Single molecule adhesion measurements reveal two homophilic neural cell adhesion molecule bonds with mechanically distinct properties

Neural cell adhesion molecule (NCAM) is a cell surface adhesion glycoprotein that plays an important role in the development and stability of nervous tissue. The homophilic binding mechanism of NCAM is still a subject of debate on account of findings that appear to support different mechanisms. This paper describes single molecule force measurements with both full-length NCAM and NCAM mutants that lack different immunoglobulin (Ig) domains. By systematically applying an external, time-dependent force to the bond, we obtained parameters that describe the energy landscape of NCAM-NCAM bonds. Histograms of the rupture forces between the full-length NCAM extracellular domains revealed two binding events, one rupturing at higher forces than the other. These bond rupture data show that the two bonds have the same dissociation rates. Despite the energetic and kinetic similarities, the bond strengths differ significantly, and are mechanically distinct. Measurements with NCAM domain deletion mutants mapped the weaker bond to the Ig1-2 segment, and the stronger bond to the Ig3 domain. Finally, the quantitative agreement between the fragment adhesion and the strengths of both NCAM bonds shows that the domain deletions considered in this study do not alter the intrinsic strengths of either of the two bonds.     

Structure and interactions of NCAM Ig1-2-3 suggest a novel zipper mechanism for homophilic adhesion

The neural cell adhesion molecule, NCAM, mediates Ca(2+)-independent cell-cell and cell-substratum adhesion via homophilic (NCAM-NCAM) and heterophilic (NCAM-non-NCAM molecules) binding. NCAM plays a key role in neural development, regeneration, and synaptic plasticity, including learning and memory consolidation. The crystal structure of a fragment comprising the three N-terminal Ig modules of rat NCAM has been determined to 2.0 A resolution. Based on crystallographic data and biological experiments we present a novel model for NCAM homophilic binding. The Ig1 and Ig2 modules mediate dimerization of NCAM molecules situated on the same cell surface (cis interactions), whereas the Ig3 module mediates interactions between NCAM molecules expressed on the surface of opposing cells (trans interactions) through simultaneous binding to the Ig1 and Ig2 modules. This arrangement results in two perpendicular zippers forming a double zipper-like NCAM adhesion complex.     

A peptide mimetic targeting trans-homophilic NCAM binding sites promotes spatial learning and neural plasticity in the hippocampus

The key roles played by the neural cell adhesion molecule (NCAM) in plasticity and cognition underscore this membrane protein as a relevant target to develop cognitive-enhancing drugs. However, NCAM is a structurally and functionally complex molecule with multiple domains engaged in a variety of actions, which raise the question as to which NCAM fragment should be targeted. Synthetic NCAM mimetic peptides that mimic NCAM sequences relevant to specific interactions allow identification of the most promising targets within NCAM. Recently, a decapeptide ligand of NCAM--plannexin, which mimics a homophilic trans-binding site in Ig2 and binds to Ig3--was developed as a tool for studying NCAM's trans-interactions. In this study, we investigated plannexin's ability to affect neural plasticity and memory formation. We found that plannexin facilitates neurite outgrowth in primary hippocampal neuronal cultures and improves spatial learning in rats, both under basal conditions and under conditions involving a deficit in a key plasticity-promoting posttranslational modification of NCAM, its polysialylation. We also found that plannexin enhances excitatory synaptic transmission in hippocampal area CA1, where it also increases the number of mushroom spines and the synaptic expression of the AMPAR subunits GluA1 and GluA2. Altogether, these findings provide compelling evidence that plannexin is an important facilitator of synaptic functional, structural and molecular plasticity in the hippocampal CA1 region, highlighting the fragment in NCAM's Ig3 module where plannexin binds as a novel target for the development of cognition-enhancing drugs.     

Unfolding of titin immunoglobulin domains by steered molecular dynamics simulation.

