Monoclonal antibodies raised against membrane glycoproteins from mouse brain recognize N-linked oligomannosidic glycans

Monoclonal L3 and L4 antibodies have been shown to recognize carbohydrate epitopes on several neural cell adhesion molecules; these epitopes can be released by treatment with endoglycosidase H. In the present study, we have identified the oligosaccharides released by endoglycosidase H from the cell adhesion molecules AMOG and L1 by fast-atom bombardment mass spectrometry as being solely of the oligomannosidic type. Using neoglycolipids of oligomannosidc glycans, we also report that both antibodies show the highest reactivity with the α-manno-pentaose Manα1-3-[Manα1-6(Manα1-3)Manα1-6]-Man, but decreasing reactivity with the α-manno-hexaose, heptaose, octaose and nonaose glycans. Thus, to our knowledge, we describe here for the first time monoclonal antibodies recognizing N-glycosidically linked oligomannosidic glycan

Structural characterization of gangliosides from resting and endotoxin-stimulated murine B lymphocytes

Pörtner A, Peter-Katalinic J, Brade H, Unland F, Büntemeyer H, Müthing J. Structural characterization of gangliosides from resting and endotoxin-stimulated murine B lymphocytes. Biochemistry. 1993;32(47):12685-12693.B lymphocytes from CBA/J mice were stimulated in splenocyte cultures for 72 h with various endotoxins. Bisphosphoryl lipid A from Escherichia coli had the highest stimulatory effect followed by LPS of Citrobacter freundii and Salmonella minnesota as measured by [H-3]thymidine uptake. Gangliosides of stimulated B cells (metabolically labeled with D-[1-C-14]galactose and D-[1-C-14]glucosamine) and unlabeled gangliosides from resting B cells (prepared from spleens without stimulus) were analyzed by high-performance TLC, DEAE anion-exchange HPLC, and immunostaining procedures. Contents of ganglioside-derived sialic acids, quantified by HPLC as their fluorescent derivatives, decreased from stimulated to resident B lymphocytes in the following order: LPS S. minnesota > LPS C.freundii > bisphosphoryl lipid A E. coli > resting B cells. Gangliosides of resting B cells contained more N-glycolyl- than N-acetylneuraminic acid, whereas inverse ratios were found in activated cells, indicating a shift from N-glycolyl- to N-acetylneuraminic acid due to stimulation. Furthermore, a higher disialoganglioside content was characteristic for activated B cells. Fast atom bombardment mass spectrometry was performed with permethylated mono- and disialoganglioside fractions of LPS S. minnesota and LPS C.freundii stimulated B cells. Major gangliosides were G(M1a) and G(D1a) beside minute amounts of G(D1b). The structural heterogeneity in the gangliosides was caused by (a) N-substitution of the sialic acids with either acetyl or glycolyl groups, (b) variation in the long-chain base (sphingosine, sphinganine), and (c) substitution of the ceramide moiety by fatty acids of different chain length and degree of unsaturation (C-16:0, C24:0,24:1). In summary, these findings indicate the predominance of the G(M1a) pathway in murine B lymphocytes whereas G(M1b)-type gangliosides are preferentially expressed in T lymphocytes as well as macrophages

Galactosaminoglycan Function and Oligosaccharide Structure Determination

This review will discuss the importance of sequencing long chondroitin sulfate and dermatan sulfate chains specifically derived from decorin. Decorin is a member of the small leucine-rich repeat proteoglycans and ubiquitously expressed primarily in the skin. Sequence information and diverse function of glycosaminoglycans is further influenced by variable expression through the core protein indicating the importance to analyse glycosaminoglycans from specific proteoglycans

THE EXPRESSION OF ONCOFETAL ANTIGENS ON MUCINS OF HUMAN AMNIOTIC FLUID

The relative high abundance of O-linked oligosaccharides on the mucin-type glycoproteins has opened a favorable perspective for detailed studies of carbohydrate epitope structures, which may be involved in processes relevant for cellular differentiation in development and oncogenesis. Immunological experiments, in particular those with monoclonal antibodies raised against the cell surfaces, have been sucessfully performed in order to monitor qualitatively oncofetal carbohydrate antigens and the changes in their patterns during such processes (1). On the other hand the biosynthetic studies on glycosyltransferases responsible for assembly of complex carbohydrates bound to proteins and their substrates lead to the conclusion, that several enzymes compete for a common substrate of specific structure available in the respective cellular compartment at the certain phase of biosynthesis (2). Therefore some carbohydrate microheterogeneity arising from the presence of different terminal epitope structures as well as the different branch length and different branching patterns on the single glycan attachment site should be expected. The structures of the O-linked glycans, liberated from the mucin-type glycoproteins by reductive elimination can be determined by spectroscopic methods, using different techniques of nuclear magnetic resonance (NMR) (3) and mass spectrometry (4). By combining the immunological and spectroscopic approach numerous oncofetal carbohydrate antigen structures have been found not only on mucins of developing systems (5), but on mucins of human body fluids of normal individuals like in seminal plasma (6) and milk (7) as well. Oncofetal antigens, recognized by a number of monoclonal antibodies like C-50, NS 19-9, OC 125, Leu Ml, 49 H 8 and 115 C 2, are strongly expressed also in the mucine fraction of human amniotic fluid (8). These were analysed by fast atom bombardment mass spectrometry (FAB-MS) (9), in particular Suitable for the determination of carbohydrate sequences, their branching patterns and their molecular size in native and derivatized samples, even in complex mixtures

Structural characterization of gangliosides from B cell derived cell lines

Steuer H, Peter-Katalinic J, Bethke U, Neumann U, Büntemeyer H, Müthing J. Structural characterization of gangliosides from B cell derived cell lines. Biological chemistry Hoppe-Seyler. 1992;373(9):851

Siglec-F ligand expression in normal mouse lung does not require the Gal-6-O-Sulfotransferases, C6ST-1 Or Ksgal6st

Protein O-GlcNAcation is a stress-induced post-translational modification of intracellular proteins. A variety of neuronal proteins have been shown to harbour O-GlcNAc modifications, which are dynamically regulated by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). We examined the effect of blocking OGT and OGA on oxygen glucose deprivation (OGD) induced cell death in primary cortical neurons. Western blot analysis revealed that OGD did not produce a pronounced effect of O-GlcNAcation. However, OGA inhibition with Pugnac induced a marked increase in protein O-GlcNAcation but did not impart a neuroprotective effect against OGD. OGT inhibition with alloxan produced a moderate decrease in protein O-GlcNAcation, but produced a significant neuroprotective effect against OGD and NMDA toxicity. This neuroprotective effect was not associated with inhibition of NMDA channel activity, as alloxan did not inhibit NMDA-stimulated Ca++ currents. Furthermore, alloxan did not inhibit either staurosporin- or H2O2-induced cell death. Lastly, multi-electrode array (MEA) analysis of cultured neurons demonstrated that alloxan and Pugnac treatment modulated synaptic signalling. This study demonstrates that protein O-GlcNAcation modulates synaptic function, and that alterations in synaptic responsiveness are responsible for alloxanmediated neuroprotection.Peer reviewed: YesNRC publication: Ye