cholesterylethanolaminolactitol, 1-Deoxy-1-cholesteryl (N-acetyl)-ethanolaminolactitol, 1-Deoxy-1-cholesterylphosphoethanolaminolactitol, and 1-Deoxy-1-cholesterylphospho (N-acetyl)-ethanolaminolactitol were synthesized and used as acceptors for sialytransferases from rat liver to Golgi vesicles. Relative activities with the neoglycolipids as acceptors varied from 28 to 163% compared to those obtained with the authentic acceptor lactosylceramide. Product identification by thin layer chromatography and fast atom bombardment mass spectrometry showed that the neoglycolipids yielded mono- and disialylated products. The results of competition experiments suggested that lactosylceramide and the neoglycolipids were sialylated by the same enzymes.

n-alkylglucosides serve as acceptors for galactosyltransferases from rat liver Golgi vesicles.

n-Alkyl alpha- and beta-D-glucopyranosides with different alkyl chain lengths (Glc-O-CxH2x+1) and n-octyl beta-D-thioglucopyranoside (Glc-S-C8H17) were synthesized, and used as acceptors for galactosyltransferases from rat liver Golgi vesicles. Only the beta-anomers were galactosylated and at constant substrate concentration, the reaction rates reached a maximum for medium alkyl chain lengths (C6, C8 and C10). Apparent Km and Vmax values decreased with increasing alkyl chain length. The reaction products were identified as n-alkyl beta-lactosides by means of thin layer chromatography, fast atom bombardment mass spectrometry and 1H-NMR spectroscopy. Competition experiments showed that UDP-Gal: N-acetylglucosamine beta 1-4-galactosyltransferase (EC 2.4.1.38) and not UDP-Gal: glucosylceramide beta 1-4-galactosyltransferase (lactosylceramide synthase, GalT-2) was responsible for the galactosylation of alkyl glucosides.

Sialylation of n-alkyllactosides, galactosides and glucosides by sialyltransferases from rat liver Golgi vesicles.

n-Alkyl alpha- and beta-lactosides, galactosides and glucosides with different alkyl chain lengths (C2, C8, C14 and C20) were synthesized and used as acceptors for sialyltransferases from rat liver Golgi vesicles. The beta-galactosides, beta-glucosides, and both alpha- and beta-lactosides, were sialylated. Keeping the acceptor concentration constant, sialylation rates reached a maximum for the n-octyl alpha- and beta-lactosides, n-octyl beta-galactoside and n-octyl beta-glucoside, respectively. n-Octyl alpha-galactoside and n-octyl alpha-glucoside were not sialylated. The reaction products were characterized by TLC. With n-octyl lactoside and galactoside as acceptors, two major sialylation products were formed. They could be separated by preparative TLC, and their structures were identified as 2-3 and 2-6 sialylated acceptors, respectively, by a combination of periodate oxidation, NaBD4 reduction, permethylation and subsequent analysis by fast atom bombardment mass spectrometry (FAB-MS). The structure of the single product obtained from n-octyl beta-glucoside was determined to be the 2-6 sialylated glucoside. Competition experiments with n-octyl lactoside and lactosylceramide and ganglioside Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1Cer (GM1) as acceptors for sialyltransferases suggested that SAT-I [NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1Cer (GM3) synthase] was at least in part responsible for the 2-3 sialylation of n-octyl lactoside.

Structural determination of N-2'-hydroxyoctadecenoyl-1-O-beta-D-glucopyranosyl-9-methyl-4, 8-sphingadienine from species of Aspergillus.

Ceramide monohexosides from Aspergillus fumigatus 2140 and 2109 strains and Aspergillus versicolor 550 strain, obtained by silica gel 60, and Iatrobeads chromatography were analysed using high-resolution 1D-, 2D-1H-NMR and 13C-NMR spectroscopy and fast atom bombardment mass spectrometry (FAB-MS). The ceramide monohexoside fraction (CMH) from A. fumigatus 2140 and A. versicolor 550 was identified as glucosylceramide, whereas glucose and galactose were present at a ratio of 1:1 in the CMH of A. fumigatus 2109. The major glycosphingolipid has a particular ceramide composition consisting of 9-methyl-4,8-sphingadienine linked to a 2-hydroxyoctadec-3-enoic acid. Although the structures presently described are similar to those of monohexosylceramides from other fungi, including edible ones, this is the first report on their occurrence in species pathogenic in humans.

