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J Biol Chem, Vol. 273, Issue 44, 28557-28559, October 30, 1998
,,
From the Department of Medical Biochemistry,
University of Turku, FIN-20520 Turku, Finland, the
§ Department of Bio-Organic Chemistry, Bijvoet Research
Center, University of Utrecht, NL-3584 CH Utrecht, The Netherlands, and
the ¶ Laboratory of Electron Microscopy, University of Turku,
FIN-20520 Turku, Finland
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ABSTRACT |
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Abstract
Introduction
Procedures
Results
Discussion
References
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Polysialic acid is a developmentally regulated
component in the neural cell adhesion molecule N-CAM which also occurs
as the capsular polysaccharide of bacteria causing meningitis.
Polysialic acid has been considered as a repulsive element that
regulates intermolecular and intercellular adhesion. Using atomic force microscopy we unexpectedly find that oligomers of polysialic acid assemble with each other into filament bundle networks. Filaments were
formed from oligomers containing 12 or more
N-acetylneuraminic acid residues, and they were sensitive
to sialidase digestion. The networks were also formed by the polysialic
acid-containing carbohydrate units of N-CAM. The formation of filament
bundles is a novel and unexpected property of polysialic acid and of
short carbohydrate oligomers in general and represents a previously unrecognized molecular interaction mechanism which impacts both eukaryotic and prokaryotic cell-cell adhesions.
Polysialic acid is a unique sialic acid polymer that occurs in the
neural cell adhesion molecule N-CAM (1-3). During development the
polysialylated embryonic N-CAM is replaced by its adult, normally sialylated form (4). Polysialic acid also occurs as the capsular polysaccharide of bacteria causing meningitis (5, 6). The molecular
mechanisms by which polysialic acid modulates cell adhesion or
participates in the pathogenesis of meningitis are not known. The main
function has been thought to be to serve as a repulsive element
contributing to the net negative charge of the cell surface.
Atomic force microscopy has become a new method to visualize filaments
of biological origin under conditions close to their native state
(7-10). Using this technique to image oligomers of polysialic acid we
unexpectedly find formation of filament bundles that assemble into
networks. This finding suggests an explanation for the unusual
properties of polysialic acid and suggests a novel molecular mechanism
in the interactions of cell adhesion molecules.
Materials--
Oligomers consisting of 6, 9, 12, 15, or 18 residues of Atomic Force Microscopy--
Aliquots of 2 µl containing 2 pmol of polysialic acid oligomer were applied together with 40 pmol of
CaCl2 onto freshly cleaved mica surfaces and allowed to dry
at 20 °C. The samples were imaged with no further treatment using
Digital Instruments Nanoscope 2 atomic force microscope with a
Nanoprobe cantilever with a quoted force constant of 0.38 newton
m-1.
Neuraminidase Digestion--
Digestion of preformed filament
networks bundles was carried out with 5 µl of 0.1 milliunit/ml of
Vibrio cholerae neuraminidase (Behringwerke AG) in 50 mM sodium acetate buffer, pH 5.5, containing 9 mM CaCl2 and 154 mM NaCl at
20 °C for 10 min. Control incubations were carried out under
identical conditions but without enzyme.
Oligomers of
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
2-8-linked N-acetylneuraminic acid were
purified by high performance liquid chromatography from colominic acid
(11). The purity of the oligomers was assessed by gel electrophoresis
(12) and staining with Alcian blue-silver. Embryonic polysialic
acid-containing glycopeptides and normal sialylated embryonic
glycopeptides without polysialyl units were isolated as described
before (13).
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
2-8-linked N-acetylneuraminic acid
residues of defined polymer length, purified from colominic acid by
high performance liquid chromatography, were placed on mica surfaces and allowed to dry. Atomic force microscopy revealed the presence of
filamentous structures in samples of oligomers of 12 or more sialyl
residues, whereas oligomers of 9 residues or shorter did not display
these structures (Fig. 1). Individual
filaments had a minimum thickness of ~1 nm (Fig.
2) but tended to occur as filament bundles. With increasing chain length, extensive branching of the
filament bundles into networks was observed. The filament networks were
degraded by sialidase, showing that they consisted of sialic acid (Fig.
3).
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Fig. 1.
Formation of filament network bundles by
oligomers of sialic acid. Oligomers consisting of 6, 9, 12, 15, or
18 residues of 2-8-linked N-acetylneuraminic acid were
imaged with atomic force microscopy. The oligomers were also analyzed
by gel electrophoresis and staining with Alcian blue-silver (staining
intensity of hexasaccharide low due to diffusion out from the gel); the
ladder obtained from colominic acid is shown for reference
(scale bar, 200 nm).
