Structure of sulfated glucuronyl glycolipids in the nervous system reacting with HNK-1 antibody and some IgM paraproteins in neuropathy

Novel sulfated glucuronic acid-containing glycolipids have been identified in the nervous system. These glycolipids are highly antigenic and share antigenic determinants with several nervous system glycoproteins, such as neural cell adhesion molecules, myelin-associated glycoprotein, and ependymins. The structure of the major antigenic glycolipid from human peripheral nerve was determined by chemical and enzymatic degradation, incorporation studies, sugar analysis after permethylation, pertrimethylsilylation, and gas liquid chromatography-mass spectrometry techniques as well as fast atom bombardment-mass spectrometry of the native antigen. The following structure was established for the major antigenic glycolipid. sulfate-3-GlcA beta(1---3)Gal beta(1----4)GlcNAc beta(1----3)Gal beta(1----4)Glc beta(1----1)-ceramide. The major fatty acids in the ceramide were 18:0, 18:1, 24:0, and 24:1, with C18-sphingenine as the long chain base.     

Biosynthesis of Lewis antigenic glycolipid by cell-free extracts of human intestinal tumor cells cultured in serum-free medium

Extracts of the human intestinal tumor cell line SW1116 were able to stimulate the incorporation of (14C) fucose from GDP-(14C) fucose into organically soluble glycolipid. The reaction required a purified glycolipid preparation from human meconium as lipid acceptor. The active glycolipid co-migrated with standard globoside on high performance thin-layer chromatography (HPTLC) and had molecular species (M + H) under fast-atom bombardment mass spectrometry of 1199, 1245 and 1269. Globoside itself was inactive and asialo GM1b had low activity. The radioactive products co-purified with Lewis a and Lewis b and co-migrated principally (60-90%) with Lewis b monoclonal antibody binding cellular glycolipids on HPTLC. Analysis of fucosidase digests suggested the presence of two different fucosyl-hexose linkages one of which was susceptible to cleavage. We conclude that the data are consistent with fucosylation of lactotetraosyl ceramide to Lewis a and Lewis b antigenic glycolipids.     

Chemical and immunological identification of glycolipid-based blood group ABH and Lewis antigens in human kidney

A polar non-acid glycolipid fraction has been isolated from human kidney. It was shown by thin-layer chromatography to be a mixture of glycolipids having more than four carbohydrate residues. Immunological testing revealed strong blood group Lea and A activity together with weak Leb, P1 and B activity. Mass spectrometry of the permethylated and permethylated-reduced (LiAlH4) glycolipid fraction showed that the two major components were a five sugar fucolipid (isomer of Lea) and a glycolipid having four hexoses and one N-acetylhexosamine. In addition, blood group Leb, B and A type hexaglycosylceramides were present. Evidence for small amounts of more complex glycolipids was also found. Acid degradation and gas chromatography of the native fraction revealed fucose, glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine. This is the first chemical isolation and characterization of complex blood group active glycolipids in human kidney. The existence of these molecules is discussed in view of their possible role as transplantation antigens.     

Forssman glycolipid variants of dog gastric mucosa. Structure of a branched ceramide octasaccharide.

A fucose-containing ceramide octasaccharide exhibiting Forssman antigenic activity, and reacting in human H anti-H and anti-A systems, was isolated from water-soluble glycolipids of dog gastric mucosa. Defucosylation of the glycolipid resulted in the loss of H-activity, but had no effect on its Forssman nor blood-group A antigenic activity. The branched structure of glycolipid was identified by partial acid hydrolysis, sequential degradation with specific glycosidases and comparison of the permethylation products of the native and enzyme-degraded compound. The structure of this glycolipid is proposed to be: formula.     

