Direct Evidence That Neural Cell Adhesion Molecule (NCAM) Polysialylation Increases Intermembrane Repulsion and Abrogates Adhesion
Molecular force measurements quantified the impact of polysialylation on the adhesive properties both of membrane-bound neural cell adhesion molecule (NCAM) and of other proteins on the same membrane. These results show quantitatively that NCAM polysialylation increases the range and magnitude of intermembrane repulsion. The repulsion is sufficient to overwhelm both homophilic NCAM and cadherin attraction at physiological ionic strength, and it abrogates the protein-mediated intermembrane adhesion. The steric repulsion is ionic strength dependent and decreases substantially at high monovalent salt concentrations with a concomitant increase in the intermembrane attraction. The magnitude of the repulsion also depends on the amount of polysialic acid (PSA) on the membranes, and the PSA-dependent attenuation of cadherin adhesion increases with increasing PSA-NCAM:cadherin ratios. These findings agree qualitatively with independent reports based on cell adhesion studies and reveal the likely molecular mechanism by which NCAM polysialylation regulates cell adhesion and intermembrane space. Molecular force measurements quantified the impact of polysialylation on the adhesive properties both of membrane-bound neural cell adhesion molecule (NCAM) and of other proteins on the same membrane. These results show quantitatively that NCAM polysialylation increases the range and magnitude of intermembrane repulsion. The repulsion is sufficient to overwhelm both homophilic NCAM and cadherin attraction at physiological ionic strength, and it abrogates the protein-mediated intermembrane adhesion. The steric repulsion is ionic strength dependent and decreases substantially at high monovalent salt concentrations with a concomitant increase in the intermembrane attraction. The magnitude of the repulsion also depends on the amount of polysialic acid (PSA) on the membranes, and the PSA-dependent attenuation of cadherin adhesion increases with increasing PSA-NCAM:cadherin ratios. These findings agree qualitatively with independent reports based on cell adhesion studies and reveal the likely molecular mechanism by which NCAM polysialylation regulates cell adhesion and intermembrane space. Direct evidence that neural cell adhesion molecule (NCAM) polysialylation increases intermembrane repulsion and abrogates adhesion. Vol. 280 (2005) 137–145Journal of Biological ChemistryVol. 280Issue 24PreviewPage 137: The grant number listed in the footnote at the bottom of the page was misprinted. The correct citation should read as follows: “This work was supported by National Institutes of Health Grant 1RO1 GM63536.” Full-Text PDF Open Access Polysialic acid (PSA) 1The abbreviations used are: PSA, polysialic acid; NCAM, neural cell adhesion molecule; endo-N, endoneuraminindase; SFA, surface force apparatus; NTA-TRIG-DLGE, 6-(9-(2,3-bis(dodecyloxy)propyl)-3,6,9-trioxanonyl-1-oxycarboxylamino)-2-(di(carboxylmethyl)amino)-hexanoic acid; FCS, fluorescence correlation spectroscopy; NTA, nitrilotriacetic acid; RG, rhodamine green. is a long, linear α2,8-linked carbohydrate composed of N-acetylneuraminic acid (Neu5Ac) residues (1Bruses J.L. Rutishauser U. Biochimie (Paris). 2001; 83: 635-643Crossref PubMed Scopus (195) Google Scholar). This carbohydrate is added post-translationally to the neural cell adhesion molecule (NCAM), which is responsible for a variety of functions, including axon pathfinding, synaptogenesis, and tissue formation in the central nervous system (2Walsh F.S. Doherty P. Annu. Rev. Dev. Biol. 1997; 13: 425-456Crossref PubMed Scopus (404) Google Scholar). The expression of the polysialylated form of NCAM (PSA-NCAM) peaks early in development and decreases with age. In some exceptions, such as the hippocampus, cells continue to express PSA-NCAM throughout the life of the organism. These regions of PSA expression are also associated with neural plasticity and the remodeling of neural connections (1Bruses J.L. Rutishauser U. Biochimie (Paris). 2001; 83: 635-643Crossref PubMed Scopus (195) Google Scholar, 2Walsh F.S. Doherty P. Annu. Rev. Dev. Biol. 1997; 13: 425-456Crossref PubMed Scopus (404) Google Scholar). Aberrant expression of PSA-NCAM is associated with tumor malignancy and metastasis, and the expression of PSA-NCAM has been detected in small cell carcinoma, neuroblastomas, and Wilm's tumor (3Tanaka R. Otake Y. Nakagawa T. Kawano Y. Miyahara R. Li M. Yanagihara K. Nakayama J. Fujimoto I. Ikenaka K. Wada H. Cancer Res. 2000; 60: 3072-3080PubMed Google Scholar). Polysialic acid is thought to facilitate cell migration and plasticity by inhibiting cell adhesion to other cells and to the extracellular matrix, as a result of the large excluded volume of the polymer (4Yang P. Yin X. Rutishauser U. J. Cell. Biol. 1992; 116: 1487-1496Crossref PubMed Scopus (158) Google Scholar, 5Yang P. Major D. Yin X. Rutishauser U. J. Biol. Chem. 1994; 269: 23039-23044Abstract Full Text PDF PubMed Google