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.

[Evolution and infection biology of hemolytic-uremic syndrome (HUS) associated E. coli (HUSEC)]. / Evolution und Infektionsbiologie der mit dem hämolytisch-urämischen Syndrom (HUS) assoziierten E. coli (HUSEC).

Shiga toxin (Stx)-producing Escherichia coli (STEC), which cause hemolytic-uremic syndrome (HUS), are designated as HUSEC. Their exceptional genome variability driven by evolutionary diversification permits fast adaptation to changed environmental conditions. The HUSEC collection (http://www.ehec.org), which has been established at the Institute for Hygiene in Münster, contains 42 EHEC reference strains (HUSEC001-HUSEC042). It represents a unique repository collection of pathogens and is extremely helpful for the analysis of evolutionary changes and fixed properties in the STEC that cause the most severe host injury. Such genomic attributes include slowly evolving loci, mobile genetic elements that often encode virulence factors and are assimilated via horizontal gene transfer. Current evolutionary models indicate that numerous outbreak strains evolved recently and that highly pathogenic HUSEC descend from less pathogenic progenitors. However, additional data suggest that HUSEC have small effective population sizes. The HUSEC collection is also a valuable resource with which to study important non-Shiga toxin virulence factors.

Ganglioside expression in tissues of mice lacking beta2-microglobulin.

This study presents a comparative analysis of gangliosides from lymphoid (spleen and thymus) and other (brain, liver, lungs and muscle) tissues of C57BL/6 mice lacking the gene for beta2-microglobulin (beta2M), a constitutive component of the MHC class I molecule. Ganglioside fractions in the tissues of mice homozygous (beta2M-/-) and heterozygous (beta2M-/+) for the gene deletion were determined by high performance thin-layer chromatography (HPTLC), followed by immunostaining with specific polyclonal antibodies. Ubiquitous gangliosides GM3(Neu5Ac) and GM3(Neu5Gc) were the dominant gangliosides in the lungs of the control beta2M-/+ mice, whereas the homozygous knockout mice had substantially decreased expression of these structures. The lungs of the beta2M-/- mice also had reduced expression of T-lymphocyte-specific GM1b-type gangliosides (GM1b and GalNAc-GM1b). beta2M-deficient mice also had more GM1a and GD1a gangliosides in the liver, and several neolacto-series gangliosides were increased in the brain and lungs. This study provides in vivo evidence that the beta2M molecule can influence the acquisition of a distinct ganglioside assembly in different mouse organs, implicating its non-immunological functions.

Production and molecular characterization of clinical phase i anti-melanoma mouse IgG3 monoclonal antibody R24.

R24 is a mouse IgG3 monoclonal antibody (mab) that reacts with the ganglioside GD3 expressed by cells of neuroectodermal origin. The anti-tumor activity of R24 has been demonstrated in initial phase I and pilot trials in patients suffering from metastatic melanoma. The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important antibody. Growth, metabolism, and IgG production of R24 secreting hybridoma cells were analyzed on 1 L bioreactor bench scale using repeated-batch mode. The amount of 57 mg of pure mab was obtained from 1.6 L crude supernatant by protein A chromatography. Western blot binding assays with sugar-specific lectins revealed glycosylation of the heavy chains, whereas no carbohydrates were detectable on the light chains. Because glycosylation is essential for antibody effector functions in vivo (such as complement fixation or binding to macrophage Fc receptors), mab R24 was subjected to both enzymatic deglycosylation using PNGase F and chemical deglycosylation by hydrazinolysis. Released glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix assisted laser desorption ionization time-of-flight (MALDI-TOF), and electrospray ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry. Six major biantennary chains of the complex glycosylation phenotype were found with variations in galactosylation and core fucosylation. The predominant N-linked structure, indicating the high degree of agalactosyl glycoforms, was the agalacto biantennary chain with a relative percentage of 57% (51% core-fucosylated, 6% nonfucosylated). The second most abundant oligosaccharide was the monogalacto biantennary chain amounting to 30% (26% core- and 4% nonfucosylated). The antibody contained 0.46 microg sialic acid per mg protein, which splits into 0.243 microg Neu5Gc and 0.217 microg Neu5Ac, corresponding to a Neu5Ac:Neu5Gc ratio of 1:1.06. Furthermore, the antigen specificity of R24 was determined by immunodetection of GD3 on thin-layer chromatograms, and real time GD3-antibody binding interactions were measured with an optical biosensor (BIAcore). From the structural data obtained in this study it is concluded that glycosylation of the antibody may be important in the clinical outcome of targeted anti-cancer immunotherapy.

