Core structures of polysialylated glycans present in neural cell adhesion molecule from newborn mouse brain

Geyer, Hildegard; Bahr, U.; Liedtke, Steffen; Schachner, Melitta; Geyer, Rudolf

doi:10.1046/j.0014-2956.2001.02613.x

Cited by 31 publications

(14 citation statements)

References 66 publications

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“…Obtained mass profiles confirmed the presence of a vast variety of compositional species corresponding to partially incomplete diantennary, triantennary, and tetraantennary glycans carrying, in part, additional fucose, sulfate and uronic acid residues. Similar species have already been detected and analyzed in detail in earlier studies on murine or bovine polySia-NCAM (4,6,61). The oligosaccharide pattern obtained from wild-type and mutant mouse polySia-NCAM were identical with comparable signal intensities in all three cases.…”

Section: St8siaii and St8siaivsupporting

confidence: 60%

“…The core structures of wild-type NCAM polySia-N-glycans detected in the present study displayed by far a greater variability than those described in our earlier analyses of murine NCAM (4,61). In addition to the already known panel of neutral, partially truncated di-, tri-, and tetraantennary fucosylated oligosaccharides, the present preparation comprised also unfu- cosylated as well as a more diverse pattern of acidic species.…”

Section: Discussioncontrasting

confidence: 40%

“…Possibly due to enhanced sensitivity of mass spectrometry a number of glycans could be now identified carrying 1-2 sulfate groups with or without glucuronic acid. In conjunction with previous data, it can be assumed that these glycans exhibit, at least in part, an HNK1 epitope (4,61). In this context, it is also interesting to note that some of these species have been already observed as components of bovine polySia-NCAM (6).…”

Section: Discussionmentioning

confidence: 51%

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Enzyme-dependent Variations in the Polysialylation of the Neural Cell Adhesion Molecule (NCAM) in Vivo

Galuska

Geyer

Gerardy‐Schahn

et al. 2008

Journal of Biological Chemistry

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Polysialic acid (polySia), an ␣2,8-linked polymer of N-acetylneuraminic acid, represents an essential regulator of neural cell adhesion molecule (NCAM) functions. Two polysialyltransferases, ST8SiaII and ST8SiaIV, account for polySia synthesis, but their individual roles in vivo are still not fully understood. Previous in vitro studies defined differences between the two enzymes in their usage of the two NCAM N-glycosylation sites affected and suggested a synergistic effect. Using mutant mice, lacking either enzyme, we now assessed in vivo the contribution of ST8SiaII and ST8SiaIV to polysialylation of NCAM. PolySia-NCAM was isolated from mouse brains and trypsinized, and polysialylated glycopeptides as well as glycans were analyzed in detail. Our results revealed an identical glycosylation and almost complete polysialylation of N-glycosylation sites 5 and 6 in polySia-NCAM irrespective of the enzyme present. The same sets of glycans were substituted by identical numbers of polySia chains in vivo, the length distribution of which, however, differed with the enzyme setting. Expression of ST8SiaIV alone led to higher amounts of short polySia chains and gradual decrease with length, whereas exclusive action of ST8SiaII evoked a slight reduction in long polySia chains only. These variations were most pronounced at N-glycosylation site 5, whereas the polysialylation pattern at N-glycosylation site 6 did not differ between NCAM from wild-type and ST8SiaII-or ST8SiaIV-deficient mice. Thus, our fine structure analyses suggest a comparable quality of polysialylation by ST8SiaII and ST8SiaIV and a distinct synergistic action of the two enzymes in the synthesis of long polySia chains at N-glycosylation site 5 in vivo.

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Section: St8siaii and St8siaivsupporting

confidence: 60%

Section: Discussioncontrasting

confidence: 40%

Section: Discussionmentioning

confidence: 51%

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Enzyme-dependent Variations in the Polysialylation of the Neural Cell Adhesion Molecule (NCAM) in Vivo

Galuska

Geyer

Gerardy‐Schahn

et al. 2008

Journal of Biological Chemistry

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“…Whereas chemical methods of analysis employing fluorescent derivatization with DMB have provided the most promising approach (46,49,56,57), they underestimate chain length because of the intramolecular self-cleavage of internal Sia residues resulting from the lability of ␣2,8-ketosidic linkages to even mildly acidic conditions (58). Unfortunately, acidic conditions (pH ϳ1.9) are required for DMB fluorescent tagging, a prerequisite for enhanced sensitivity required for chromatographic detection in the femtomol to picomol range.…”

Section: The C 7 /C 9 Methods For Dp Analysis and The Structural Complmentioning

confidence: 99%

Degree of Polymerization (DP) of Polysialic Acid (PolySia) on Neural Cell Adhesion Molecules (N-CAMs)