Expression and localization of Lewisx glycolipids and GD1a ganglioside in human glioma cells

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 µg of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewis^x (fucosylneolactonorpentaosyl ceramide; Le^x), difucosylneolactonorhexaosyl ceramide (dimeric Le^x), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 µg of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Le^x and the complex type of sialyl-Le^x derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Le^x glycolipids and sialyl-Le^x were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Le^x glycolipid was located on the tip of fine cellular processes. The unique localization of the Le^x glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.     

Glycolipid antigens with blood group I and i specificities from human adult and umbilical cord erythrocytes

Neutral glycolipids and gangliosides of umbilical cord and adult human erythrocytes were separated by high performance liquid chromatography, and each fraction was analyzed by direct binding of anti-I (Ma) and anti-i (Den) on solid phase glycolipid-lecithin-cholesterol. The I- and i-active glycolipids were isolated and their structures were determined by methylation analysis and direct probe mass spectrometry. The major I antigen in adult erythrocytes, showing a remarkable binding activity with anti-I(Ma), was found in one neutral glycolipid fraction, designated fraction y4, which was identified as a mixture of two glycolipids of a new type, lactoisooctaosylceramide and monofucosyllactoisooctaosylceramide (for structures, see Table I). In addition, two gangliosides displaying direct binding activity with anti-I(Ma) were identified as monosialoganglioside G8, as previously described and disialosyllactoisooctaosylceramide, which showed the same level of I activity as the y4 glycolipid. The same ganglioside was recently isolated and characterized by Kundu and co-workers. The major i-active glycolipid antigen in umbilical cord erythrocytes, showing a strong binding activity with anti-i(Den), was a neutral glycolipid, x4a, which was identified as lactonorhexaosylceramide. This glycolipid without fucosyl or sialosyl substitution has not been isolated previously and was present as an obvious normal component of umbilical cord erythrocytes, but an extremely minor component of adult erythrocytes. Sialosyllactonorhexaosylceramide (G6) was isolated and characterized as a second i antigen of umbilical cord erythrocytes, but showed a very weak binding activity with the anti-i antibody. Although these sialosyl derivatives displayed only weak activity, the chemical quantity of the sialosyl derivatives is significantly large in fetal erythrocytes; therefore, Ii activity of human erythrocytes, in general, must be significantly dependent on sialosyl derivatives in addition to unsubstituted structures.     

Sulfated glucuronic acid-containing glycoconjugates are temporally and spatially regulated antigens in the developing mammalian nervous system

Monoclonal antibody 4F4, which was raised against a cell suspension of embryonic rat forebrain, reacts with acidic glycolipids and several high-molecular-weight glycoproteins in rodent brain. The major reactive glycolipid is maximally expressed at Embryonic Day 15 (E15) and is no longer detectable at Postnatal Day 14 (P14) in the rat. 4F4 antibody reacts with a glucuronic acid- and sulfate-containing lipid isolated from human sciatic nerve as well as with lipids from mouse and rat embryonic brain tissue. Although the glycolipid disappears postnatally, the immunoreactive glycoproteins continue to be expressed in brain until adulthood. Both sciatic nerve and embryonic brain glycolipids are hydrolyzed by glucuronidase/sulfatase treatment but are insensitive to all other glycosidases tested. In addition, the observed 4F4 reactivity with extracted glycolipids, glycoproteins, and tissue sections of embryonic brain is identical to the reactivity demonstrated by HNK-1 antibodies. Immunocytochemical studies in developing brain showed stage-specific distribution of this carbohydrate antigen. At E10 in the mouse, immunoreactivity is associated with the mantle layer of the neural tube. At E15 in the cortex, the most intense staining is associated with the molecular layer and the subplate, and weaker staining is seen in the intermediate zone and cortical plate, suggesting that the antigen is highly concentrated on postmigratory cells in the embryonic nervous system.     

