Homophilic adhesion of the myelin P0 protein requires glycosylation of both molecules in the homophilic pair

The myelin P0 protein is glycosylated at a single site, asparagine 93, within its only immunoglobulin (Ig)-like domain. We have previously shown that P0 behaves like a homophilic adhesion molecule (Filbin, M. T., F. S. Walsh, B. D. Trapp, J. A. Pizzey, and G. I. Tennekoon. 1990. Nature (Lond.). 344:871-872). To determine if the sugar residues of this molecule contribute to its adhesiveness, the glycosylation site was eliminated by replacing asparagine 93 with an alanine, through site- directed mutagenesis of the P0 cDNA. The mutated P0 cDNA was transfected into CHO cells and surface expression of the mutated P0 was assessed by immunofluorescence, limited trypsinization and an ELISA. A cell line was chosen which expressed approximately equivalent amounts of the unglcosylated P0 (UNGP0) at the cell surface as did a cell line expressing the fully glycosylated P0 (GPo); the adhesive properties of these two cell lines were compared. It was found that when a single cell suspension of the UNGPo cells were incubated, by 60 min, unlike the GP0 cells, they had not formed large aggregates; they were indistinguishable from the control transfected cells. This suggests that the UNGP0 protein does not behave like an adhesion molecule. To establish if only one molecule in the P0:P0 homophilic pair must be glycosylated for adhesion to occur, the ability of UNGP0 cells to adhere to GP0 cells was assessed both qualitatively and quantitatively. The results of both types of assay imply that, indeed, both P0 molecules in the homophilic pair must be glycosylated for adhesion to take place.     

Functional cooperation between the neural adhesion molecules L1 and N- CAM is carbohydrate dependent

The neural cell adhesion molecules L1 and N-CAM have been suggested to interact functionally by formation of a complex between the two molecules (Kadmon, G., A. Kowitz, P. Altevogt, and M. Schachner. 1990. J. Cell Biol. 110:193-208). To determine the molecular mechanisms underlying this functional cooperation, we have studied the contribution of carbohydrates to the association of the two molecules at the cell surface. Aggregation or adhesion between L1- and N-CAM-positive neuroblastoma N2A cells was reduced when the synthesis of complex and/or hybrid glycans was modified by castanospermine. Fab fragments of polyclonal antibodies to L1 inhibited aggregation and adhesion of castanospermine-treated cells almost completely, whereas untreated cells were inhibited by approximately 50%. Fab fragments of polyclonal antibodies to N-CAM did not interfere with the interaction between castanospermine-treated cells, whereas they inhibited aggregation or adhesion of untreated cells by approximately 50%. These findings indicate that cell interactions depending both on L1 and N-CAM ("assisted homophilic" binding) can be reduced to an L1-dominated interaction ("homophilic binding"). Treatment of cells with the carbohydrate synthesis inhibitor swainsonine did not modify cell aggregation in the absence or presence of antibodies compared with untreated cells, indicating that castanospermine-sensitive, but swainsonine-insensitive glycans are involved. To investigate whether the appropriate carbohydrate composition is required for an association of L1 and N-CAM in the surface membrane (cis-interaction) or between L1 on one side and L1 and N-CAM on the other side of interacting partner cells (trans-interaction), an L1-positive lymphoid tumor cell line was coaggregated with and adhered to neuroblastoma cells in the various combinations of castanospermine-treated and untreated cells. The results show that it is the cis-interaction between L1 and N-CAM that depends on the appropriate carbohydrate structures.     

Induction of neuronal differentiation by a peptide corresponding to the homophilic binding site of the second Ig module of the neural cell adhesion molecule

NCAM plays a key role in neural development and plasticity-mediating cell adhesion and differentiation mainly through homophilic binding. Until recently, attempts to modulate neuronal differentiation and plasticity through NCAM have been impeded by the absence of small synthetic agonists mimicking homophilic interactions of NCAM. We show here that a peptide, P2, corresponding to a 12-amino acid sequence localized in the FG loop of the second Ig module of NCAM, binds to the first Ig module, which is the natural binding partner of the second Ig module, with an apparent K(d) of 4.7 +/- 0.9 x 10(-6) m. P2 inhibits cell aggregation and induces neurite outgrowth from hippocampal neurons, maximal neuritogenic effect being obtained at a concentration of 0.8 microm. The neuritogenic effect was inhibited by preincubation of P2 with the recombinant NCAM-IgI. Both the length of P2 and the basic amino acid residues at the N and C termini are important for its neuritogenic activity. Treatment of hippocampal cultures with P2 results in induction of phosphorylation of the mitogen-activated protein kinases ERK1 and ERK2. Thus, P2 is a potent mimetic of NCAM, and therefore, an attractive compound for the development of drugs for the treatment of neurodegenerative diseases.     