Gangliosides from human granulocytes: a nano-ESI QTOF mass spectrometry fucosylation study of low abundance species in complex mixtures.

Nano-ESI QTOF MS was used for sensitive mapping and sequencing of single molecular species in complex ganglioside mixtures obtained from human granulocytes, where the fucosylated carbohydrate chains of granulocyte gangliosides carry sLex and VIM-2 epitopes postulated to interact with E-selectin of the blood vessel wall in the early phase of the inflammation process. Functionally relevant components are expressed only at a low level, but using the negative ion detection it is possible to trace and identify such species, which were not detectable even by TLC. Advantage of the low-energy CID fragmentation for low abundance components of the complex ganglioside mixtures in the negative ion mode is to produce clear-cut series of fragment ions for sequencing. Fucosylation analysis carried out for each molecular species by MS/MS permits the clear distinction between sLex and VIM-2 epitope. VIM-2 epitope was expressed in all species with a longer sugar core, while in the short oligosaccharide chain with a sLex only, using biological material at a mid-femtomol level detection.

Anencephaly: structural characterization of gangliosides in defined brain regions.

Gangliosides from histopathologically-defined human cerebrum-resembling remnant and cerebellum from 37 and 30 gestational week-old anencephaluses were identified using mass spectrometry and high performance thin layer chromatography combined with immunochemical analysis in comparison to respective normal newborn/fetal and adult brain regions. A novel strategy of nano-electrospray ionization quadrupole time-of-flight tandem MS has been developed for identification of ganglioside components in complex mixtures. By morphoanatomical and histological investigation the anencephalic cerebral remnant was found to be aberrant, while the anencephalic cerebellum was defined as normal. Total ganglioside concentrations in the anencephalic cerebral remnant and the cerebellum were 34% and 13% lower in relation to the age-matched controls. In the cerebral remnant, GD3, GM2 and GT1b were elevated, while GD1a was decreased in the anencephalic cerebral remnant, but enriched in anencephalic cerebellum. GQ1b was reduced in both anencephalic regions. Gg4Cer, GM1b and GD1alpha, members of the alpha-series biosynthetic pathway, and neolacto-series gangliosides were found to be present in anencephalic, as well as in normal, fetal and adult brain tissues, indicating the occurrence of these biosynthetic pathways in human brain. In both cerebral and cerebellar anencephalic tissues, GM1b, GD1alpha, nLM1 and nLD1 were expressed at a higher rate in relation to normal tissue. It can be demonstrated that the anencephalic cerebral remnant, as a primitive brain structure, represents a naturally-occurring model to study the ganglioside involvement in induction of aberrant brain development.

Characterization of cytosolic sialidase from Chinese hamster ovary cells: part I: cloning and expression of soluble sialidase in Escherichia coli.