New-found phenolic glycolipids in Mycobacterium bovis BCG. Presence of a diglycosylated glycolipid

A crude phenolic glycolipid extract from Mycobacterium bovis bacille Calmette-Guerin (BCG) was fractionated until homogeneity at the intact level into four phenolic glycolipids called B, B-1, B-2, and B-3 according to their polarity. The apolar one, which is the most abundant was assigned to the well-known mycoside B. The B-2 and B-3 phenolic glycolipids were purified by direct-phase high performance liquid chromatography using a 5 micron Spherisorb column but were only recovered in small amounts (3 mg). A linear gradient of 0-20% methanol in chloroform was used. The B-1, B-2, and B-3 glycolipids were subjected to suitable modern analytical techniques selected for their potential to elucidate the structure at the intact level. Desorption chemical ionization-mass spectrometry allowed the molecular mass of B-3 to be determined as 1652 Da for the major homolog establishing the molecular formula as C103H192O14. Thus, the B-3 polar phenolic glycolipid contained two deoxyhexoses, one molecule of phenolphthiocerol esterified by two molecules of mycocerosic acid. Using two-dimensional 1H NMR (correlated chemical shift and nuclear Overhauser effect spectroscopy) at the intact level the B-3 oligosaccharide structure was determined as an alpha-L-Rhap-(1----3)-2-O-Me-alpha-L-Rhap. This is the first report of a diglycosylated phenolic glycolipid in a nonpathogenic mycobacteria. The disaccharide unit, the antigenic determinant, appears to be characteristic of M. bovis BCG. This polar glycolipid B-3 and the apolar ones, B-1 and B-2, were reactive in enzyme-linked immunosorbent assay against serum from rabbit hyperimmunized with M. bovis BCG.     

Specificity of binding of a strain of uropathogenic Escherichia coli to Gal alpha 1----4Gal-containing glycosphingolipids

A strain of Escherichia coli originally isolated from urine of a patient with acute pyelonephritis was studied in detail for binding to glycosphingolipids. Bacteria labeled metabolically with [14C]glucose were layered over a glycolipid chromatogram and bound bacteria were detected by autoradiography. The detection was down to a few ng of glycolipid (pmol level) under these assay conditions. At a test level of 500 ng all glycolipids (more than a dozen molecular species analyzed) with Gal alpha 1----4Gal as an internal or terminal part bound the bacteria strongly while glycolipids known to lack this sequence were negative. Conformational analysis using hard sphere calculations including the exo-anomeric effect showed a bend in the saccharide chain at this disaccharide with a largely hydrophobic surface of the convex side, probably being part of the binding epitope. Mixtures of glycolipids isolated from a human ureter scraping and from urinary sediments bound bacteria in the 2- to 7-sugar interval. Thus, this infectious strain of E. coli recognizes glycolipids being present in epithelial cells lining the urinary tract.     

A processive lipid glycosyltransferase in the small human pathogen Mycoplasma pneumoniae: involvement in host immune response

The human pathogen Mycoplasma pneumoniae has a very small genome but with many yet not identified gene functions, e.g. for membrane lipid biosynthesis. Extensive radioactive labelling in vivo and enzyme assays in vitro revealed a substantial capacity for membrane glycolipid biosynthesis, yielding three glycolipids, five phosphoglycolipids, in addition to six phospholipids. Most glycolipids were synthesized in a cell protein/lipid-detergent extract in vitro; galactose was incorporated into all species, whereas glucose only into a few. One (MPN483) of the three predicted glycosyltransferases (GTs; all essential) was both processive and promiscuous, synthesizing most of the identified glycolipids. These enzymes are of a GT-A fold, similar to an established structure, and belong to CAZy GT-family 2. The cloned MPN483 could use both diacylglycerol (DAG) and human ceramide acceptor substrates, and in particular UDP-galactose but also UDP-glucose as donors, making mono-, di- and trihexose variants. MPN483 output and processitivity was strongly influenced by the local lipid environment of anionic lipids. The structure of a major beta1,6GlcbetaGalDAG species was determined by NMR spectroscopy. This, as well as other purified M. pneumoniae glycolipid species, is important antigens in early infections, as revealed from ELISA screens with patient IgM sera, highlighting new aspects of glycolipid function.     