Critical role of the fifth domain of E-cadherin for heterophilic adhesion with alpha E beta 7, but not for homophilic adhesion

The integrin alpha(E)beta(7) is expressed on intestinal intraepithelial T lymphocytes and CD8(+) T lymphocytes in inflammatory lesions near epithelial cells. Adhesion between alpha(E)beta(7)(+) T and epithelial cells is mediated by the adhesive interaction of alpha(E)beta(7) and E-cadherin; this interaction plays a key role in the damage of target epithelia. To explore the structure-function relationship of the heterophilic adhesive interaction between E-cadherin and alpha(E)beta(7), we performed cell aggregation assays using L cells transfected with an extracellular domain-deletion mutant of E-cadherin. In homophilic adhesion assays, L cells transfected with wild-type or a domain 5-deficient mutant formed aggregates, whereas transfectants with domain 1-, 2-, 3-, or 4-deficient mutants did not. These results indicate that not only domain 1, but domains 2, 3, and 4 are involved in homophilic adhesion. When alpha(E)beta(7)(+) K562 cells were incubated with L cells expressing the wild type, 23% of the resulting cell aggregates consisted of alpha(E)beta(7)(+) K562 cells. In contrast, the binding of alpha(E)beta(7)(+) K562 cells to L cells expressing a domain 5-deficient mutant was significantly decreased, with alpha(E)beta(7)(+) K562 cells accounting for only 4% of the cell aggregates, while homophilic adhesion was completely preserved. These results suggest that domain 5 is involved in heterophilic adhesion with alpha(E)beta(7), but not in homophilic adhesion, leading to the hypothesis that the fifth domain of E-cadherin may play a critical role in the regulation of heterophilic adhesion to alpha(E)beta(7) and may be a potential target for treatments altering the adhesion of alpha(E)beta(7)(+) T cells to epithelial cells in inflammatory epithelial diseases.     

The cytoplasmic domain of the myelin P0 protein influences the adhesive interactions of its extracellular domain

The extracellular domain of the myelin P0 protein is believed to engage in adhesive interactions and thus hold the myelin membrane compact. We have previously shown that P0 can behave as a homophilic adhesion molecule through interactions of its extracellular domains (Filbin, M. T., F. S. Walsh, B. D. Trapp, J. A. Pizzey, and G. I. Tennekoon. 1990. Nature (Lond.) 344:871-872). To determine if the cytoplasmic domain of P0 must be intact for the extracellular domains to adhere, we compared the adhesive capabilities of P0 proteins truncated at the COOH-terminal to the full-length P0 protein. P0 cDNAs lacking nucleotides coding for the last 52 or 59 amino acids were transfected into CHO cells, and surface expression of the truncated proteins was assessed by immunofluorescence, surface labeling followed by immunoprecipitation, and an ELISA. Cell lines were chosen that expressed at least equivalent amounts of the truncated P0 proteins at the surface as did a cell line expressing the full-length P0. The adhesive properties of these three cell lines were compared. It was found that when a suspension of single cells was allowed to aggregate for a period of 60 min, only the cells expressing the full-length P0 had formed large aggregates, while the cells expressing the truncated P0 molecules were still mostly single cells indistinguishable from the control cells. Furthermore, 25-30% of the full-length P0 was insoluble in NP40, indicative of an interaction with the cytoskeleton, whereas only 5-10% of P0 lacking 52 amino acids and none of P0 lacking 59 amino acids were insoluble. These results suggest that for the extracellular domain of P0 to behave as a homophilic adhesion molecule, its cytoplasmic domain must be intact, and most probably, it is interacting with the cytoskeleton.     

The receptor tyrosine kinase ARK mediates cell aggregation by homophilic binding.

