Brigitte Schmitz

The Lex carbohydrate sequence is recognized by antibody to L5, a functional antigen in early neural development

Abstract: The L5 antigenic determinant was previously suggested to be a carbohydrate epitope present on murine cell recognition molecules in the developing brain and to be an early neural marker in the chick embryo. Here, we show that L5 immunoreactivity is associated with complex‐type N‐glycosidic oligosaccharides. To identify the carbohydrate structure recognized by the L5 antibody, we investigate its binding to N‐linked oligosaccharides derived from L5 glycoproteins and to known glycans. Results of mass spectrometric analyses of L5‐positive neoglycolipids prepared from L5 glycoproteins are consistent with those for N‐glycans containing a 3‐fucosyl N‐acetyllactosamine sequence. We also investigate L5 binding to structurally defined, lipid‐linked oligosaccharides based on the blood group type I and II backbones. Chromatogram binding assays, ELISA, and inhibition studies show that the antibody reacts strongly with carbohydrate chains presenting the 3‐fucosyl N‐acetyllactosamine sequence [Lewisx (Lex) or X‐hapten] also recognized by anti‐SSEA‐1 and anti‐CD15. Histochemical studies with different antibodies recognizing the Lex sequence show partially overlapping patterns of immunoreactivity during early neural development in the chick embryo. Therefore, we suggest that the epitope recognized by L5 antibody is closely related to those for anti‐SSEA‐1 and anti‐CD15.

Galectin-3 promotes neural cell adhesion and neurite growth

Galectin‐3 is a member of the galectin family and belongs to a group of soluble β‐galactoside‐binding animal lectins. The molecule is expressed by neural and nonneural cells intra‐ (cytoplasm and nucleus) as well as extra‐cellularly (plasma membrane and extracellular space). By using an in vitro cell‐substratum adhesion assay, we have addressed the question whether galectin‐3 present in the extracellular milieu may support the adhesion and/or neurite outgrowth of neural cells in a manner analogous to cell adhesion molecules. Galectin‐3 was immobilized as a substratum and various cell types, N2A (neuroblastoma), PC12 (pheochromocytoma), and TSC (transformed Schwann cells) cell lines, neural cells from early postnatal mouse cerebellum, and dorsal root ganglion neurons from newborn mice were allowed to adhere to the lectin. Here we show that all cell types studied specifically adhered to galectin‐3 by the following criteria: 1) the number of adherent cells was dependent on the galectin‐3 concentration used for coating; 2) adhesion of cells to galectin‐3, but not to collagen type I or laminin was inhibited by polyclonal antibodies to galectin‐3; 3) upon addition of asialofetuin (a polyvalent carrier of terminal β‐galactosides) to the cell suspension prior to the adhesion assay, cell adhesion to galectin‐3 was inhibited in a dose‐dependent manner; and 4) cell adhesion to galectin‐3 was abolished by treatment of cells with endo‐β‐galactosidase. In addition, the adhesion of dorsal root ganglion neurons to galectin‐3 could be inhibited by lactose. Notably, substratum‐bound galectin‐3 promoted the outgrowth of neurites from dorsal root ganglia explants and this neurite outgrowth promoting activity could be inhibited by polyclonal antibodies to galectin‐3. J. Neurosci. Res. 54:639‐654, 1998.

NCAM is ubiquitylated, endocytosed and recycled in neurons

The neural cell adhesion molecule NCAM plays an important role during neural development and in the adult brain. To study the intracellular trafficking of NCAM in neurons, two major isoforms, NCAM140 or NCAM180, were expressed in primary cortical neurons and in the rat B35 neuroblastoma cell line. NCAM was endocytosed and subsequently recycled to the plasma membrane, whereas only a minor fraction was degraded in lysosomes. In cortical neurons, endocytosis of NCAM was detected in the soma, neurites and growth cones in a developmentally regulated fashion. Furthermore, we found that NCAM is mono-ubiquitylated at the plasma membrane and endocytosis was significantly increased in cells overexpressing ubiquitin. Therefore, we propose that ubiquitylation represents an endocytosis signal for NCAM.