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Fig. 2.
Surface contour display of polysialic acid
filaments in high resolution. Purified oligomers of polysialic
acid (degree of polymerization 18) were imaged by atomic force
microscopy under conditions of Fig. 1. The surface scan contour image
displays the filaments as two ridges, cross-sectioned by the display
program in the front and their anastomosis farther back in the
image.
View larger version (97K):
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Fig. 3.
Degradation of polysialic acid filaments by
sialidase. Filament bundle networks were formed from polysialic
acid oligomers (degree of polymerization 18). A, digestion
with V. cholerae neuraminidase. B, control
incubation under identical conditions but without enzyme. After
incubation the samples were rinsed lightly with distilled water,
allowed to dry, and examined by atomic force microscopy. The large
particles in both panels are crystals of precipitated buffer
(scale bar, 200 nm).
The purified polysialylated glycopeptides of the neural cell adhesion molecule N-CAM of embryonic brain also formed extensive branched filament bundle networks (Fig. 4A) similar to those obtained from polysialic acid. Control specimens of glycopeptides without polysialic acid from the same source did not form these structures (Fig. 4B).
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DISCUSSION |
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Abstract
Introduction
Procedures
Results
Discussion
References
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Polysialic acid is an exceptional polysaccharide in that a long oligosialyl fragment, ~8-10 sialyl residues, is needed for the interaction of polysialic acid with most of its antibodies (13-15). Similarly, a minimum of 8 residues is required for the most efficient cleavage of polysialic acid by endosialidases (14, 16). The requirement of the unusually long segment of polysialic acid for the interaction with these proteins is suggested to be related to conformational factors (15, 17). A helical coil structure may be stabilized for oligomers of sufficient chain length, as supported by NMR studies (18). It is possible that the attaining of the helical coil epitope is also a prerequisite for the formation of the filament structure observed in the present study. Thus, only fragments of sufficient length would fulfill the physical requirements for the association with each other into filaments. With regard to the interaction mechanisms of polysialic acid with proteins, the possibility remains that the interaction in fact is with the filamentous form of polysialic acid. Alternatively, the filamentous form may represent an additional form of polysialic acid. The filamentous form could also correspond to the postulated "intermolecular epitope" of polysialic acid, implicated as a potential vaccine epitope against group B meningococci (19).
Polysialic acid occurs abundantly at the cell surface of developing neural and other cells (2). One of its biological roles has been suggested to be associated with a barrier function between cells (20). Due to its negative charge, polysialic acid has been viewed mainly as a repulsive element between cells and molecules (21, 22). The potential of association into bundle networks suggests that polysialic acid could also participate in associative interactions. In developing brain, the length of the polysialic acid chains undergoes temporal and topical modulation. The observed length-dependent formation of filament bundle networks may offer a molecular mechanism for the modulation of cell interactions involved in these processes.
The formation of filament networks by polysialic acid could also be a mechanism to enhance its postulated barrier function. In addition to its presence on the eukaryotic cell surface, polysialic acid forms the polysaccharide capsule of some important bacterial pathogens causing sepsis and meningitis, such as Neisseria meningitidis group B, Escherichia coli K1, and Pasteurella hemolytica A2 (6, 23). It is conceivable that the stability of the surface layer of the cells would be enhanced significantly by a filamentous network.
Filaments of protein and nucleic acid have been examined by atomic
force microscopy (7-10). Some polysaccharides have also been reported
to form filamentous networks (24, 25). In addition hyaluronic acid, an
abundant extracellular component, may form filaments (26). However,
polysialic acid and other cell surface carbohydrates have mainly been
considered as inert or repulsive molecules in cellular and molecular
interactions. Although carbohydrate-carbohydrate interactions have in
some cases been proposed to mediate cell adhesion (27, 28), no data are
available on the supramolecular organization of the complexes involved.
The results of the present study unexpectedly suggest that carbohydrate
oligomers as short as 12 residues long may associate into long
filamentous structures. The filament network formation has to be
considered as a novel potential mechanism by which N-CAM and other cell
surface components may mediate their interactions with other molecules
and cells.
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ACKNOWLEDGEMENTS |
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We thank Drs. M. Radmacher and J. Mäki for decisive guidance and technical help.
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FOOTNOTES |
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* This work was supported by the European Commission, the Academy of Finland, the Sigrid Jusélius Foundation, and the Turku University Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Medical
Biochemistry, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku,
Finland. Tel.: 358-2-333-7240; Fax: 358-2-333-7229; E-mail: finne{at}utu.fi.
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REFERENCES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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