The ARK (AXL, UFO) receptor is a member of a new family of receptor tyrosine kinases whose extracellular domain contains a combination of fibronectin type III and immunoglobulin motifs similar to those found in many cell adhesion molecules. ARK mRNA is expressed at high levels in the mouse brain, prevalently in the hippocampus and cerebellum, and this pattern of expression resembles that of adhesion molecules that are capable of promoting cell aggregation through homophilic or heterophilic binding. We report here the ability of the murine ARK receptor to mediate homophilic binding. Expression of the ARK protein in Drosophila S2 cells induces formation of cell aggregates consisting of ARK-expressing cells, and aggregation leads to receptor activation, with an increase in receptor phosphorylation. Homophilic binding does not require ARK tyrosine kinase activity, since S2 cells expressing a receptor in which the intracellular domain was deleted were able to undergo aggregation as well as cells expressing the wild-type ARK receptor. Similar results were obtained with NIH 3T3 and CHO cells expressing high levels of ARK, although in this case ARK expression appeared to be accompanied by constitutive activation. The purified recombinant extracellular domain of ARK can induce homotypic aggregation of coated fluorescent beads (Covaspheres), and this protein can also function as a substrate for adhesion by S2 and NIH 3T3 cells expressing ARK. These results suggest that ARK represents a new cell adhesion molecule that through its homophilic interaction may regulate cellular functions during cell recognition.     

Glycosyl phosphatidylinositol--anchored T-cadherin mediates calcium-dependent, homophilic cell adhesion.

Cadherins are a family of cell adhesion molecules that exhibit calcium-dependent, homophilic binding. Their function depends on both an HisAlaVal sequence in the first extracellular domain, EC1, and the interaction of a conserved cytoplasmic region with intracellular proteins. T-cadherin is an unusual member of the cadherin family that lacks the HisAlaVal motif and is anchored to the membrane through a glycosyl phosphatidylinositol moiety (Ranscht, B., and M. T. Dours-Zimmermann. 1991. Neuron. 7:391-402). To assay the function of T-cadherin in cell adhesion, we have transfected T-cadherin cDNA into CHO cells. Two proteins, mature T-cadherin and the uncleaved T-cadherin precursor, were produced from T-cadherin cDNA. The T-cadherin proteins differed from classical cadherins in several aspects. First, the uncleaved T-cadherin precursor was expressed, together with mature T-cadherin, on the surface of the transfected cells. Second, in the absence of calcium, T-cadherin was more resistant to proteolytic cleavage than other cadherins. Lastly, in contrast to classical cadherins, T-cadherin was not concentrated into cell-cell contacts between transfected cells in monolayer cultures. In cellular aggregation assays, T-cadherin induced calcium-dependent, homophilic adhesion which was abolished by treatment of T-cadherin-transfected cells with phosphatidylinositol-specific phospholipase C. These results demonstrate that T-cadherin is a functional cadherin that differs in several properties from classical cadherins. The function of T-cadherin in homophilic cell recognition implies that the mechanism of T-cadherin-induced adhesion is distinct from that of classical cadherins.     

Adhesion-induced receptor segregation and adhesion plaque formation: A model membrane study.

A model system to study the control of cell adhesion by receptor-mediated specific forces, universal interactions, and membrane elasticity is established. The plasma membrane is mimicked by reconstitution of homophilic receptor proteins into solid supported membranes and, together with lipopolymers, into giant vesicles with the polymers forming an artificial glycocalix. The homophilic cell adhesion molecule contact site A, a lipid-anchored glycoprotein from cells of the slime mold Dictyostelium discoideum, is used as receptor. The success of the reconstitution, the structure and the dynamics of the model membranes are studied by various techniques including film balance techniques, micro fluorescence, fluorescence recovery after photobleaching, electron microscopy, and phase contrast microscopy. The interaction of the functionalized giant vesicles with the supported bilayer is studied by reflection interference contrast microscopy, and the adhesion strength is evaluated quantitatively by a recently developed technique. At low receptor concentrations adhesion-induced receptor segregation in the membranes leads to decomposition of the contact zone between membranes into domains of strong (receptor-mediated) adhesion and regions of weak adhesion while continuous zones of strong adhesion form at high receptor densities. The adhesion strengths (measured in terms of the spreading pressure S) of the various states of adhesion are obtained locally by analysis of the vesicle contour near the contact line in terms of elastic boundary conditions of adhesion: the balance of tensions and moments. The spreading pressure of the weak adhesion zones is S approximately 10(-9) J/m(2) and is determined by the interplay of gravitation and undulation forces whereas the spreading pressure of the tight adhesion domains is of the order S approximately 10(-6) J/m(2).     