Titin, a 1-microm-long protein found in striated muscle myofibrils, possesses unique elastic and extensibility properties in its I-band region, which is largely composed of a PEVK region (70% proline, glutamic acid, valine, and lysine residue) and seven-strand beta-sandwich immunoglobulin-like (Ig) domains. The behavior of titin as a multistage entropic spring has been shown in atomic force microscope and optical tweezer experiments to partially depend on the reversible unfolding of individual Ig domains. We performed steered molecular dynamics simulations to stretch single titin Ig domains in solution with pulling speeds of 0.5 and 1.0 A/ps. Resulting force-extension profiles exhibit a single dominant peak for each Ig domain unfolding, consistent with the experimentally observed sequential, as opposed to concerted, unfolding of Ig domains under external stretching forces. This force peak can be attributed to an initial burst of backbone hydrogen bonds, which takes place between antiparallel beta-strands A and B and between parallel beta-strands A' and G. Additional features of the simulations, including the position of the force peak and relative unfolding resistance of different Ig domains, can be related to experimental observations.     

Backbone dynamics of the first, second, and third immunoglobulin modules of the neural cell adhesion molecule (NCAM)

The neural cell adhesion molecule (NCAM) is a cell surface multimodular protein, which plays an important role in cell-cell adhesion by homophilic (NCAM-NCAM) and heterophilic (NCAM-non-NCAM molecules) binding. In the present study, the backbone dynamics of the first three immunoglobulin-like (Ig) modules of NCAM have been investigated by NMR spectroscopy. Ig1, Ig2, and Ig3 share low sequence identity but possess the same fold and have very similar three-dimensional structures. (15)N longitudinal and transverse relaxation rates and heteronuclear NOEs have been measured and subsequently analyzed by the axial symmetric Lipari-Szabo modelfree formalism to characterize fast (pico- to nanosecond) and slow (micro- to millisecond) motions in the three protein modules. We found that backbone motions of residues located in the beta-strand regions are generally restricted, while increased flexibility is observed in turns and loops. In all three modules, residues located in the segments connecting the C- and D-strand plus residues located in the segment connecting the E- and F-strand show significant chemical exchange on the micro- to millisecond time scale. In addition, a number of residues with small chemical exchange contribution seem to form contiguous regions in the beta sheets, suggesting that these motions might be correlated. Only few residues in the homophilic binding sites in the NCAM Ig1 and Ig2 modules show increased flexibility, indicating that the Ig1-Ig2-mediated NCAM homophilic binding does not depend on the local backbone mobility of the interacting modules.     

The cytosolic domain of protein-tyrosine kinase 7 (PTK7), generated from sequential cleavage by a disintegrin and metalloprotease 17 (ADAM17) and γ-secretase, enhances cell proliferation and migration in colon cancer cells

Protein-tyrosine kinase 7 (PTK7) is a member of the defective receptor protein-tyrosine kinases and is known to function as a regulator of planar cell polarity during development. Its expression is up-regulated in some cancers including colon carcinomas. A 100-kDa fragment of PTK7 was detected in the culture media from colon cancer cells and HEK293 cells. The shed fragment was named sPTK7-Ig1-7 because its molecular mass was very similar to that of the entire extracellular domain of PTK7 that contains immunoglobulin-like loops 1 to 7 (Ig1-7). The shedding of sPTK7-Ig1-7 was enhanced by treatment with phorbol 12-myristate 13-acetate. In addition to the sPTK7-Ig1-7 found in the culture medium, two C-terminal fragments of PTK7 were detected in the cell lysates: PTK7-CTF1, which includes a transmembrane segment and a cytoplasmic domain, and PTK7-CTF2, which lacks most of the transmembrane segment from PTK7-CTF1. Analysis of PTK7 processing in the presence of various protease inhibitors or after knockdown of potential proteases suggests that shedding of PTK7 into sPTK7-Ig1-7 and PTK7-CTF1 is catalyzed by ADAM17, and further cleavage of PTK7-CTF1 into PTK7-CTF2 is mediated by the γ-secretase complex. PTK7-CTF2 localizes to the nucleus and enhances proliferation, migration, and anchorage-independent colony formation. Our findings demonstrate a novel role for PTK7 in the tumorigenesis via generation of PTK7-CTF2 by sequential cleavage of ADAM17 and γ-secretase.     