The cDNA of Chinese hamster ovary (CHO) cell cytosolic sialidase was amplified by RT-PCR and cloned into the pGEX-2T plasmid vector encoding for glutathione S-transferase (GST). Screening revealed transformed Escherichia coli clones with the constructed plasmid encoding the CHO cell sialidase sequence. After isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, SDS-PAGE of the total protein extracts revealed a new protein of about 70 kDa, correlating with the molecular weight of a fusion protein composed of the GST (26 kDa) and the cloned cytosolic CHO cell sialidase (43 kDa). A soluble fusion protein was purified from sonified E. coli homogenates by one-step affinity chromatography on Glutathione Sepharose 4B, which showed sialidase activity towards 4-methyl-umbelliferyl-alpha-D-N-acetylneuraminic acid (MUF-Neu5Ac) substrate. Induction of cells with 0.1, 0.5, and 1.0 mM IPTG revealed highest total protein amounts after induction with 1.0 mM IPTG, but highest specific activity for affinity chromatography purified eluates from cultures induced with 0.1 mM IPTG. Therefore, large scale production was performed by inducing cells during exponential growth in a 25 L bioreactor for 3 h with 0.1 mM IPTG after chilling the cell suspension to 25 degrees C. The amount of 26.46 mg of 40-fold purified GST-sialidase with a specific activity of 0.999 U/mg protein was obtained from crude protein extracts by one-step affinity chromatography. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) and Neu5Ac were competitive inhibitors for the sialidase, the former being the more effective one using MUF-Neu5Ac as the substrate. The cytosolic sialidase is capable of desialylating a wide spectrum of different types of gangliosides using a thin-layer chromatography overlay kinetic assay without detergents. This is the subject of the accompanying paper (Müthing, J.; Burg, M. Carbohydr. Res. 2001, 330, 347-356).

Characterization of cytosolic sialidase from Chinese hamster ovary cells: part II. Substrate specificity for gangliosides.

Cytosolic Chinese hamster ovary (CHO) cell sialidase has been cloned as a soluble glutathione S-transferase (GST)-sialidase fusion protein with an apparent molecular weight of 69 kD in Escherichia coli. The enzyme has then been produced in mg quantities at 25-L bioreactor scale and purified by one-step affinity chromatography on glutathione sepharose (Burg, M.; Müthing, J. Carbohydr. Res. 2001, 330, 335-346). The cloned sialidase was probed for desialylation of a wide spectrum of different types of gangliosides using a thin-layer chromatography (TLC) overlay kinetic assay. Different gangliosides were separated on silica gel precoated TLC plates, incubated with increasing concentrations of sialidase (50 degreesU/mL up to 1.6 mU/mL) without detergents, and desialylated gangliosides were detected with specific anti-asialoganglioside antibodies. The enzyme exhibited almost identical hydrolysis activity in degradation of GM3(Neu5Ac) and GM3(Neu5Gc). A slightly enhanced activity, compared with reference Vibrio cholerae sialidase, was detected towards terminally alpha(2-3)-sialylated neolacto-series gangliosides IV3-alpha-Neu5Ac-nLc4Cer and VI3-alpha-Neu5Ac-nLc6Cer. The ganglio-series gangliosides G(D1a), G(D1b), and G(T1b), the preferential substrates of V. cholerae sialidase for generating cleavage-resistant G(M1), were less suitable targets for the CHO cell sialidase. The increasing evidence on colocalization of gangliosides and sialidase in the cytosol strongly suggests the involvement of the cytosolic sialidase in ganglioside metabolism on intracellular level by yet unknown mechanisms.

Immunohistological and flow cytometric analysis of glycosphingolipid expression in mouse lymphoid tissues.

Expression of neutral glycosphingolipids (GSLs) and gangliosides in normal lymphoid tissues and cells has been studied mostly by biochemical and immunochemical analysis of lipid extracts separated by thin-layer chromatography. GSLs and gangliosides involved in the GM1b biosynthetic pathway were assigned to T-lymphocytes, whereas B-cell gangliosides and GSLs have been poorly characterized in former publications. We used specific polyclonal antibodies in immunohistochemistry and flow cytometry to analyze the distribution of globotriaosylceramide (Gb(3)Cer), globoside (Gb(4)Cer), gangliotriaosylceramide (Gg(3)Cer), gangliotetraosylceramide (Gg(4)Cer), and gangliosides GM3 and GalNAc-GM1b in the mouse thymus, spleen, and lymph node. Immature thymocytes expressed epitopes recognized by all antibodies, except for anti-Gb(4)Cer. Mature thymocytes bound only antibodies to GalNAc-GM1b, Gg(4)Cer, and Gb(4)Cer. In secondary lymphoid organs, antibodies to globo-series GSLs bound to vascular spaces of secondary lymphoid organs, whereas the ganglio-series GSL antibodies recognized lymphocyte-containing regions. In a Western blotting analysis, only GalNAc-GM1b antibody recognized a specific protein band in all three organs. Flow cytometric analysis of spleen and lymph node cells revealed that B-cells carried epitopes recognized by all antibodies, whereas the T-cell GSL repertoire was mostly oriented to ganglio-series-neutral GSLs and GM1b-type gangliosides. The results of immunohistochemistry and flow cytometry were not always identical, possibly because of crossreactivity to glycoprotein-linked oligosaccharides and/or differences between cell surface carbohydrate profiles of isolated cells and cells in a tissue environment.

Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers.

Cultivation of the new immortalized hepatocyte cell line HepZ was performed with a 1:1 mixture of DMEM and Ham's F12 media containing 5% FCS. The cells were grown in their 40th passage in 100 mL and 1 L volumes in spinner flasks and in a bioreactor, respectively. For the production of adherently growing HepZ cells macroporous CultiSpher G gelatin microcarriers were used in various concentrations from 1 to 3 g/L. The cells were seeded in a density of 2 x 10(5) cells/mL when using a microcarrier concentration of 1 g/L and 5 x 10(5) cells/mL at a microcarrier concentration of 3 g/L. After 7 days of cultivation a maximum cell concentration of 4.5 x 10(6) cells/mL was obtained in the spinner culture using a microcarrier concentration of 1 g/L. With bubble-free aeration and daily medium exchange from day 7, 7.1 x 10(6) cells/mL were achieved in the bioreactor using a microcarrier concentration of 3 g/L. The cells exhibited a maximum specific growth rate of 0.84 per day in the spinner system and 1.0 per day in the bioreactor, respectively. During the growth phase the lactate dehydrogenase (LDH) activity rose slightly up to values of 200 U/L. At the end of cultivation the macroporous carriers were completely filled with cells exhibiting a spherical morphology whereas the hepatocytes on the outer surface were flat-shaped. Concerning their metabolic activity the cells predominantly consumed glutamine and glucose. During the growth phase lactate was produced up to 19.3 mM in the spinner culture and up to 9.1 mM in the bioreactor. Maximal oxygen consumption was 1950 nmol/(10(6) cells. day). HepZ cells resisted a 4-day long chilling period at 9.5 degrees C. The cytochrome P450 system was challenged with a pulse of 7 microgram/mL lidocaine at a cell density of 4.5 x 10(6) cells/mL. Five ng/mL monoethylglycinexylidide (MEGX) was generated within 1 day without phenobarbital induction compared to 26 ng/mL after a preceded three day induction period with 50 microgram/mL of phenobarbital indicating hepatic potency. Thus, the new immortalized HepZ cell line, exhibiting primary metabolic functions and appropriate for a mass cell cultivation, suggests its application for a bioartificial liver support system.

Ganglioside expression in tissues of mice lacking the tumor necrosis factor receptor 1.

This study presents a comparative analysis of gangliosides from lymphoid (spleen and thymus) and other tissues (brain, liver, lung, muscle) of C57BL/6 mice homozygous (-/-) and heterozygous (+/-) for the tumor necrosis factor receptor 1 (TNFRp55). Quantitative and qualitative differences in the expression of the lipid-bound N-acetylneuraminic (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) and of various ganglioside biosynthesis pathways were detected between the tissues of the TNFRp55 -/- and the control TNFRp55 +/- mice. Sialic acid profiles showed a strong decrease in the absolute amount of sialic acids (Neu5Ac + Neu5Gc) in the lungs and thymus of homozygous (1.41 and 0.3 ng/mg wet weight, respectively) compared with control heterozygous animals (7.18 and 2.05 ng/mg wet weight, respectively). Considerable differences of Neu5Ac/Neu5Gc ratios in the lungs, muscle, spleen, and thymus were also detected. The gangliosides GM3(Neu5Ac) and GM3(Neu5Gc) were the dominant gangliosides in the lungs of the control animals, whereas the knockout mice almost completely lacked these structures in this organ. Reduced expression of GM1b-type gangliosides (GM1b and GalNAc-GM1b) was also found in the lungs, spleen, and thymus of the TNFRp55 knockout mice. On the other hand, neolacto-series gangliosides were more abundant in the lungs, brain, and muscle of the knockout mice, whereas their expression in the liver, spleen, and thymus was similar in both groups of animals. This study provides in vivo evidence that TNF signaling via the TNFRp55 is involved in the acquisition of a distinct ganglioside assembly in different mouse organs. TNFRp55 signaling seems to be especially important for the activation of the GM1b-type ganglioside biosynthetic pathway that is a unique characteristic of the mouse lymphoid tissues.