Tyrosine Residue in Exon 14 of the Cytoplasmic Domain of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1/CD31) Regulates Ligand Binding Specificity

Platelet/endothelial cell adhesion molecule (PECAM-1) is a cell adhesion molecule of the immunoglobulin superfamily that plays a role in a number of vascular processes including leukocyte transmigration through endothelium. The presence of a specific 19– amino acid exon within the cytoplasmic domain of PECAM-1 regulates the binding specificity of the molecule; specifically, isoforms containing exon 14 mediate heterophilic cell–cell aggregation while those variants missing exon 14 mediate homophilic cell–cell aggregation. To more precisely identify the region of exon 14 responsible for ligand specificity, a series of deletion mutants were created in which smaller regions of exon 14 were removed. After transfection into L cells, they were tested for their ability to mediate aggregation. For heterophilic aggregation to occur, a conserved 5–amino acid region (VYSEI in the murine sequence or VYSEV in the human sequence) in the mid-portion of the exon was required. A final construct, in which this tyrosine was mutated into a phenylalanine, aggregated in a homophilic manner when transfected into L cells. Inhibition of phosphatase activity by exposure of cells expressing wild type or mutant forms of PECAM-1 to sodium orthovanadate resulted in high levels of cytoplasmic tyrosine phosphorylation and led to a switch from heterophilic to homophilic aggregation. Our data thus indicate either loss of this tyrosine from exon 14 or its phosphorylation results in a change in ligand specificity from heterophilic to homophilic binding. Vascular cells could thus determine whether PECAM-1 functions as a heterophilic or homophilic adhesion molecule by processes such as alternative splicing or by regulation of the balance between tyrosine phosphorylation or dephosphorylation. Defining the conditions under which these changes occur will be important in understanding the biology of PECAM-1 in transmigration, angiogenesis, development, and other processes in which this molecule plays a role.     

Identification of a Homophilic Binding Site in Immunoglobulin-Like Domain 2 of the Cell Adhesion Molecule L1

Abstract: The cell adhesion molecule L1 plays an important role in neural development, and mutations in human L1 have been implicated in X-linked hydrocephalus and related neurological diseases. We have previously demonstrated that recombinant proteins containing the second immunoglobulin-like domain (Ig2) of L1 contain both homophilic binding and neuritogenic activities. In this report, the involvement of L1 Ig2 in cell-cell adhesion and neuritogenesis was further evaluated in cell transfection studies. Transfectants expressing intact L1 were capable of undergoing L1-dependent self-aggregation and promoting neurite outgrowth from neural retinal cells. However, both activities were abolished in transfectants expressing L1Δ2, a mutant L1 with Ig2 deleted. In competition experiments, the wild-type Ig2 fusion protein inhibited L1-dependent cell aggregation, whereas an Ig2 fusion protein containing the hydrocephalus mutation R184Q did not. Oligopeptides flanking Arg¹⁸⁴ were therefore synthesized and assayed for their effects on L1-mediated cell-cell binding and neuritogenesis. The peptide L1-A, spanning the residues His¹⁷⁸ and Gly¹⁹¹, inhibited both L1- and Ig2 fusion protein-mediated homophilic binding. When neural retinal cells were cultured on substrate-coated Ig2 fusion protein, peptide L1-A also abolished L1-dependent neurite outgrowth. Substitutions of several charged residues and hydrophobic residues with alanine in peptide analogues led to the loss of inhibitory effects, suggesting that multiple amino acids might be involved in L1-L1 binding. Taken together, these results identify an L1 homophilic binding site within the sequence HIKQDERVTMGQNG of Ig2 and demonstrate the requirement of L1 homophilic binding in the promotion of neurite outgrowth.     

Multiple cadherin extracellular repeats mediate homophilic binding and adhesion.