Interaction between Src homology 2 domain bearing protein tyrosine phosphatase substrate-1 and CD47 mediates the adhesion of human B lymphocytes to nonactivated endothelial cells

CD47 modulates a variety of cell functions such as adhesion, spreading, and migration. Using a fusion protein consisting of the extracellular region of Src homology 2 domain bearing protein tyrosine phosphatase substrate-1 (SHPS-1) and the Fc portion of human Ig (SHPS-1-Ig) we investigated the effects of SHPS-1 as a ligand for CD47 on B lymphocytes. Although SHPS-1-Ig binding to human B cell lines was solely mediated via CD47, their binding capacity for soluble and immobilized SHPS-1-Ig varied among cell lines irrespective of the similar expression levels of CD47, suggesting that distinctive affinity/avidity states exist during B cell maturation. Nalm6 cell line and tonsilar B lymphocytes adhered to immobilized SHPS-1-Ig and showed polarization-like morphology. These effects of SHPS-1-Ig were blocked by anti-CD47 mAbs (B6H12 and SE5A5). Wortmannin, a phosphatidylinositol-3 kinase inhibitor, but not pertussis toxin significantly inhibited the polarization induced by the immobilized SHPS-1-Ig. Thus, SHPS-1 acts as an adhesive substrate via CD47 in human B lymphocyte. Immunohistochemical analyses indicated that SHPS-1 is expressed on high endothelial venule as well as macrophages in human tonsils. HUVECs also express SHPS-1 in the absence of any stimuli, and the adhesion of tonsilar B lymphocytes to nonactivated HUVECs was significantly inhibited by SE5A5, indicating that SHPS-1/CD47 interaction is involved in the adhesion. Our findings suggest that SHPS-1/CD47 interaction may contribute to the recruitment of B lymphocytes via endothelial cells under steady state conditions.     

PC12 cells utilize the homophilic binding site of L1 for cell-cell adhesion but L1-alphavbeta3 interaction for neurite outgrowth

Treatment of PC12 cells with nerve growth factor induces their differentiation into sympathetic neuron-like cells and the concomitant expression of the neural cell adhesion molecule L1, a member of the Ig superfamily. To investigate the mechanism of L1-stimulated neurite outgrowth in PC12 cells, substrate-immobilized fusion proteins containing different extracellular domains of L1 were assayed for their neuritogenic activity. Surprisingly, domain Ig2 of L1, which was previously found to contain both homophilic binding and neuritogenic activities, failed to promote neurite outgrowth. In contrast, L1-Ig6 stimulated neurite outgrowth from PC12 cells. Despite this, homotypic binding of PC12 cells was significantly inhibited by antibodies against L1-Ig2, indicating that L1-L1 binding contributed to the intercellular adhesiveness of PC12 cells, but L1-stimulated neurite outgrowth depends on heterophilic interactions. Thus, PC12 cells provide a valuable model for the study of these two distinct functions of L1. Mutagenesis of L1-Ig6 highlighted the importance of the Arg-Gly-Asp motif in this domain for neuritogenesis. Inhibition studies using cyclic Arg-Gly-Asp-containing peptide and anti-integrin antibodies suggested the involvement of alphavbeta3 integrin. Furthermore, neurite outgrowth stimulated by L1-Ig6 was inhibited by lavendustin A and the MEK inhibitor PD98059, suggesting a signaling pathway that involves tyrosine kinase activation and the mitogen-activated protein kinase cascade.     

Molecular mechanics of cardiac titin's PEVK and N2B spring elements.

Titin is a giant elastic protein that is responsible for the majority of passive force generated by the myocardium. Titin's force is derived from its extensible I-band region, which, in the cardiac isoform, comprises three main extensible elements: tandem Ig segments, the PEVK domain, and the N2B unique sequence (N2B-Us). Using atomic force microscopy, we characterized the single molecule force-extension curves of the PEVK and N2B-Us spring elements, which together are responsible for physiological levels of passive force in moderately to highly stretched myocardium. Stretch-release force-extension curves of both the PEVK domain and N2B-Us displayed little hysteresis: the stretch and release data nearly overlapped. The force-extension curves closely followed worm-like chain behavior. Histograms of persistence length (measure of chain bending rigidity) indicated that the single molecule persistence lengths are approximately 1.4 and approximately 0.65 nm for the PEVK domain and N2B-Us, respectively. Using these mechanical characteristics and those determined earlier for the tandem Ig segment (assuming folded Ig domains), we modeled the cardiac titin extensible region in the sarcomere and calculated the extension of the various spring elements and the forces generated by titin, both as a function of sarcomere length. In the physiological sarcomere length range, predicted values and those obtained experimentally were indistinguishable.     