Nakata

Troy

2005

Journal of Biological Chemistry

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␣2,8-Linked polysialic acid (polySia) is a structurally unique antiadhesive glycotope that covalently modifies N-linked glycans on neural cell adhesion molecules (N-CAMs). These sugar chains play a key role in modulating cell-cell interactions, principally during embryonic development, neural plasticity, and tumor metastasis. The degree of polymerization (DP) of polySia chains on N-CAM is postulated to be of critical importance in regulating N-CAM function. There are limitations, however, in the conventional methods to accurately determine the DP of polySia on N-CAM, the most serious being partial acid hydrolysis of internal ␣2,8-ketosidic linkages that occur during fluorescent derivatization, a step necessary to enhance chromatographic detection. To circumvent this problem, we have developed a facile method that combines the use of Endo-␤-galactosidase to first release linear polySia chains from N-CAM, with high resolution high pressure liquid chromatography profiling. This strategy avoids acid hydrolysis prior to chromatographic profiling and thus provides an accurate determination of the DP and distribution of polySia on N-CAM. The potential of this new method was evaluated using a nonpolysialylated construct of N-CAM that was polysialylated in vitro using a soluble construct of ST8Sia II or ST8Sia IV. Whereas most of the oligosialic acid/polySia chains consisted of DPs ϳ50 -60 or less, a subpopulation of chains with DPs ϳ150 to ϳ180 and extending to DP ϳ400 were detected. The DP of this subpopulation is considerably greater than reported previously for N-CAM. Endo-␤-galactosidase can also release polySia chains from polysialylated membranes expressed in the neuroblastoma cell line, Neuro2A, and native N-CAM from embryonic chick brains.

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“…poly-N-acetyllactosamine stretches for cleavage (38,39), which are not found in polysialylated N-glycans of perinatal mouse brain (13,40 Ϫ/Ϫ ST8SiaIV ϩ/ϩ ) were used. After DMB derivatization, fluorescently labeled polySia species were separated according to DP by anion-exchange HPLC (32).…”

Section: St8siaivmentioning

confidence: 99%

Polysialic Acid Profiles of Mice Expressing Variant Allelic Combinations of the Polysialyltransferases ST8SiaII and ST8SiaIV

Galuska¹,

Oltmann-Norden²,

Geyer³

et al. 2006

J. Biol. Chem.

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The post-translational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) represents a remarkable example of dynamic modulation of homo-and heterophilic cell interactions by glycosylation. The synthesis of this unique carbohydrate polymer depends on the polysialyltransferases ST8SiaII and ST8SiaIV. Aiming to understand in more detail the contributions of ST8SiaII and ST8SiaIV to polySia biosynthesis in vivo, we used mutant mouse lines that differ in the number of functional polysialyltransferase alleles. The 1,2-diamino-4,5-methylenedioxybenzene method was used to qualitatively and quantitatively assess the polySia patterns. Similar to the wild-type genotype, long polySia chains (>50 residues) were detected in all genotypes expressing at least one functional polysialyltransferase allele. However, variant allelic combinations resulted in distinct alterations in the total amount of polySia; the relative abundance of long, medium, and short polymers; and the ratio of polysialylated to non-polysialylated NCAM. In ST8SiaII-null mice, 45% of the brain NCAM was non-polysialylated, whereas a single functional allele of ST8SiaII was sufficient to polysialylate ϳ90% of the NCAM pool. Our data reveal a complex polysialylation pattern and show that, under in vivo conditions, the coordinated action of ST8SiaII and ST8SiaIV is crucial to fine-tune the amount and structure of polySia on NCAM.Carbohydrate modifications of proteins and lipids play an important role in the development and maintenance of the nervous system as mediators of cell recognition events (1). One striking example is polysialic acid (polySia), 2 a linear homopolymer of ␣2,8-linked N-acetylneuraminic acid. In vertebrates, polySia is found almost exclusively as a post-translational modification of the neural cell adhesion molecule (NCAM), a member of the immunoglobulin superfamily. Attachment of polySia to NCAM was demonstrated to double the hydrodynamic radius of NCAM, thereby increasing the intermembrane space and disrupting the adhesive properties of NCAM and other cell adhesion molecules such as L1, integrins, and cadherins (2-4). PolySia promotes migration of neuronal precursor cells, axonal outgrowth, and synaptic plasticity (for review, see Ref. 5). In addition to its function as a negative regulator of cell adhesion, polySia was shown to bind heparan sulfate proteoglycans (6), forming a complex that supports synaptogenesis and activity-dependent remodeling of synapses (7). In addition, polySia can bind brain-derived neurotrophic factor to enhance brain-derived neurotrophic factor-dependent survival of cortical neurons (8, 9) and appears to be involved in the regulation of neurotransmitter receptor activity (10). Whereas polySia levels are high during embryonic development, expression in the adult is restricted to brain areas of persistent neurogenesis and synaptic plasticity (11).Interestingly, the biosynthesis of polySia depends on two enzymes, the Golgi-resident polysialyltransferases ST8SiaII and ST8SiaIV, which s...

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Core structures of polysialylated glycans present in neural cell adhesion molecule from newborn mouse brain

Cited by 31 publications

References 66 publications

Enzyme-dependent Variations in the Polysialylation of the Neural Cell Adhesion Molecule (NCAM) in Vivo

Enzyme-dependent Variations in the Polysialylation of the Neural Cell Adhesion Molecule (NCAM) in Vivo

Degree of Polymerization (DP) of Polysialic Acid (PolySia) on Neural Cell Adhesion Molecules (N-CAMs)

Polysialic Acid Profiles of Mice Expressing Variant Allelic Combinations of the Polysialyltransferases ST8SiaII and ST8SiaIV

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