Cultivation and characterization of a new immortalized human hepatocyte cell line, HepZ, for use in an artificial liver support system.

The new human hepatocyte cell line HepZ was investigated with regard to use it for a mass cell cultivation. The cells were originally derived from a human liver biopsy and immortalized through lipofectamine-mediated transfection of albumin-promotor-regulated antisense constructions against the negative controlling cell cycle proteins Rb and p53 (pAlb asRb, pAIb asp53). Furthermore, plasmids including genes coding for the cellular transcription factor E2F and D1 cyclin (pCMV E2F, pSV2neo D1) were cotransfected to overcome the G1-restriction point. Cell cultivation was performed in a 2-liter bioreactor with a working volume of 1 liter. With CultiSpher G microcarriers used in a concentration of 3 g/l a maximal density of 7.1 x 10(6) cells/ml was achieved in a cultivation period of 20 days. The cells exhibited a maximal specific growth rate of 1.0 per day in the first 4 days. After 9 days of cultivation the stationary growth phase was reached with an average cell density of 5.5 x 10(6) cells/ml. The viability status of the culture was determined indirectly by measuring of the lactate dehydrogenase activity (LDH) at 37 degrees C. During the growth phase the activity rose slightly up to a value of 200 U/l. The cells were flat after first attachment on the gelatine microcarriers and spherical after growing into the three-dimensional inner matrix--both of which characteristics were verified by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The liver-specific cytochrome P450 activity was challenged with a pulse of 7 micrograms/ml lidocaine at a cell density of 4.5 x 10(6) cells/ml. After an induction period of 3 days with 50 micrograms/ml of phenobarbital, 26 ng/ml MEGX were generated within one day compared to 5 ng/ml without induction. The new cell line HepZ has proven to retain liver-specific qualities and to be appropriate for mass cell cultivation for bioartificial devices.

Isolation and structural characterization of glycosphingolipids of in vitro propagated human umbilical vein endothelial cells.

To investigate in detail the expression of glycosphingolipids (GSLs) on endothelial cells, 4.85 x 10(9) human umbilical vein endothelial cells (HUVECs) were cultivated in a 2 l bioreactor using microcarriers as a support for anchorage dependent growing cells. Neutral GSLs and gangliosides were isolated and their structures were determined by TLC immunostaining, fast atom bombardment-mass spectrometry (FAB-MS) of the native GSLs, and gas chromatography-electron impact mass spectrometry (GC-EIMS) of partially methylated alditol acetates. GbOse4Cer, GbOse3Cer, and LacCer, all carrying mainly C24- and C16-fatty acid beside C18-sphingosine, were detected as the major neutral GSLs (36%, 23%, and 15% of the total orcinol stain, respectively); GlcCer, nLcOse4Cer, and nLcOse6Cer were expressed to substantial minor amounts (9%, 12%, and 5% of the total orcinol stain, respectively). TLC immunostaining revealed the presence of lipid bound Lewisx antigen, whereas the isomeric Lewisa structure was detectable only in very low quantities. GM3(Neu5Ac) with C18-sphingosine was the major ganglioside constituting about 90% of the whole ganglioside fraction. The fatty acid composition was determined by GC-MS of fatty acid methyl esters, indicating the predominance of C24- and C16-substituted GM3(Neu5Ac), followed by C18- and C22-substituted species. Terminally alpha2-3 sialylated neolacto-series ganglioside IV3Neu5Ac-nLcOse4Cer was the second most abundant ganglioside in HUVECs (8% of the total resorcinol stain), and IV6Neu5Ac-nLcOse4Cer and VI3Neu5Ac-nLcOse6Cer (together less than 2% of total resorcinol stain) were found in minor quantities. Lipid bound sialyl Lewisx antigen with poly-N-acetyllactosaminyl chains, and traces of gangliotetraose-type gangliosides GM1 and GD1a were identified by TLC immunostaining. The expression of dominant neutral GSLs LacCer, GbOse3Cer, and GbOse4Cer, and of ganglioside GM3(Neu5Ac) was assayed by indirect immunofluorescence microscopy of cell layers grown in chamber slides, each showing different plasma membrane and subcellular distribution patterns. The complete structural characterization of GSLs from HUVECs contributes to our understanding about their functional role, not only of the carbohydrate but also of the lipid moiety, as receptors for bacterial toxins, as cell surface antigens of cellular interaction and as receptors for blood components and macromolecules of the extracellular matrix.