Structural and serological characterization of the major glycolipid from Rothia mucilaginosa

Structural studies on the major glycolipid isolated from Rothia mucilaginosa were carried out utilising specific chemical degradation, NMR spectroscopy and matrix-assisted laser-desorption/ionization time of flight mass spectrometry (MALDI TOF-MS). The glycolipid was found to be a dimannosylacylmonoglyceride in which the carbohydrate part was the glycerol-linked dimannoside alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-sn-Gro (Man A-Man B-Gro), of which Man B was esterified at O-6 by a fatty acid residue. A second fatty acid substituted the secondary methylene position of the glycerol residue, in contrast to the glycolipid previously found in R. dentocariosa and Saccharopolyspora strains, in which the second fatty acid esterified the primary methylene position of glycerol. Results of the ELISA experiment with rabbit specific antibacterial sera indicate that these two major glycolipids are antigenic, and the patterns of serological reactivity are similar but not identical.     

Novel type-specific lipooligosaccharides from Mycobacterium tuberculosis

Mycobacterium tuberculosis (strain Canetti) is characterized by the presence of two novel glycolipids of the alkali-labile, trehalose-containing lipooligosaccharide class. Their structures were established by permethylation, partial acid hydrolysis, infrared and high-field NMR spectroscopy, and electron-impact and fast atom bombardment mass spectrometry of the native glycolipids and hydrolysis products. The trehalose substituent is unique in that it is methylated at the 6'-position. The structure of the simpler of the two glycolipids is 2-O-Me-alpha-L-Fucp(1----3)-beta-D-Glcp(1----3)-2-O-Me- alpha-L-Rhap(1----3)-2-O-Me-alpha-L- Rhap(1----3)-beta-D-Glcp(1----3)-4-O-Me-alpha-L-Rhap(1----3) -6-O-Me-alpha-D- Glc. Further glycosylation of the octaglycosyl unit of this nonantigenic glycolipid by an incompletely defined N-acyl derivative of a 4-amino-4,6-dideoxy-Galp residue results in the second, highly antigenic nonasaccharide-containing glycolipid. Application of two-dimensional proton correlation spectroscopy demonstrated that the fatty acyl substituents are located on the 2,3,6 and 3,4,6 hydroxyl groups of the terminal glucosyl unit in the proportions of 2:3. Gas chromatography/mass spectrometry and optical rotation measurement allowed identification of the fatty acyl esters as primarily 2L-, 4L-dimethylhexadecanoate, 2L-,4L-,6L-,8L-tetramethyloctadecanoate, and 2-methyl-3-hydroxyeicosanoate. The relationship of these glycolipids to different morphological forms of M. tuberculosis and to virulence is discussed.     

A novel tumor-associated, developmentally regulated glycolipid antigen defined by monoclonal antibody ACFH-18

A mouse IgM monoclonal antibody, ACFH-18, was established after immunization of mice with the human gastric cancer cell line MKN74. The antibody reacts strongly with gastrointestinal carcinoma and showed a clear dependence on the degree of differentiation of gastric cancer cells. The antibody defines a series of glycolipid species with extremely slow TLC mobility present in both acidic and neutral glycolipid fractions of the extract from gastrointestinal adenocarcinoma and the original MKN74 cells. Isolation and structural study of the active glycolipids present in the acidic and neutral fractions and comparison of the antibody reactivity with glycolipids having related structures revealed a novel specificity. The minimum requirement for the maximal reactivity of ACFH-18 was identified as VII3Fuc-nLc10 (Structure A below and Fig. 8 in text). Since the antibody did not react with III3Fuc-nLc6 (Structure B below), which shares the same terminal sequence as VII3Fuc-nLc10, and since it cross-reacted with VII3Fuc-nLc8 (Z1 glycolipid) and VII3Fuc,V3-Fuc,III3Fuc-nLc8 (Z3 glycolipid), antibody ACFH-18 is capable of recognizing a fucosyl residue plus an internal repeating N-acetyllactosamine proximal to ceramide, as indicated by lines in Fig. 8 (Structures A, B, and E-G). (Formula: see text).     