Scholar). Electron microscopy images showed that PSA expression increased intercellular spacing by 10-15 nm (4Yang P. Yin X. Rutishauser U. J. Cell. Biol. 1992; 116: 1487-1496Crossref PubMed Scopus (158) Google Scholar). The latter could be because of the inactivation of adhesion proteins or to the increased inter-membrane repulsion resulting from the confinement of the carbohydrate chains. Light scattering studies demonstrated that NCAM polysialylation doubles the hydrodynamic radius of NCAM. However, the latter results were based on calculations, using light scattering data and the assumption that the rod-like proteins were spherical. While this indicates the approximate size of the protein, the hydrodynamic radius does not quantify the effect of the carbohydrates on NCAM-mediated adhesion. In one proposed mechanism, for example, the increased repulsive pressure between the membranes is hypothesized to push the cells apart (6Rutishauser U. Curr. Opin. Cell Biol. 1996; 8: 679-684Crossref PubMed Scopus (146) Google Scholar). Such a shift in the force balance between cells from attractive to repulsive requires the increased intermembrane repulsion to be at least as large as the protein attraction at the membrane distance at which the proteins bind. For example, if NCAM bridges two membranes at a separation of 40 nm with an adhesion energy of ∼1000 kBT/μm2, where kB is the Boltzmann constant and T is the temperature, then NCAM polysialylation would have to increase the repulsion at 40 nm by at least this amount, to disrupt the adhesive junction. Testing this, however, requires determining both the magnitudes of the intermembrane forces and their range. The impact of ionic strength on the adhesion between cells expressing PSA-NCAM further supports the view that PSA acts by increasing the repulsion between cells. The hydrodynamic volume of polyelectrolytes decreases with increasing monovalent salt concentrations (7Pincus P. Macromolecules. 1991; 24: 2912-2919Crossref Scopus (891) Google Scholar), and this would in turn reduce the repulsion between two membranes with surface-anchored chains. Consistent with this, an increase in the monovalent salt concentration from 0.15 to 0.5 m NaCl restored the adhesion between cells expressing PSA-NCAM (5Yang P. Major D. Yin X. Rutishauser U. J. Biol. Chem. 1994; 269: 23039-23044Abstract Full Text PDF PubMed Google Scholar). Investigations of cell adhesion also suggested that the effects of PSA be to a of adhesion including NCAM, and I. J.L. Rutishauser U. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). this of adhesion not NCAM for studies both and show that the carbohydrate the membrane J. and D. (2005) in These findings are with a mechanism in which PSA cell by increasing the inter-membrane repulsion. This would protein cell and shift the membrane However, is evidence intermembrane spacing and the of adhesion by In this the surface force and fluorescence correlation were used to the molecular mechanism by which polysialic acid abrogates NCAM and intermembrane adhesion. The the distance of the force between such as membranes J. J. 1992; Scopus Google Scholar, D. Annu. Rev. 2000; PubMed Scopus Google Scholar). These measurements are to a of the of The of such as PSA, as as the magnitude of the repulsive pressure between membranes is a of the (7Pincus P. Macromolecules. 1991; 24: 2912-2919Crossref Scopus (891) Google Scholar). In this molecule measurements would not The results show that the increase in the intermembrane repulsion NCAM polysialylation is sufficient to overwhelm both homophilic NCAM and cadherin attraction at physiological ionic adhesion at high monovalent salt concomitant with a in the PSA-dependent intermembrane repulsion. independent evidence that the hydrodynamic radius of PSA that of the NCAM. measurements with of NCAM and show that the of adhesive be to other cell adhesion and were from acid was by and other high were from was from Molecular was from was and as U. J. Google Scholar). and of the extracellular of NCAM, was at the of the and to a a and a acid as Fujimoto I. Rutishauser U. D. U. Scholar). This was a expression from and the protein was in cells The resulting protein of the extracellular to a Fujimoto I. Rutishauser U. D. U. Scholar). The both the of the protein and on Fujimoto I. Rutishauser U. D. U. Scholar). PSA-NCAM expression was by cells with a the NCAM and a the I. J.L. Rutishauser U. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Fujimoto I. Rutishauser U. D. U. Scholar). NCAM and PSA-NCAM were from the cell in a Fujimoto I. Rutishauser U. D. U. Scholar). the was a and the protein was with a of and the concentration of the protein with a the protein was further on a The PSA-NCAM were and the protein was to by The PSA-NCAM were in a and NaCl at at 280 nm and an of were used to the NCAM The expression and of NCAM and have been Fujimoto I. Rutishauser U. D. U. Scholar, D. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The used to the concentration at 280 nm was D. J. Full Text Full Text PDF PubMed Scopus Google Scholar). measurements were with proteins on supported The were by Fujimoto I. Rutishauser U. D. U. Scholar, D. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, D. J. Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. 24: PubMed Scopus Google Scholar). were by a of the at the of a The were to a surface pressure at temperature, and the surface of a D. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, D. J. Full Text Full Text PDF PubMed Scopus Google Scholar). The was the from the at a of The of and of was at which to The supported were then for at in a and concentrations of the The protein was to the the the were with to protein, and the supported were then in the surface force For protein the supported were with and PSA-NCAM or with NCAM and at to the PSA-NCAM:cadherin and surface forces were between two protein as a of the intermembrane with a The the force between the of two of radius J. J. 1992; Scopus Google Scholar, D. Annu. Rev. 2000; PubMed Scopus Google Scholar). in the the the force between the by the is as a of the distance with a of J. J. 1992; Scopus Google Scholar, J. J. Scopus Google Scholar). measurements quantify the between the with a of nm J. J. Scopus Google Scholar). In this the to the distance between the of the Fujimoto I. Rutishauser U. D. U. Scholar, D. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, D. J. Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. 24: PubMed Scopus Google Scholar). The radius is also quantified from the J. J. Scopus Google Scholar). of this is that for where the radius is the range of the force between the is to the energy between two by the Scopus Google Scholar). the a which the magnitude of the The force is a of the This result is in J. and Scholar). The adhesion energy is to the force to the or the by the to K. R. Scholar). of the surface of proteins on the supported the amount of protein, to D. 1997; 13: Scopus Google Scholar, T Scopus Google Scholar). measurements were with NCAM and PSA-NCAM as as with and PSA-NCAM:cadherin at ratios. For the measurements with the protein one of the protein was with and both proteins were with the supported measurements with were with NCAM and then with quantify the surface of NCAM, PSA-NCAM and cadherin from of two proteins at other quantified the fluorescence on the supported one of the proteins and the fluorescence from from the protein the in the surface from the in the The surface of proteins from and PSA-NCAM:cadherin were then by the in to protein from protein For example, a increase in the fluorescence from NCAM would a NCAM The rhodamine used does not and the were the of the the is to with the protein the fluorescence measurements quantify in fluorescence the fluorescence the amount of protein For example, if the surface of NCAM from a is then a increase in the fluorescence would to a surface of The proteins were with rhodamine to the The of to protein was at a of This a of for NCAM, for and for images were using a on a from which was to a For a supported a protein as was in a cell and on the The fluorescence was by the from a of images from regions of the supported of the in were quantified by in the fluorescence as a small The of the using an the of the to Y. PubMed Scopus Google Scholar, where is the and is the in the number of in the volume at Y. PubMed Scopus Google Scholar, J. and T. Scholar). to the of be as the and are the of the in the and is the number of and is the of the quantify in the hydrodynamic of NCAM and PSA-NCAM from their NCAM and PSA-NCAM were with as The protein was from by as and was at The for the and the for the of data are at the for at the of at Y. PubMed Scopus Google Scholar, J. and T. Scholar). the a by an was used as a were using an and the was a data was used with an The PSA-NCAM and NCAM concentrations used in the were were to the by the results to an that a Y. PubMed Scopus Google Scholar, J. and T. Scholar). The depends on the and be of in was used both for and as a for determining the size of the quantify the hydrodynamic of NCAM and measurements were using measurements were with or least of the data to an of for PSA-NCAM and for the NCAM. a the hydrodynamic radius a using the is the kB is to the is the and a is the hydrodynamic this and the the of the two hydrodynamic is PSA doubles the hydrodynamic radius of NCAM in light scattering studies of PSA-NCAM from neural cells a of (5Yang P. Major D. Yin X. Rutishauser U. J. Biol. Chem. 1994; 269: 23039-23044Abstract Full Text PDF PubMed Google Scholar). surface of NCAM, and cadherin were with The surface of on membranes and was These results are in I. In studies with the surface was at In from a of the NCAM surface was the cadherin surface was measurements of from a PSA-NCAM:cadherin surface of 0.5 and in a of protein the of the proteins from in of of in surface NCAM cadherin with with with by fluorescence with with by fluorescence by fluorescence with by fluorescence Open in a were used to quantify the protein at other of the from on from a increase in the fluorescence the was from to the by the NCAM surface from measurements an NCAM in the of The was with In this the fluorescence by the amount of cadherin in from to the cadherin on from the the cadherin surface is 0.5 measurements with and show that the was at the concentration The surface on the resulting was 0.5 The fluorescence the of cadherin from with and ratios. The surface increased by The results are in I. PSA NCAM force measurements of the homophilic NCAM adhesion that NCAM two Fujimoto I. Rutishauser U. D. U. 