Large scale purification of gangliosides GM3(Neu5Ac) and GM3(Neu5Gc) by trimethylaminoethyl-Fractogel high-performance liquid chromatography.

A preparative anion-exchange high-performance liquid chromatographic method for the separation of the closely allied monosialogangliosides GM3(Neu5Ac) and GM3(Neu5Gc) has been developed. Hybridoma cells, readily available material derived from industrial monoclonal antibody production, were used as ganglioside source and led to fractions with pure GM3(Neu5Ac) and GM3(Neu5Gc) in high milligram quantities. The crude ganglioside extract was loaded onto columns filled with the strong anion-exchanger trimethylaminoethyl (TMAE)-Fractogel. Gangliosides were eluted from the stationary phase with a gradient system of ammonium acetate in methanol. The scaled-up approach ranged over more than one order of magnitude from 20 to 500 mg batches of GM3 gangliosides. Thus, the high-resolution power of the strong anion-exchanger TMAE-Fractogel allowed the preparative isolation by one-step column chromatography of two GM3 specimens which only differ in one hydroxyl group at position 5 of the neuraminic acid (N-acetyl-versus N-glycolylneuraminic acid).

Glycosphingolipids of skeletal muscle: I. Subcellular distribution of neutral glycosphingolipids and gangliosides in rabbit skeletal muscle.

Membrane vesicles were prepared from rabbit skeletal muscle, separated by sucrose density gradient centrifugation and characterized by their specific marker enzymes, ligand binding, and ion flux activities. The fractions obtained (in the order of increasing density) were sarcolemma (SL), T-tubules (TT), sarcoplasmic reticulum (SR1 and SR2) and triads/mitochondria (Tr/M). Their glycosphingolipid compositions were analyzed by biochemical and immunochemical methods with specific antibodies (TLC immunostaining) and characteristic patterns were obtained from respective membrane fractions, expressed on a protein basis. Glucosylceramide, the main neutral glycosphingolipid of rabbit muscle, was found in SL and TT fractions, whereas SR and Tr/M vesicles lack this compound. Lactosylceramide was selectively recovered in the SR1 fraction. GM3(Neu5Ac), the main ganglioside in rabbit muscle, was found to account for 64% in the SL, 13% in the TT, 7% in the SR1, 3% in the SR2 and 13% in the Tr/M fractions. IV3Neu5Ac-nLcOse4Cer was mostly abundant in SL and decreased in the order SL > TT, Tr/M > SR1, SR2. IV6Neu5Ac-nLcOse4Cer was only detected in the SL and Tr/M fractions in noteworthy quantities. Ganglioseries gangliosides GM1, GD1a, GD1b and GT1b displayed homogeneous distribution patterns in each membrane preparation. They were expressed only in small amounts but mainly in SL, TT and Tr/M vesicles and to less extent in SR1 and SR2 fractions. The presence of GM3(Neu5Ac) in the SL as well as on subcellular level was confirmed in transverse muscle cryosections by means of indirect immunofluorescence microscopy. The SL was brightly stained, but considerable intracellular fluorescence was observed as expected from the biochemical analyses. Thus, the neutral GSL and ganglioside expression of the SL and the intracellular membraneous network is different in skeletal muscle both in terms of quantitative and qualitative GSL composition as demonstrated in details by means of biochemical and immunochemical techniques. The modulatory functions of GM3 and gangliosides of the neolacto- and ganglio-series towards the voltage dependent Ca(2+)-channel, largely preponderant in the triads-containing Tr/M fraction, is the subject of the accompanying paper (J. Müthing, U. Maurer, and S. Weber-Schürholz, Carbohydr. Res., 307 (1998) 147-157).