Glycosphingolipids of human large intestine: detailed structural characterization with special reference to blood group compounds and bacterial receptor structures

Non-acid glycosphingolipid expression was studied in the large intestines from four individuals with the A1Le(a-b+), BLe(a-b+), and OLe(a-b+) blood group phenotypes. In the A1Le(a-b+) case, specimens were taken from the ascending and sigmoid parts of the large intestine in order to compare the expression of glycolipids in the proximal and distal regions of the intestine. In one blood group OLe(a-b+) individual, epithelial cells were isolated from the residual stroma to compare the glycolipid compositions in these two tissue compartments. GlcCer, GalCer, LacCer, Gb3Cer, and Gb4Cer were the major compounds in all three individuals, as shown by mass spectrometry, proton NMR spectroscopy, and degradation studies. The Lea-5 glycolipid was the major complex blood group glycolipid in all individuals, except in the proximal ascending part of the large intestine of the A1Le(a-b+) case, in which the Leb-6 glycolipid was predominant. There were trace amounts of blood group ABH glycolipids, in agreement with the ABO blood group phenotypes of the donors, Lewis antigens with more than six sugar residues in the carbohydrate chain, and blood group X and Y glycolipid antigens. The epithelial cells were dominated by monoglycosylceramides and the Lea-5 glycolipid, while only trace amounts of di-, tri-, and tetraglycosylceramide structures were present. No reactivity was seen in the epithelial cell fraction with Gal alpha 1-4Gal specific Escherichia coli, anti-Pk, or anti-P antibodies, indicating the absence of the glycolipid-borne Gal alpha 1-4Gal sequence in human large intestinal epithelial cells.     

The parasitic trematode Fasciola hepatica exhibits mammalian-type glycolipids as well as Gal(beta1-6)Gal-terminating glycolipids that account for cestode serological cross-reactivity

Neutral glycosphingolipids from sheep-derived Fasciola hepatica liver flukes were isolated and characterized both structurally and serologically. After HPLC fractionation, glycolipids were analyzed by linkage analysis, enzymatic cleavage, and MALDI-TOF as well as electrospray ionization mass spectrometry. Obtained results revealed the presence of two types of neutral glycolipids. The first group represented mammalian-type species comprising globo- and isoglobotriaosylceramides (Gal(alpha1-4)Gal(beta1-4)Glc(1-1)ceramide and Gal(alpha1-3)Gal(beta1-4)Glc(1-1)ceramide, respectively) as well as Forssman antigen (GalNAc(alpha1-3)GalNAc(beta1-3/4)Gal(alpha1-4/3)Gal(beta1-4)Glc(1-1)ceramide). Applying Helix pomatia agglutinin, recognizing terminal alpha-linked GalNAc, to cryosections of adult flukes, the latter glycolipid could be localized to the F. hepatica gut. As Forssman antigen from the parasite and sheep host led to identical MALDI-TOF MS profiles, this glycolipid might be acquired from the definitive host. As a second group, highly antigenic glycolipids were structurally characterized as Gal(beta1-6)Gal(beta1-4)Glc(1-1)ceramide, Gal(beta1-6)Gal(alpha1-3/4)Gal(beta1-4)Glc(1-1)ceramide and Gal(beta1-6)Gal(beta1-6)Gal(alpha1-3/4)Gal(beta1-4)Glc(1-1)ceramide, the latter two structures of which exhibited both isoglobo- or globo-series core structures. Terminal Gal(beta1-6)Gal1-motifs have previously been shown to represent antigenic epitopes of neogala-series glycosphingolipids from tape worms. Using human Echinococcus granulosus infection sera, Gal(beta1-6)Gal-terminating glycolipids could be allocated to the gut in adult liver fluke cryosections. Corresponding neogala-reactive antibodies in F. hepatica infection serum were detected by their binding to E. granulosus and Taenia crassiceps neogala-glycosphingolipids. These antibodies might contribute to the known serological cross-reactivity between F. hepatica and parasitic cestode infections.     