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Biol. Chem. Full Text PDF PubMed Google Scholar). requires a of acid residues for a small J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the force measurements with PSA-NCAM These force are to with NCAM the magnitude of the repulsion was and the steric repulsion at nm to the is the of NCAM adhesion at both and that the magnitude of adhesion at nm is between NCAM Fujimoto I. Rutishauser U. D. U. Scholar). The adhesion at nm was and for and NCAM, was not a between the at nm showed that the molecular of the PSA-NCAM was the same as the protein, PSA The adhesion at nm is to the of the J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). PSA is and that the of polyelectrolytes in the are by (7Pincus P. Macromolecules. 1991; 24: 2912-2919Crossref Scopus (891) Google Scholar). The of the of surface-anchored and the range of the repulsion between with where is the concentration of monovalent (7Pincus P. Macromolecules. 1991; 24: 2912-2919Crossref Scopus (891) Google Scholar). the ionic strength would reduce the hydrodynamic radius of PSA and reduce the range and magnitude of the intermembrane repulsion. the repulsive and attractive forces between the NCAM are then the intermembrane adhesion increase with the repulsion. the forces between PSA-NCAM in m the between the PSA-dependent the of and the ionic the steric repulsion between PSA-NCAM in m to the repulsive force with In m both the range and magnitude of the repulsion are and the repulsive from to the separation of the proteins the adhesion at with a magnitude of This in the with the in the adhesion at high ionic strength, further supports the view that PSA by increasing the intermembrane repulsion (1Bruses J.L. Rutishauser U. Biochimie (Paris). 2001; 83: 635-643Crossref PubMed Scopus (195) Google Scholar, U. Curr. Opin. Cell Biol. 1996; 8: 679-684Crossref PubMed Scopus (146) Google Scholar, I. J.L. Rutishauser U. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). the studies with the adhesive at nm was not detected in m the excluded volume of the carbohydrate is in m the polymer a large repulsion at nm to the studies were also with in the the range of the repulsion to and the of the repulsion increased to The magnitude of the repulsive force is also at the ionic in to measurements with was in the that the and separation by of NCAM and of intercellular is by one of adhesion and NCAM polysialylation the adhesive of other proteins on cell membranes I. J.L. Rutishauser U. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). measurements between membranes of PSA-NCAM and were to the molecular of the impact of PSA on the of were also with of NCAM and for the between protein NCAM and cadherin at a of the repulsion increases at 40 and then increases at This from between NCAM Fujimoto I. Rutishauser U. D. U. or cadherin D. J. 2001; Full Text Full Text PDF PubMed Scopus Google separation from the at nm This distance one of the between D. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, D. J. Full Text Full Text PDF PubMed Scopus Google Scholar). The magnitude of the adhesion was NCAM at and the adhesion at nm is to homophilic cadherin between protein cadherin D. J. 2001; Full Text Full Text PDF PubMed Scopus Google Open in a for adhesion at other intermembrane force were from where the of both proteins this which is in Fujimoto I. Rutishauser U. D. U. Scholar, D. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, D. J. 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J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). force at nm detected a adhesive at nm This distance the same as the of the of both cadherin 40 and NCAM The adhesion at nm is to the of both The magnitude of the adhesion was other adhesive were The of the adhesion on the protein was in a of force measurements with an of in the in this from of NCAM or cadherin The force detected at the same However, in this the adhesion at nm from to because of the cadherin The adhesion at and nm because the in adhesion is by the increased NCAM and by the number of NCAM PSA-NCAM force measurements between of PSA-NCAM and at a are in The The range and magnitude of the repulsion are between the The of repulsion at and the repulsive force is at is also adhesion at distance The magnitude of the force at and 40 nm is and the repulsive force the attractive forces of both cadherin and NCAM and is sufficient to the protein-mediated adhesion. the of adhesion is because of the effect of steric repulsion and protein then PSA-NCAM should reduce the magnitude of