Titin Extensibility In Situ: Entropic Elasticity of Permanently Folded and Permanently Unfolded Molecular Segments

Abstract. Titin (also known as connectin) is a giant protein that spans half of the striated muscle sarcomere. In the I-band titin extends as the sarcomere is stretched, developing what is known as passive force. The I-band region of titin contains tandem Ig segments (consisting of serially linked immunoglobulin-like domains) with the unique PEVK segment in between (Labeit, S., and B. Kolmerer. 1995. Science. 270:293–296). Although the tandem Ig and PEVK segments have been proposed to behave as stiff and compliant springs, respectively, precise experimental testing of the hypothesis is still needed. Here, sequence-specific antibodies were used to mark the ends of the tandem Ig and PEVK segments. By following the extension of the segments as a function of sarcomere length (SL), their respective contributions to titin's elastic behavior were established. In slack sarcomeres (~2.0 μm) the tandem Ig and PEVK segments were contracted. Upon stretching sarcomeres from ~2.0 to 2.7 μm, the “contracted” tandem Ig segments straightened while their individual Ig domains remained folded. When sarcomeres were stretched beyond ~2.7 μm, the tandem Ig segments did not further extend, instead PEVK extension was now dominant. Modeling tandem Ig and PEVK segments as entropic springs with different bending rigidities (Kellermayer, M., S. Smith, H. Granzier, and C. Bustamante. 1997. Science. 276:1112–1116) indicated that in the physiological SL range (a) the Ig-like domains of the tandem Ig segments remain folded and (b) the PEVK segment behaves as a permanently unfolded polypeptide. Our model provides a molecular basis for the sequential extension of titin's different segments. Initially, the tandem Ig segments extend at low forces due to their high bending rigidity. Subsequently, extension of the PEVK segment occurs only upon reaching sufficiently high external forces due to its low bending rigidity. The serial linking of tandem Ig and PEVK segments with different bending rigidities provides a unique passive force–SL relation that is not achievable with a single elastic segment.     

Crystal structure of the Ig1 domain of the neural cell adhesion molecule NCAM2 displays domain swapping

The crystal structure of the first immunoglobulin (Ig1) domain of neural cell adhesion molecule 2 (NCAM2/OCAM/RNCAM) is presented at a resolution of 2.7 Å. NCAM2 is a member of the immunoglobulin superfamily of cell adhesion molecules (IgCAMs). In the structure, two Ig domains interact by domain swapping, as the two N-terminal β-strands are interchanged. β-Strand swapping at the terminal domain is the accepted mechanism of homophilic interactions amongst the cadherins, another class of CAMs, but it has not been observed within the IgCAM superfamily. Gel-filtration chromatography demonstrated the ability of NCAM2 Ig1 to form dimers in solution. Taken together, these observations suggest that β-strand swapping could have a role in the molecular mechanism of homophilic binding for NCAM2.     

Identification of a neuritogenic ligand of the neural cell adhesion molecule using a combinatorial library of synthetic peptides.

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and learning. In this study, we identified a synthetic peptide-ligand of the NCAM Ig1 module by combinatorial chemistry and showed it could modulate NCAM-mediated cell adhesion and signal transduction with high potency. In cultures of dissociated neurons, this peptide, termed C3, stimulated neurite outgrowth by activating a signaling pathway identical to that activated by homophilic NCAM binding. A similar effect was shown for the NCAM Ig2 module, the endogenous ligand of NCAM Ig1. By nuclear magnetic resonance spectroscopy, the C3 binding site in the NCAM Ig1 module was mapped and shown to be different from the binding site of the NCAM Ig2 module. The C3 peptide may prove useful as a lead in development of therapies for neurodegenerative disorders, and the C3 binding site of NCAM Ig1 may represent a target for discovery of nonpeptide drugs.     