The specific glycosphingolipid composition of human ureteral epithelial cells

Total non-acid and acid glycolipid fractions were isolated from epithelial cell scrapings and the non-epithelial residue of a human upper ureter. The glycolipid fractions were structurally characterized as total mixtures by thin-layer chromatography, mass spectrometry, and proton NMR spectroscopy. Selected structural information was also obtained on binding of monoclonal antibodies and bacteria to the thin-layer chromatograms. The major epithelial cell glycolipids were Glc beta 1-1ceramide (75%), dihexosylceramide (10%) and NeuAcLacceramide (10%). In addition, 8 minor glycolipids belonging to the blood group P, Lewis and ABO systems were identified. The major glycolipids of the non-epithelial residues were mono- and dihexosylceramides together with globotriaosyl- and globotetraosylceramides. The epithelial mono- and diglycosylceramide compounds had an unusual ceramide composition with mainly C18 and C20 trihydroxy long chain bases in combination with C22-C24 hydroxy fatty acids in contrast to the non-epithelial glycolipids which contained mainly C18 dihydroxy long chain bases in combination with C16-C24 non-hydroxy fatty acids.     

Glycolipid antigens of human polymorphonuclear neutrophils and the inducible HL-60 myeloid leukemia line

Glycolipid and cell surface carbohydrate antigens of human polymorphonuclear neutrophils (PMN) and of HL-60 myeloid leukemia cells were analyzed with a panel of defined, monoclonal anti-carbohydrate antibodies. Antigenicities of intact PMN, HL-60, and retinoic acid-induced HL-60 (r.a.-HL-60) were studied by flow cytofluorometry. These three cell populations displayed quantitative differences, some of which were induction dependent, in their expression of lactosyl, N-acetyllactosaminyl, Y-hapten (Fuc alpha 1----2Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----R), and sialosyl-X-hapten (SA alpha 2----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----R) specificities. Structures reactive with antibodies specific for long-chain mono-, and di- or tri- alpha 1----3 fucosylated lacto-series glycolipids were also detected. Glycosphingolipids purified from organic extracts of these cells were analyzed to seek information concerning the chemical basis for these surface antigenic differences, to assess the structural and antigenic diversity of PMN and HL-60 glycolipids, and to quantitate chemically and antigenically prominent glycolipids. Binding of monoclonal antibodies to thin-layer chromatograms demonstrated that each of the specificities on intact cells was carried by one or more distinct glycolipids. The abundance of immunoreactive glycolipids in the extracts paralleled the relative staining intensities of the intact cell populations. Several "cryptic" glycolipid antigens, including alpha 2----6 sialosylated structures enriched five- to 10-fold in PMN extracts, were not detected on intact cells. Lactosylceramide accounted for two-thirds of the approximately 1.5 X 10(9) glycolipid molecules contained in each PMN. The remaining glycolipid antigens appeared to include structurally diverse fucolipids, fucogangliosides, and neutral and sialosylated glycolipids with Gal beta 1----4GlcNAc beta 1----R terminal core structure. The abundance, diversity, and induction-dependent expression of these structures suggest that they may participate in PMN maturation and function.     