Glycosphingolipids of skeletal muscle: II. Modulation of Ca2(+)-flux in triad membranes by gangliosides.

Membrane vesicles of rabbit skeletal muscle were prepared and separated by sucrose density gradient centrifugation. The fractions obtained (in the order of increasing density) were sarcolemma (SL), T-tubules (TT), sarcoplasmic reticulum (SR1 and SR2) and triads/mitochondria (Tr/M) as characterized by their specific marker enzymes, ligand binding, and ion flux activities. The distribution of neutral glycosphingolipids and gangliosides in these membrane preparations has been documented in the preceding paper (J. Müthing, U. Maurer, U. Neumann, B. Kniep, and S. Weber-Schürholz, Carbohydr, Res., (1988) 135-145). GM3(Neu5Ac) is the dominant ganglioside, neolacto-series gangliosides are moderately expressed and ganglio-series gangliosides were found in minor quantities, however, all showing different qualitative and quantitative membrane-type specific patterns. The voltage dependent Ca(2+)-channels of skeletal muscle reside prevalently in the triad enriched membrane fractions deduced from highest binding capacity of 1,4-dihydropyridines. Calcium channel complexes of triads were reconstituted into unilamellar phospholipid vesicles of 400 nm defined size and the active 45Ca(2+)-uptake into intravesicular space was measured after incorporation of muscle specific gangliosides into the outer vesicle lipid bilayer in parallel to control liposomes without gangliosides. GM3(Neu5Ac) strongly increased the uptake of 45Ca2+ (+285%) whereas GM3(Neu5Gc) severely inhibited the ion flux (-61%). Neolacto-series gangliosides evoked miscellaneous effects upon 45Ca(2+)-flux depending on isomeric sialic acid configuration, oligosaccharide size and fatty acid chain length of the ceramide portion. VI3Neu5Ac-nLcOse6Cer (C24-fatty acid), IV3Neu5Ac-nLcOse4Cer (C16-fatty acid) and IV6Neu5Ac-nLcOse4Cer (C16-fatty acid) strongly enhanced the 45Ca(2+)-flux (+208, +162, and +120%, respectively, whereas IV3Neu5Ac-nLcOse4Cer (C24-fatty acid), VI3Neu5Ac-nLcOse6Cer (C16-fatty acid) and IV6Neu5Ac-nLcOse4Cer (C24-fatty acid) slightly reduced 45Ca(2+)-flux (-3, -6, and -17%, respectively). Out of all gangliosides tested in this study, GM1 showed the strongest stimulatory effect (+327%). GD1a and GT1b gave rise to remarkable flux-stimulation of +283 and +255%, respectively, whereas GD1b exhibited only a slightly positive effect (+38%). This data suggest a functional role of gangliosides in subcellular muscle membranes giving strong evidence that gangliosides are capable of modulating the cytosolic calcium level of muscle, which regulates muscle contraction.

Nanogram detection of phospholipids on thin-layer chromatograms.

An extremely sensitive method for the quantitative determination of phospholipids on silica gel-precoated thin-layer chromatography plates is reported. The procedure is based on the successive application of two lipid spray reagents. Firstly, phospholipid chromatograms are sprayed with the lipophilic fluorochrome 1,6-diphenyl-1,3,5-hexatriene, followed by application of the phosphorus-specific molybdenum blue (Dittmer-Lester) reagent. Compared to the single use of the respective dyes, the consecutive utilization of both spray reagents resulted in an enormous enhancement in sensitivity of at least one order of magnitude. The procedure was proved with eight phospholipids of animal origin, and visual detection limits of down to 10 ng were achieved. The described technique provides a sensitive and specific means for the analysis of phospholipids on the nanogram scale by augmenting the molybdenum blue staining with the fluorochrome 1,6-diphenyl-1,3,5-hexatriene operating as an enhancer.