The fourth immunoglobulin-like domain of NCAM contains a carbohydrate recognition domain for oligomannosidic glycans implicated in association with L1 and neurite outgrowth

We have previously shown that the neural adhesion molecules L1 and NCAM interact with each other to form a complex which binds more avidly to L1 than L1 to L1 alone (Kadmon, G., A. Kowitz, P. Altevogt, and M. Schachner. 1990a. J. Cell Biol. 110:193-208). This cis-association between L1 and NCAM is carbohydrate-dependent (Kadmon, G., A. Kowitz, P. Altevogt, and M. Schachner. 1990b. J. Cell Biol. 110:209-218). In the present study, we report that L1 and NCAM bind to each other via oligomannosidic carbohydrates expressed by L1, but not by NCAM, as shown in several experiments: (a) complex formation between L1 and NCAM is inhibited by a mAb to oligomannosidic carbohydrates and by the oligosaccharides themselves; (b) NCAM binds to oligomannosidic carbohydrates; (c) within the L1/NCAM complex, the oligomannosidic carbohydrates are hidden from accessibility to a mAb against oligomannosidic carbohydrates; (d) the recombinant protein fragment of NCAM containing the immunoglobulin-like domains and not the fragment containing the fibronectin type III homologous repeats binds to oligomannosidic glycans. Furthermore, the fourth immunoglobulin-like domain of NCAM shows sequence homology with carbohydrate recognition domains of animal C-type lectins and, surprisingly, also with plant lectins. A peptide comprising part of the C-type lectin consensus sequence in the fourth immunoglobulin-like domain of NCAM interferes with the association between L1 and NCAM. The functional importance of oligomannosidic glycans at the cell surface was shown for neurite outgrowth in vitro. When neurons from early postnatal mouse cerebellum were maintained on laminin or poly-L-lysine, neurite outgrowth was inhibited by oligomannosidic glycans, by glycopeptides, glycoproteins, or neoglycolipids containing oligomannosidic glycans, but not by nonrelated oligosaccharides or oligosaccharide derivates. Neurite outgrowth was also inhibited by the peptide comprising part of the C- type lectin consensus sequence in the fourth immunoglobulin-like domain of NCAM. The combined results suggest that carbohydrate-mediated cis- associations between adhesion molecules at the cell surface modulate their functional properties.     

Extended flexible linker structures in the complement chimaeric conjugate CR2-Ig by scattering, analytical ultracentrifugation and constrained modelling: implications for function and therapy

Complement receptor 2 (CR2; CD21) is a membrane-bound regulator of complement activation, being comprised of 15 or 16 short complement repeat (SCR) domains. A recombinant glycosylated human CR2 SCR 1-2 domain pair was engineered with the Fc fragment of a mouse IgG1 antibody to create a chimaera CR2-Ig containing the major ligand binding domains. Such a chimaera has therapeutic potential as a complement inhibitor or immune modulator. X-ray and neutron scattering and analytical ultracentrifugation identified its domain structure in solution, and provided a comparison with controversial folded-back crystal structures for deglycosylated CR2 SCR 1-2. The radius of gyration R(G) of CR2-Ig was determined to be 5.39(+/-0.14) nm and 5.29(+/-0.01) nm by X-ray and neutron scattering, respectively. The maximum dimension of CR2-Ig was determined to be 17 nm. The molecular mass of CR2-Ig ranged between 101,000 Da and 107,000 Da as determined by neutron scattering and sedimentation equilibrium, in good agreement with the sequence-derived value of 106,600 Da. Sedimentation velocity gave a sedimentation coefficient of 4.49(+/-0.11) S. Stereochemically complete models for CR2-Ig were constructed from crystal structures for the CR2 SCR 1-2 and mouse IgG1 Fc fragments. The two SCR domains and the Fc fragment were joined by randomised conformational peptides. The analysis of 35,000 possible CR2-Ig models showed that only those models in which the two SCR domains were arranged in an open V-shape in random orientations about the Fc fragment accounted for the scattering and sedimentation data. It was not possible to define one single conformational family of Fab-like fragment relative to the Fc fragment. This flexibility is attributed to the relatively long linker sequence and the absence of the antibody light chain from CR2-Ig. The modelling also confirmed that the structure of CR2 SCR 1-2 is more extended in solution than in its crystal structure.     