The extracellular homophilic-binding domain of the cadherins consists of 5 cadherin repeats (EC1-EC5). Studies on cadherin specificity have implicated the NH(2)-terminal EC1 domain in the homophilic binding interaction, but the roles of the other extracellular cadherin (EC) domains have not been evaluated. We have undertaken a systematic analysis of the binding properties of the entire cadherin extracellular domain and the contributions of the other EC domains to homophilic binding. Lateral (cis) dimerization of the extracellular domain is thought to be required for adhesive function. Sedimentation analysis of the soluble extracellular segment of C-cadherin revealed that it exists in a monomer-dimer equilibrium with an affinity constant of approximately 64 microm. No higher order oligomers were detected, indicating that homophilic binding between cis-dimers is of significantly lower affinity. The homophilic binding properties of a series of deletion constructs, lacking successive or individual EC domains fused at the COOH terminus to an Fc domain, were analyzed using a bead aggregation assay and a cell attachment-based adhesion assay. A protein with only the first two NH(2)-terminal EC domains (CEC1-2Fc) exhibited very low activity compared with the entire extracellular domain (CEC1-5Fc), demonstrating that EC1 alone is not sufficient for effective homophilic binding. CEC1-3Fc exhibited high activity, but not as much as CEC1-4Fc or CEC1-5Fc. EC3 is not required for homophilic binding, however, since CEC1-2-4Fc and CEC1-2-4-5Fc exhibited high activity in both assays. These and experiments using additional EC combinations show that many, if not all, the EC domains contribute to the formation of the cadherin homophilic bond, and specific one-to-one interaction between particular EC domains may not be required. These conclusions are consistent with a previous study on direct molecular force measurements between cadherin ectodomains demonstrating multiple adhesive interactions (Sivasankar, S., W. Brieher, N. Lavrik, B. Gumbiner, and D. Leckband. 1999. PROC: Natl. Acad. Sci. USA. 96:11820-11824; Sivasankar, S., B. Gumbiner, and D. Leckband. 2001. Biophys J. 80:1758-68). We propose new models for how the cadherin extracellular repeats may contribute to adhesive specificity and function.     

Crystal Structure of the cis-Dimer of Nectin-1: implications for the architecture of cell-cell junctions

In multicellular organisms, cells are interconnected by cell adhesion molecules. Nectins are immunoglobulin (Ig)-like cell adhesion molecules that mediate homotypic and heterotypic cell-cell adhesion, playing key roles in tissue organization. To mediate cell-cell adhesion, nectin molecules dimerize in cis on the surface of the same cell, followed by trans-dimerization of the cis-dimers between the neighboring cells. Previous cell biological studies deduced that the first Ig-like domain of nectin and the second Ig-like domain are involved in trans-dimerization and cis-dimerization, respectively. However, to understand better the steps involved in nectin adhesion, the structural basis for the dimerization of nectin must be determined. In this study, we determined the first crystal structure of the entire extracellular region of nectin-1. In the crystal, nectin-1 formed a V-shaped homophilic dimer through the first Ig-like domain. Structure-based site-directed mutagenesis of the first Ig-like domain identified four essential residues that are involved in the homophilic dimerization. Upon mutating the four residues, nectin-1 significantly decreased cis-dimerization on the surface of cultured cells and abolished the homophilic and heterophilic adhesion activities. These results indicate that, in contrast with the previous notion, our structure represents a cis-dimer. Thus, our findings clearly reveal the structural basis for the cis-dimerization of nectins through the first Ig-like domains.     

Expression and involvement of gicerin, a cell adhesion molecule, in the development of chick optic tectum

Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. It has both a homophilic binding activity and a heterophilic binding activity to neurite outgrowth factor (NOF) a molecule belonging to the laminin family. We have reported many studies on the heterophilic activity of gicerin and NOF, but the function of its homophilic binding activity in vivo had been unclear. In the retina, gicerin is expressed in retinal ganglion cells only when they extend neurites to the optic tectum. In this report we have found that gicerin is also transiently expressed in the optic tectum during this time. First, cell aggregation assays were used to show that gicerin expressed in the optic tectum displays homophilic binding activity. Then, explant cultures of embryonic day 6 chick optic tectum on gicerin-Fc chimeric protein-coated dishes and NOF-coated dishes were carried out. It was found that gicerin-gicerin homophilic interactions promoted cell migration, whereas heterophilic interactions with NOF induced neurite formation. Furthermore, when anti-gicerin antibodies were injected in order to examine the effect of gicerin protein in the formation of the tectal layer in ovo, cell migration was strongly inhibited. These data suggest that homophilic interaction of gicerin participates in the migration of neural cells during the layer formation and plays a crucial role in the organization of the optic tectum.     

The 80L5C4 epitope overlaps with the homophilic binding site of the cell adhesion molecule gp80 of Dictyostelium.