the repulsion and increase the attraction. measurements with a PSA-NCAM:cadherin the PSA-NCAM surface the repulsive as the magnitude of the repulsion the protein attractive and was adhesion was between the and force and this to some protein However, between from a PSA-NCAM:cadherin the range and magnitude of the repulsion were and the protein at and nm The magnitude of adhesion at nm was and the magnitude of the adhesion at nm was was adhesion at nm the between ionic strength and PSA in cadherin force measurements were with m In to the measurements with PSA-NCAM:cadherin in the range and magnitude of the repulsive force are the of the repulsion from to and the repulsive force from to two adhesive at nm and at nm was adhesion at measurements with NCAM and cadherin in m showed that high monovalent salt concentrations not of and that the adhesive at and 40 nm are because of the effects of both the cadherin was by the concentration to J. J. PubMed Scopus Google Scholar, J. 1997; PubMed Scopus Google Scholar). adhesion the is at this concentration J. J. PubMed Scopus Google Scholar). cadherin in was adhesion at and 40 However, the proteins at with an adhesion of in a the adhesion at nm was to In the of cadherin this to between NCAM Fujimoto I. Rutishauser U. D. U. Scholar). The adhesion at 40 nm also to and is to homophilic NCAM In measurements between PSA-NCAM:cadherin in m and the at nm was The adhesion at nm to and the evidence for at 40 nm was the in the These findings at the both cadherin and NCAM to the adhesion at and 40 of the force between PSA-NCAM in the the PSA The salt not the ionic strength, because is the The effect on the force The force measurements for the proposed mechanism for the of NCAM-mediated adhesion by PSA (6Rutishauser U. Curr. Opin. Cell Biol. 1996; 8: 679-684Crossref PubMed Scopus (146) Google Scholar). This that the of attractive protein forces and repulsive steric forces the separation between cells. For PSA to NCAM adhesion by increasing the repulsive force between the force balance requires that the magnitude of the repulsion the NCAM attraction at both membrane at which NCAM These measurements show quantitatively that the of NCAM substantially increases the magnitude of the repulsive pressure between membranes, sufficient to both and adhesion. The increased excluded volume of NCAM was by both and force the latter demonstrated the impact of this on protein-mediated intermembrane adhesion. PSA has a effect on adhesion including For PSA to other adhesion the magnitude of the steric repulsion would also have to be large to for example, cadherin at cadherin The quantified magnitude of the steric repulsion at and 40 the cadherin this with a of the repulsion the cadherin and adhesion. The of NCAM adhesion at nm the of the of the PSA However, it is to that the effects of be because of steric repulsion between or to polymer repulsion between the NCAM adhesion at nm requires the on would likely this by steric repulsion. However, the force would not the and this is the if this attenuation is because of repulsion from then the in PSA that would the repulsion from to For at a in the repulsion with the of the of (7Pincus P. Macromolecules. 1991; 24: 2912-2919Crossref Scopus (891) Google Scholar), where and are the magnitudes of the repulsive forces at nm and PSA The of PSA is to be J.L. J. Biol. Chem. Full Text PDF PubMed Google and that it could be as as P. I. U. and D. then the of of the would this is a the PSA-NCAM does not a molecular range with this amount of This that the adhesion is likely because of steric repulsion by the is to that this PSA-dependent repulsion is a of the and is independent of the of other proteins on the membrane. For this the repulsive pressure between the PSA on membranes have a effect on the of other proteins to intermembrane adhesion at membrane the range of the steric repulsion. The ionic strength of the range and magnitude of the intermembrane repulsion further evidence that PSA cell adhesion large excluded These data show that increasing the ionic strength decreases the steric of This in turn the intermembrane and the intercellular adhesion. The repulsion between also with the of the (7Pincus P. Macromolecules. 1991; 24: 2912-2919Crossref Scopus (891) Google Scholar). The of PSA on homophilic cadherin adhesion depends on the of PSA-NCAM to cadherin on the and on the balance between the steric repulsion and cadherin attraction. this is a with the magnitude of cadherin adhesion increasing with PSA-NCAM and with the in the intermembrane repulsion. the PSA-NCAM:cadherin the which was by at was a protein attraction was as adhesion at a of This correlation between the PSA-NCAM the magnitude of the and the adhesion further supports the that the impact of PSA is to the range and magnitude of the associated intermembrane repulsion. These findings show quantitatively that the magnitude of PSA-dependent repulsion with the homophilic adhesion by NCAM and is to that the polymer a are data that PSA or the steric by the excluded volume could also NCAM This be in