Different molecular mechanics displayed by titin's constitutively and differentially expressed tandem Ig segments

Titin is a giant elastic protein responsible for passive force generated by the stretched striated-muscle sarcomere. Passive force develops in titin's extensible region which consists of the PEVK segment in series with tandemly arranged immunoglobulin (Ig)-like domains. Here we studied the mechanics of tandem Ig segments from the differentially spliced (I65-70) and constitutive (I91-98) regions by using an atomic force microscope specialized for stretching single molecules. The mechanical stability of I65-70 domains was found to be different from that of I91-98 domains. In the range of stretch rates studied (0.05-1.00 microm/s) lower average domain unfolding forces for I65-70 were associated with a weaker stretch-rate dependence of the unfolding force, suggesting that the differences in the mechanical stabilities of the segments derive from differences in the zero force unfolding rate (K(0)(u)) and the characteristic distance (location of the barrier) along the unfolding reaction coordinate (DeltaX(u)). No effect of calcium was found on unfolding forces and persistence length of unfolded domains. To explore the structural basis of the differences in mechanical stabilities of the two fragment types, we compared the amino acid sequence of I65-70 domains with that of I91-98 domains and by using homology modeling analyzed how sequence variations may affect folding free energies. Simulations suggest that differences in domain stability are unlikely to be caused by variation in the number of hydrogen bonds between the force-bearing beta-strands at the domain's N- and C-termini. Rather, they may be due to differences in hydrophobic contacts and strand orientations.     

CD84 functions as a homophilic adhesion molecule and enhances IFN-gamma secretion: adhesion is mediated by Ig-like domain 1

CD84 is a member of the CD2 subset of the Ig superfamily of cell surface molecules. Its cytoplasmic tail binds to Src homology 2 domain-containing protein 1A (signaling lymphocytic activation molecule-associated protein), a protein encoded by the X-linked lymphoproliferative disease gene. It is preferentially expressed on B lymphocytes, monocytes, and platelets. We show that it is also expressed on thymocytes and T cells. CD84 was positive on CD4-CD8- thymocytes, and its expression decreased with cell maturation. It is expressed on mature T cells preferentially on CD45RO+. To identify the CD84 ligand, we generated a soluble Ig fusion protein containing the human CD84 extracellular domains (CD84-Ig). Because receptor-ligand interactions occur between several members of this subfamily, we assayed CD84-Ig binding with all members of the CD2 family. CD84-Ig bound to CD84-transfected cells, whereas no binding was detected with cells expressing other CD2 subfamily receptors, showing that CD84 binds to itself. Anti-CD84 mAbs recognizing epitopes wholly within domain 1 of CD84 blocked the binding of the CD84-Ig fusion protein to CD84-transfected cells and platelets. Data from CD84 domain human/mouse chimeras further revealed that only the first extracellular domain of the molecule is involved in the ligand receptor recognition. The CD84-CD84 interaction was independent of its cytoplasmic tail. Finally, concurrent ligation of human CD84 with mAbs or CD84-Ig and CD3 enhanced IFN-gamma secretion in human lymphocytes. Thus, CD84 is its own ligand and acts as a costimulatory molecule.     

The retinal specific CD147 Ig0 domain: from molecular structure to biological activity

CD147 is a type I transmembrane protein that is involved in inflammatory diseases, cancer progression, and multiple human pathogens utilize CD147 for efficient infection. CD147 expression is so high in several cancers that it is now used as a prognostic marker. The two primary isoforms of CD147 that are related to cancer progression have been identified, differing in their number of immunoglobulin (Ig)-like domains. These include CD147 Ig1-Ig2, which is ubiquitously expressed in most tissues, and CD147 Ig0-Ig1-Ig2, which is retinal specific and implicated in retinoblastoma. However, little is known in regard to the retinal specific CD147 Ig0 domain despite its potential role in retinoblastoma. We present the first crystal structure of the human CD147 Ig0 domain and show that the CD147 Ig0 domain is a crystallographic dimer with an I-type domain structure, which maintained in solution. Furthermore, we have utilized our structural data together with mutagenesis to probe the biological activity of CD147-containing proteins, both with and without the CD147 Ig0 domain, within several model cell lines. Our findings reveal that the CD147 Ig0 domain is a potent stimulator of interleukin-6 and suggest that the CD147 Ig0 domain has its own receptor distinct from that of the other CD147 Ig-like domains, CD147 Ig1-Ig2. Finally, we show that the CD147 Ig0 dimer is the functional unit required for activity and can be disrupted by a single point mutation.     