The monoclonal antibody (mAb) 80L5C4 is a potent inhibitor of the cell adhesion molecule gp80 of Dictyostelium discoideum. To map the exact location of the epitope recognized by mAb 80L5C4, overlapping hexapeptides were synthesized on plastic pins and the binding p6 mAb 80L5C4 to these peptides was monitored by enzyme-linked immunosorbent assay. The 80L5C4 epitope is mapped to a single hexapeptide sequence GYKLNV, which shares five amino acid residues with the octapeptide sequence YKLNVNDS involved in gp80 homophilic binding. Analogue studies indicate that the hydrophobic residues within this sequence are crucial for antigen recognition.     

Differences in aggregation properties and levels of the neural cell adhesion molecule (NCAM) between islet cell types

Cells within rat islets of Langerhans are typically organized as a core of B-cells, surrounded by the other cell types. When mixed in culture, primary islet cells and insulinoma (RIN2A) cells form aggregates where B-cells are centrally located, surrounded by non-B-cells, while RIN-cells segregate as the outermost layer. To gain insight into the molecular basis underlying this nonrandom cellular organization, the aggregation properties of the three cell populations were studied. Isolated islet cells were separated into B-cells and non-B-cells by autofluorescence-activated cell sorting (FACS). In a short-term aggregation assay, primary B-cell aggregation in the absence of calcium was only 19 +/- 3.7%, compared to the 67 +/- 2.9% seen in the presence of calcium (mean +/- SEM; P less than 0.001; n = 7). By contrast, non-B-cell aggregation and RIN cell aggregation in the absence of calcium (62 +/- 2 and 66 +/- 2%, respectively) were only slightly less than with calcium (70 +/- 3 and 76 +/- 3%). The surface density of the Ca2(+)-independent neural CAM (NCAM) was therefore measured by flow cytometry and found to be 2.64 +/- 0.82-fold higher in non-B-cells, compared to that in B-cells (P less than 0.01; n = 3). Even higher levels were found on RIN cells. In the three cell types, NCAM-140 was the only molecular form detected by immunoblotting. In conclusion, differences in the calcium dependency of aggregation and in the levels of NCAM are demonstrated among islet B-cells, non-B-cells, and RIN cells. Because cell-cell adhesion is crucial for the maintenance of adult tissue, these aggregation specificities might contribute to the concentric segregation of islet cell types in culture and to the nonrandom distribution of cells within rat islets.     

The role of complex carbohydrates in adhesion of the myelin protein, P0.

The most abundant protein of peripheral nerve myelin, a glycoprotein termed P0, is believed to be involved in the compaction of the myelin sheath and is postulated to be the closest relative to the ancestral gene for the immunoglobulin superfamily. Recently, P0 has indeed been shown to behave like a homophilic adhesion molecule via interactions of its extracellular domains. Here we demonstrate the importance of the oligosaccharide moieties of P0 in its functioning as a homophilic adhesion molecule. Expression of the complex form of P0 glycoprotein in transfected Chinese hamster ovary cells greatly increased the adhesiveness of those cells, whereas expression of the high-mannose form of P0 glycoprotein did not. This is the first step in the dissection of P0-P0 interaction at the molecular level.     

Axonin-1/TAG-1 mediates cell-cell adhesion by a cis-assisted trans-interaction.

The neural cell adhesion molecule axonin-1/TAG-1 mediates cell-cell interactions via homophilic and heterophilic contacts. It consists of six Ig and four fibronectin type III domains anchored to the membrane by glycosylphosphatidylinositol. The recently solved crystal structure indicates a module composed of the four N-terminal Ig domains as the contact site between trans-interacting axonin-1 molecules from apposed membranes. Here, we have tested domain-specific monoclonal antibodies for their capacity to interfere with homophilic binding in a cell aggregation assay. The results confirmed the existence of a binding region within the N-terminal Ig domains and identified a second region contributing to homophilic binding on the third and fourth fibronectin domains near the C terminus. The perturbation of each region alone resulted in a complete loss of cell aggregation, suggesting that axonin-1-mediated cell-cell contact results from a cooperative action of two homophilic binding regions. The data support that axonin-1-mediated cell-cell contact is formed by cis-assisted trans-binding. The N-terminal binding regions of axonin-1 establish a linear zipper-like string of trans-interacting axonin-1 molecules alternately provided by the two apposed membranes. The C-terminal binding regions strengthen the cell-cell contact by enhancing the expansion of the linear string into a two-dimensional array via cis-interactions. Cis-assisted trans-binding may be a basic binding mechanism common to many cell adhesion molecules.     