Characterization of sulfated glucuronic acid containing glycolipids reacting with IgM M-proteins in patients with neuropathy

In some patients with neuropathy and plasma cell dyscrasia, the serum IgM M-proteins are known to bind to the myelin associated glycoprotein and to peripheral nerve glycolipids. We have isolated two acidic glycolipids which bind to the M-protein from human cauda equina by DEAE-Sephadex, Iatrobeads, and high performance liquid column chromatographies. The major acidic glycolipid migrated between GM1 and GD1a and the minor acidic glycolipid migrated between GD1a and GD1b. Their structures were elucidated by sugar analysis, enzymatic digestion, mild acid hydrolysis, permethylation, fast atom bombardment mass spectrometry, and NMR studies. Their core structure was confirmed to be paragloboside by high performance thin-layer chromatography-immunostaining using anti-paragloboside monoclonal antibody. Both acidic glycolipids lacked sialic acid but contained sulfated glucuronic acid as their acidic moiety. The sulfate group in the glucuronic acid was established by periodate oxidation and permethylation studies to be attached to the 3 position. The structures of the two acidic glycolipids are therefore consistent with the following: IV3GlcUA(3-sulfate)nLcOse4Cer and VI3GlcUA(3-sulfate)nLcOse6Cer. Additionally, the free carboxyl group on the glucuronic acid residue was shown to be necessary to bind the IgM M-proteins from neuropathy patients.     

Characterization of the binding of Actinomyces naeslundii (ATCC 12104) and Actinomyces viscosus (ATCC 19246) to glycosphingolipids, using a solid-phase overlay approach

Actinomyces naeslundii (ATCC 12104) and Actinomyces viscosus (ATCC 19246) were radiolabeled externally (125I) or metabolically (35S) and analyzed for their ability to bind glycosphingolipids separated on thin layer chromatograms or coated in microtiter wells. Two binding properties were found and characterized in detail. (i) Both bacteria showed binding to lactosylceramide (LacCer) in a fashion similar to bacteria characterized earlier. The activity of free LacCer was dependent on the ceramide structure; species with 2-hydroxy fatty acid and/or a trihydroxy base were positive, while species with nonhydroxy fatty acid and a dihydroxy base were negative binders. Several glycolipids with internal lactose were active but only gangliotriaosylceramide and gangliotetraosylceramide were as active as free LacCer. The binding to these three species was half-maximal at about 200 ng of glycolipid and was not blocked by preincubation of bacteria with free lactose or lactose-bovine serum albumin. (ii) A. naeslundii, unlike A. viscosus, showed a superimposed binding concluded to be to terminal or internal GalNAc beta and equivalent to a lactose-inhibitable specificity previously analyzed by other workers. Terminal Gal beta was not recognized in several glycolipids, although free Gal and lactose were active as soluble inhibitors. The binding was half-maximal at about 10 ng of glycolipid. A glycolipid mixture prepared from a scraping of human buccal epithelium contained an active glycolipid with sites for both binding specificities.     

Characterization of SSEA-1 glycolipids from the brain of a patient with fucosidosis

Neutral glycolipids from the brain of a patient with Fucosidosis were analyzed and two complex glycolipids containing five and eight sugars were isolated from the cortical grey matter. These two glycolipids reacted with antibodies recognizing the SSEA-1 [Le^x(X)] carbohydrate determinant. SSEA-1 glycolipids are normally expressed in human embryonic brain but are found in only small amounts in postnatal human brain. The accumulation of the two SSEA-1 glycolipids in Fucosidosis brain thus represents a defect which affects the normal developmentally regulated decrease in postnatal, expression of these glycolipids, and may be a contributing factor in the abnormal brain development associated with the disease. Chemical characterization of the two isolated glycolipids by gas chromatographic and mass spectrometric analyses has identified the two glycolipids as lacto-N-fucopentaosylceramide (III) and difucosyl-neolactonorhexaosylceramide.Abbreviations DCl direct chemical ionization - FAB tastatiom bombardment - GC gas chromatography - GSLs glycosphingolipids - MS mass spectrometry - SSEA-1 stage specific embryonic antigen-1 - TLC thin layer chromatographys