Triple effect of mimetic peptides interfering with neural cell adhesion molecule homophilic cis interactions

The neural cell adhesion molecule (NCAM) is pivotal in neural development, regeneration, and learning. Here we characterize two peptides, termed P1-B and P2, derived from the homophilic binding sites in the first two N-terminal immunoglobulin (Ig) modules of NCAM, with regard to their effects on neurite extension and adhesion. To evaluate how interference of these mimetic peptides with NCAM homophilic interactions in cis influences NCAM binding in trans, we employed a coculture system in which PC12-E2 cells were grown on monolayers of fibroblasts with or without NCAM expression and the rate of neurite outgrowth subsequently was analyzed. P2, but not P1-B, induced neurite outgrowth in the absence of NCAM binding in trans. When PC12-E2 cells were grown on monolayers of NCAM-expressing fibroblasts, the effect of both P1-B and P2 on neurite outgrowth was dependent on peptide concentrations. P1-B and P2 acted as conventional antagonists, agonists, and reverse agonists of NCAM at low, intermediate, and high peptide concentrations, respectively. The demonstrated in vitro triple pharmacological effect of mimetic peptides interfering with the NCAM homophilic cis binding will be valuable for the understanding of the actions of these mimetics in vivo.     

Homophilic adhesion between Ig superfamily carcinoembryonic antigen molecules involves double reciprocal bonds

Both carcinoembryonic antigen (CEA) and neural cell adhesion molecule (NCAM) belong to the immunoglobulin supergene family and have been demonstrated to function as homotypic Ca(++)-independent intercellular adhesion molecules. CEA and NCAM cannot associate heterotypically indicating that they have different binding specificities. To define the domains of CEA involved in homotypic interaction, hybrid cDNAs consisting of various domains from CEA and NCAM were constructed and were transfected into a CHO-derived cell line; stable transfectant clones showing cell surface expression of CEA/NCAM chimeric-proteins were assessed for their adhesive properties by homotypic and heterotypic aggregation assays. The results indicate that all five of the Ig(C)-like domains of NCAM are required for intercellular adhesion while the COOH-terminal domain containing the fibronectin-like repeats is dispensable. The results also show that adhesion mediated by CEA involves binding between the Ig(V)-like amino-terminal domain and one of the Ig(C)-like internal repeat domains: thus while transfectants expressing constructs containing either the N domain or the internal domains alone were incapable of homotypic adhesion, they formed heterotypic aggregates when mixed. Furthermore, peptides consisting of both the N domain and the third internal repeat domain of CEA blocked CEA-mediated cell aggregation, thus providing direct evidence for the involvement of the two domains in adhesion. We therefore propose a novel model for interactions between immunoglobulin supergene family members in which especially strong binding is effected by double reciprocal interactions between the V-like domains and C-like domains of antiparallel CEA molecules on apposing cell surfaces.     

The minimal structural domains required for neural cell adhesion molecule polysialylation by PST/ST8Sia IV and STX/ST8Sia II

A limited number of mammalian proteins are modified by polysialic acid, with the neural cell adhesion molecule (NCAM) being the most abundant of these. We hypothesize that polysialylation is a protein-specific glycosylation event and that an initial protein-protein interaction between polysialyltransferases and glycoprotein substrates mediates this specificity. To evaluate the regions of NCAM required for recognition and polysialylation by PST/ST8Sia IV and STX/ST8Sia II, a series of domain deletion proteins were generated, co-expressed with each enzyme, and their polysialylation analyzed. A protein consisting of the fifth immunoglobulin-like domain (Ig5), which contains the reported sites of polysialylation, and the first fibronectin type III repeat (FN1) was polysialylated by both enzymes, whereas a protein consisting of Ig5 alone was not polysialylated by either enzyme. This demonstrates that the Ig5 domain of NCAM and FN1 are sufficient for polysialylation, and suggests that the FN1 may constitute an enzyme recognition and docking site. Two other NCAM mutants, NCAM-6 (Ig1-5) and NCAM-7 (FN1-FN2), were weakly polysialylated by PST/ST8Sia IV, suggesting that a weaker enzyme recognition site may exist within the Ig domains, and that glycans in the FN region are polysialylated. Further analysis indicated that O-linked oligosaccharides in NCAM-7, and O-linked and N-linked glycans in full-length NCAM, are polysialylated when these proteins are co-expressed with the polysialyltransferases in COS-1 cells. Our data support a model in which the polysialyltransferases bind to the FN1 of NCAM to polymerize polysialic acid chains on appropriately presented glycans in adjacent regions.