NCAM140 stimulates integrin-dependent cell migration by ectodomain shedding

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration and synaptic plasticity. This study describes a novel function of NCAM140 in stimulating integrin‐dependent cell migration. Expression of NCAM140 in rat B35 neuroblastoma cells resulted in increased migration toward the extracellular matrix proteins fibronectin, collagen IV, vitronectin, and laminin. NCAM‐potentiated cell migration toward fibronectin was dependent on β1 integrins and required extracellular‐regulated kinase (ERK)1/2 mitogen‐activated protein kinase (MAPK) activity. NCAM140 in B35 neuroblastoma cells was subject to ectodomain cleavage resulting in a 115 kDa soluble fragment released into the media and a 30 kDa cytoplasmic domain fragment remaining in the cell membrane. NCAM140 ectodomain cleavage was stimulated by the tyrosine phosphatase inhibitor pervanadate and inhibited by the broad spectrum metalloprotease inhibitor GM6001, characteristic of a metalloprotease. Moreover, treatment of NCAM140‐B35 cells with GM6001 reduced NCAM140‐stimulated cell migration toward fibronectin and increased cellular attachment to fibronectin to a small but significant extent. These results suggested that metalloprotease‐induced cleavage of NCAM140 from the membrane promotes integrin‐ and ERK1/2‐dependent cell migration to extracellular matrix proteins.

Tyrosine and serine phosphorylation of the neural cell adhesion molecule L1 is implicated in its oligomannosidic glycan dependent association with NCAM and neurite outgrowth

We have previously shown that a cis interaction between the cell adhesion molecules L1 and NCAM is mediated by N-linked oligomannosidic glycans carried by L1 and that this L1/NCAM association is involved in basal neurite outgrowth from early postnatal cerebellar neurons of mouse brain [R. Horstkorte et al., J. Cell Biol. 121, 1409-1421 (1993)]. Extending these earlier studies we investigated signal transduction mechanisms elicited by this molecular interaction. We show here that phosphorylation of L1 is reduced concomitant with reduced neurite outgrowth when the L1/NCAM interaction is inhibited by oligomannosidic glycopeptides. Similarly, when a peptide of the 4th immunoglobulin (Ig)-like domain of NCAM - representing part of NCAMs carbohydrate-binding site - was added to the culture medium of the cells, neurite outgrowth and phosphorylation of L1 was strongly reduced. No effect on neurite outgrowth and phosphorylation of L1 was observed when cells were maintained in the presence of a peptide comprising part of the 1st Ig-like domain of NCAM or in the presence of the peptide encoded by the variable alternative spliced exon (VASE), which is also located in the 4th Ig-like domain of NCAM. Furthermore, phosphorylation of tyrosine and serine residues of L1 is reduced when the L1/NCAM interaction at the cell surface of cerebellar neurons is perturbed. Our observations suggest that a signal transduction mechanism is implicated in basal neurite outgrowth in which both tyrosine and serine phosphorylation of L1 represent a possible proximal step. Some of these results were presented at the International Glycoconjugate Symposium in Seattle, USA [P. C. Heiland et al., Glycoconj. J. 12, 521(1995)].

O-GlcNAc expression in developing and ageing mouse brain

In order to understand whether there is a specific role for the posttranslational N-acetylglucosamine modification linked O-glycosidically (O-GlcNAc) to serine and threonine residues of proteins during development and/or ageing of the brain, we investigated the O-GlcNAc expression of early postnatal cerebellar neurons as well as of mouse brain of different ages. In all cells either in culture or of cryosections mainly the nuclei and nuclear membranes were stained with an O-GlcNAc specific monoclonal antibody. In cerebellar neurons in culture the level of expression could be manipulated by directly interfering with either the biosynthesis of GlcNAc or the removal of O-GlcNAc from proteins confirming the dynamic nature of this protein modification. O-GlcNAc was ubiquitously expressed in mouse brains from embryonic day 10 until late adulthood with some variations in expression strength from cell to cell. In addition, no significant difference in O-GlcNAc expression of subcellular fractions from brains of mice which age at an accelerated rate could be detected compared to normal mice. Taken together these observations support the view that the O-GlcNAc modification has important functional roles for physiological processes of neural cell throughout development, in adulthood and ageing.

Determination of double bond position in tri- to hexaenoic fatty acids by mass spectrometry

Abstract Methylesters of polyenoic fatty acids (FAE) with up to six double bonds can be transformed to polyhydroxy derivatives by treatment with OsO4. Under low energy electron impact the trimethylsilyloxy (O-TMS) derivatives of these polyenoic fatty acids exhibit mass spectrometric fragmentation patterns that are straightforward due to the almost exclusive cleavage of CC bonds in between vicinal O-TMS groups. Thus it is possible to localise original double bond positions and also to predict fragmentation patterns of hitherto unknown fatty acid species. The method is applicable to polyenoic compounds with isolated as well as conjugated double bonds.