A complex of Protocadherin-19 and N-cadherin mediates a novel mechanism of cell adhesion

During embryonic morphogenesis, adhesion molecules are required for selective cell-cell interactions. The classical cadherins mediate homophilic calcium-dependent cell adhesion and are founding members of the large and diverse cadherin superfamily. The protocadherins are the largest subgroup within this superfamily, yet their participation in calcium-dependent cell adhesion is uncertain. In this paper, we demonstrate a novel mechanism of adhesion, mediated by a complex of Protocadherin-19 (Pcdh19) and N-cadherin (Ncad). Although Pcdh19 alone is only weakly adhesive, the Pcdh19-Ncad complex exhibited robust adhesion in bead aggregation assays, and Pcdh19 appeared to play the dominant role. Adhesion by the Pcdh19-Ncad complex was unaffected by mutations that disrupt Ncad homophilic binding but was inhibited by a mutation in Pcdh19. In addition, the complex exhibited homophilic specificity, as beads coated with Pcdh19-Ncad did not intermix with Ncad- or Pcdh17-Ncad-coated beads. We propose a model in which association of a protocadherin with Ncad acts as a switch, converting between distinct binding specificities.     

The measurement of specific cell: cell interactions by dual-parameter flow cytometry

The Fc receptor-mediated aggregation of antibody-coated spleen cells with cells from the P388D1 mouse macrophage line was followed using a novel flow cytometric technique. P388D1 and spleen cells were directly labeled with green-emitting (fluorescein isothiocyanate) and red-emitting (substituted rhodamine isothiocyanate) fluorophores, respectively. They were mixed, incubated in suspension at 4 degrees C, and analyzed for aggregation with a dual laser flow cytometer. Unconjugated cells appeared as particles which were either red or green, while conjugates were detected as particles which were both red and green. Using this assay procedure, 5 X 10(4) cells were analyzed in 2-3 min for the percentages of conjugates, free spleen cells, and free P388D1 cells. Intercellular aggregation required both antibody on the spleen cells and free Fc receptors on the P388D1 cells; nonspecific aggregates accounted for 1% or less of the total particles analyzed. Measurements of the fluorescence distributions within conjugates indicated that the majority of conjugates contained a single P388D1 cell bound to 1-3 spleen cells, and that only heterophilic aggregation occurred. The flow cytometric technique described here should be applicable for the measurement of the initial events of intercellular aggregation in other systems as well.     

Epitope diversity of N-glycans from bovine peripheral myelin glycoprotein P0 revealed by mass spectrometry and nano probe magic angle spinning 1H NMR spectroscopy.

The carbohydrate structures present on the glycoproteins in the central and peripheral nerve systems are essential in many cell adhesion processes. The P0 glycoprotein, expressed by myelinating Schwann cells, plays an important role during the formation and maintenance of myelin, and it is the most abundant constituent of myelin. Using monoclonal antibodies, the homophilic binding of the P0 glycoprotein was shown to be mediated via the human natural keller cell (HNK)-1 epitope (3-O-SO(3)H-GlcUA(beta1-3)Gal(beta1-4)GlcNAc) present on the N-glycans. We recently described the structure of the N-glycan carrying the HNK-1 epitope, present on bovine peripheral myelin P0 (Voshol, H., van Zuylen, C. W. E. M., Orberger, G., Vliegenthart, J. F. G., and Schachner, M. (1996) J. Biol. Chem. 271, 22957-22960). In this study, we report on the structural characterization of the detectable glycoforms, present on the single N-glycosylation site, using state-of-the-art NMR and mass spectrometry techniques. Even though all structures belong to the hybrid- or biantennary complex-type structures, the variety of epitopes is remarkable. In addition to the 3-O-sulfate present on the HNK-1-carrying structures, most of the glycans contain a 6-O-sulfated N-acetylglucosamine residue. This indicates the activity of a 6-O-sulfo-GlcNAc-transferase, which has not been described before in peripheral nervous tissue. The presence of the disialo-, galactosyl-, and 6-O-sulfosialyl-Lewis X epitopes provides evidence for glycosyltransferase activities not detected until now. The finding of such an epitope diversity triggers questions related to their function and whether events, previously attributed merely to the HNK-1 epitope, could be mediated by the structures described here.