Fast atom bombardment mass spectrometry of N-acetylated neoglycolipids of the 1-deoxy-1-phosphatidylethanolamino-lactitol-type.

N-Acetylated neoglycolipids (neoGL) of the 1-deoxy-1-phosphatidylethanolamino-lactitol-type (Lac-PtdEtn) carrying lactose, sialyllactose, disialyllactose, II3sialylgangliotetraose, II3,IV3disialylgangliotetraose, lacto-N-tetraose, IV6sialyllacto-N-tetraose, lacto-N-triaose, and bloodgroup A determinant as carbohydrate moieties were synthesized either chemically or enzymatically by glycosylation or deglycosylation of the parent compounds. The neoGL were then analyzed by fast atom bombardment mass spectrometry (FAB MS) with positive (FAB(+)) and negative ion (FAB(-)) detection. The resulting spectra showed intense pseudomolecular ions and characteristic fragmentations. FAB(-) spectra of the N-acetylated Lac-PtdEtn-type neoGL showed pseudomolecular ions (M-H)- of one magnitude higher intensity compared to those from the corresponding non-acetylated compounds. The main fragment ions were obtained from successive cleavage of the sugar units, thereby indicating the monosaccharide sequence. In FAB(+) spectra of the title compounds clearly detectable pseudomolecular ions were observed. The most prominent peaks, however, were obtained from cleavage of phosphatidic acid. The N-acetyl-ethyleneamine moieties of the corresponding glycosyl-Etn-fragments most probably formed five membered rings and thereby mesomery-stabilized cations. Secondary ions resulting from loss of the respective terminal sugars demonstrated the monosaccharide sequence.

Synthesis of 2-deoxy-D-galactose containing gangliosides in vivo.

Incorporation of 2-deoxy-D-galactose into the oligosaccharide moieties of different gangliosides of rat liver was examined. After intraperitoneal administration of 2-deoxy-D-galactose it was shown by GLC/MS analysis that this hexose analogue is metabolized and incorporated into all the gangliosides investigated, and predominantly into GM3 and GD3. In both of these gangliosides, 25-55% of the galactose residues were substituted by 2-deoxy-D-galactose. The epimer, 2-deoxy-D-glucose, was not detectable.

An investigation into the glycolipid metabolism of alpha-N-acetylgalactosaminidase-deficient fibroblasts using native and artificial glycolipids.

Deficient activity of lysosomal alpha-N-acetylgalactosaminidase represents a recently recognized lysosomal disorder whose neurologic manifestation in infancy is infantile neuroaxonal dystrophy. The lysosomal enzyme defect, inherited as an autosomal recessive trait, was first identified in the two brothers, GD and BD. Metabolic modification of glycolipids with terminal alpha-GalNAc was studied in fibroblasts from these patients. [Ceramide-3H]Forssman-glycosphingolipid (GSL), the fluorescent C6-NBD-lyso-Forssman-glycolipid (GL) and a 14C-labelled neoglycolipid containing the blood group A trisaccharide were synthesized and used as probes in degradation studies with cell homogenates and with cells in culture. Assays of each of these substrates with fibroblast homogenates of the patients demonstrated the profound deficiency of alpha-N-acetylgalactosaminidase activity compared with controls. Residual activities in the patients' fibroblast homogenates were detected with all glycolipid substrates; those amounted to 6.3 +/- 3.7% (BD) and 12.8 +/- 6.3% (GD) of the mean activity in controls for [3H]Forssman-GSL, and to 2.2 +/- 0.8% (BD) and 3.6 +/- 1.8% (GD) for C6-NBD-lyso-Forssman GL, respectively. alpha-N-Acetylgalactosaminidase deficiency in intact cells was confirmed by TLC analyses, which showed impaired glycolipid modification in cell extracts obtained following addition of [3H]Forssman GSL and C6-NBD-lyso-Forssman GL to the culture media of fibroblasts from the patients.