Increased O-GlcNAc levels correlate with decreased O-GlcNAcase levels in Alzheimer disease brain

The potential role of the posttranslational modification of proteins with O-linked N-acetyl-β-d-glucosamine (O-GlcNAc) in the pathogenesis of Alzheimer disease (AD) has been studied extensively, yet the exact function of O-GlcNAc in AD remains elusive. O-GlcNAc cycling is facilitated by only two highly conserved enzymes: O-GlcNAc transferase (OGT) catalyzes the addition, while O-GlcNAcase (OGA) catalyzes the removal of GlcNAc from proteins. Studies analyzing global O-GlcNAc levels in AD brain have produced inconsistent results and the reasons for altered O-GlcNAcylation in AD are still poorly understood. In this study, we show a 1.2-fold increase in cytosolic protein O-GlcNAc modification in AD brain when compared to age-matched controls. Interestingly, O-GlcNAc changes seem to be attributable to differential modification of a few individual proteins. While our finding of augmented O-GlcNAcylation concurs with some reports, it is contrary to others demonstrating decreased O-GlcNAc levels in AD brain. These conflicting results emphasize the need for further studies providing conclusive evidence on the subject of O-GlcNAcylation in AD. We further demonstrate that, while OGT protein levels are unaffected in AD, OGA protein levels are significantly decreased to 75% of those in control samples. In addition, augmented protein O-GlcNAc modification correlates to decreased OGA protein levels in AD subjects. While OGA inhibitors are already being tested for AD treatment, our results provide a strong indication that the general subject of O-GlcNAcylation and specifically its regulation by OGA and OGT in AD need further investigation to conclusively elucidate its potential role in AD pathogenesis and treatment.

Positional isomers of unsaturated fatty acids in rat liver lipids.

The fatty acids of liver lipids from rats raised on a fat free diet from the 30th to the 90th day after birth were analyzed with special regard to the detection of positional isomers of mono-, di-, tri-, and tetraenoic fatty acids. The methyl esters obtained after transesterification of total lipids were separated by argentation chromatography into five fractions: I saturated, II monoenoic, III dienoic, IV dienoic nonmethylene interrupted, V triand tetraenoic fatty acid esters. After hydroxylation of the double bonds with osmium tetroxide, the analysis of the poly-O-trimethylsilyl derivatives by gas liquid chromatography on S.C.O.T. columns combined with mass spectrometry revealed the presence of 19 monoenoic, 15 dienoic, and 9 trienoic as well as 3 tetraenoic fatty acid isomers including the normally occurring representatives of the (n−3), (n−6), (n−7), and (n−9) fatty acid families. The majority of the identified isomers can be coordinated to one of these families like 7–16∶1; 11–20∶1; 6,9–18∶2; 8,11–20∶2; 5,11–20∶2; 5,8,11–20∶3; 7,10,13–22∶3 to the (n−9) family, 11–18∶1; 13–20∶1; 5,11–18∶2; 7,13–20∶2; 6,11–18∶2; 6,9–16∶2; 8, 11–18∶2; 10,13–20∶2; 5,8,11–18∶3; 7,10,13–20∶3; 4,7,10,13–20∶4 to the (n−7) family and 11,14–20∶2; 5,11,14–20∶3; 6,9,12–18∶3; 8,11,14–20∶3; 5,8,11,14–20∶4; 7,10,13,16–22∶4 to the (n−6) family. All these naturally occuring isomers can be placed into a network of desaturation and chain elongation steps which allows certain conclusions about the substrate specificity of the Δ6-, Δ5-and Δ4-desaturase systems. The great number of isomers found in the (n−7) family indicates that the members of this family are actively metabolized in partial essential fatty acid deficiency.

Do rodent and human brains have different N-glycosylation patterns?

Abstract A large number of studies on the structure of Nglycosidically linked oligosaccharides from glycoproteins of different organs and/or different species have been carried out in the past using various combinations of techniques such as monosaccharide analysis, permethylation, peracteylation, exoglycosidase sequencing, normal and reversed phase HPLC, mass spectrometry and nuclear magnetic resonance spectroscopy. Although it is widely accepted that the processing of Nglycans in the ER and Golgi of mammalian cells follows the same principal metabolic rules, analyses have revealed that the glycosylation pattern of a particular protein may differ depending on the cell type in which it is expressed. Nglycans from brain glycoproteins have been shown to include a variety of hybrid and complextype structures with structural features that are not so commonly found on glycoproteins from other organs and which have, therefore, been classified as brainspecific. Comparison of the Nglycans of glycoproteins from homogenates of rat, mouse and human brains confirm that, in general, glycoproteins from human brain show a similar profile of brainspecific Nglycans as glycoproteins from mouse and rat brain.