Electron microscopy of ECADCOMP, a recombinant E-cadherin ectodomain pentamerized by the assembly domain of cartilage oligomeric matrix protein, has been used to analyze the role of cis-dimerization and trans-interaction in the homophilic association of this cell adhesion molecule. The Ca 2⍣ dependency of both interactions was investigated. Low Ca 2⍣ concentrations (50 μM) stabilized the rod-like structure of E-cadherin. At medium Ca 2⍣ concentration (500 μM), two adjacent ectodomains in a pentamer formed cis-dimers. At high Ca 2⍣ concentration (>1 mM), two cis-dimers from different pentamers formed a trans-interaction. The X-ray structure of an N-terminal domain pair of E-cadherin revealed two molecules per asymmetric unit in an intertwisted X-shaped arrangement with closest contacts in the Ca 2⍣ -binding region between domains 1 and 2. Contrary to previous data, Trp2 was docked in the hydrophobic cavity of its own molecule, and was therefore not involved in cis-dimerization of two molecules. This was supported further by W2A and A80I (a residue involved in the hydrophobic cavity surrounding Trp2) mutations in ECADCOMP which both led to abrogation of the trans-but not the cisinteraction. Structural and biochemical data suggest a link between Ca 2⍣ binding in the millimolar range and Trp2 docking, both events being essential for the trans-association.

Cluster formation of E‐cadherin on the cell surface is believed to be of major importance for cell‐cell adhesion. To mimic this process the extracellular part of mouse E‐cadherin (ECAD) was recombinantly fused to the assembly domain of rat cartilage oligomeric matrix protein (COMP), resulting in the chimeric protein ECAD‐COMP. The COMP domain formed a five‐stranded alpha‐helical coiled‐coil. This enabled the formation of a pentameric ECAD with bundled C‐termini and free N‐termini. The pentameric protein construct ECAD‐COMP and the monomeric ECAD were expressed in human embryonal kidney 293 cells. Electron microscopy, analytical ultracentrifugation, solid phase binding and cell attachment assays revealed that pentamers showed strong self‐association and cell attachment, whereas monomers exhibited no activity. At the high internal concentration in the pentamer the N‐terminal EC1 domains of two E‐cadherin arms interact to form a ring‐like structure. Then the paired domains interact with a corresponding pair from another pentamer. None of the four other extracellular domains of E‐cadherin is involved in this interaction. Based on these results, an in vivo mechanism is proposed whereby two N‐terminal domains of neighbouring E‐cadherins at the cell surface first form a pair, which binds with high affinity to a similar complex on another cell. The strong dependence of homophilic interactions on C‐terminal clustering points towards a regulation of E‐cadherin mediated cell‐cell adhesion via lateral association.

Agrin is a large, multidomain heparan sulfate proteoglycan that is associated with basement membranes of several tissues. Particular splice variants of agrin are essential for the formation of synaptic structures at the neuromuscular junction. The binding of agrin to laminin appears to be required for its localization to synaptic basal lamina and other basement membranes. Here, electron microscopy was used to determine the structure of agrin and to localize its binding site in laminin‐1. Agrin appears as an ∼95 nm long particle that consists of a globular, N‐terminal laminin‐binding domain, a central rod predominantly formed by the follistatin‐like domains and three globular, C‐terminal laminin G‐like domains. In a few cases, heparan sulfate glycosaminoglycan chains were seen emerging from the central portion of the core protein. Moreover, we show that agrin binds to the central region of the three‐stranded, coiled‐coil oligomerization domain in the long arm of laminin‐1, which mediates subunit assembly of the native laminin molecule. In summary, our data show for the first time a protein–protein interaction of the extracellular matrix that involves a coiled‐coil domain, and they assign a novel role to this domain of laminin‐1. Based on this, we propose that agrin associates with basal lamina in a polarized way.

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Mac-2-binding protein (M2BP) is a secreted glycoprotein suggested to have a role in host defense. It forms linear and ring-shaped oligomers, with each ring segment being composed of two monomers. We have produced recombinant human M2BP fragments comprising domains 1 and 2 (M2BP-1,2) and domains 3 and 4 (M2BP-3,4) in 293 human kidney cells to characterize structural and functional properties of M2BP. Both fragments were obtained in a native and glycosylated form, as analyzed by CD spectroscopy, trypsin susceptibility, and enzymatic deglycosylation. These results strongly suggest that both fragments are autonomous folding units. All three potential N-glycosylation sites in M2BP-1,2 and all four in M2BP-3,4 were found to be occupied. M2BP-1,2 expressed in tunicamycin-treated cells contained no glycosyl residues, indicating that O-glycosylation is not occurring. Ultracentrifugation revealed that M2BP-1,2 is homogeneously dimeric in the nanomolar range reflecting the properties of intact M2BP. Domain 2 (BTB/ POZ domain) is thus identified as the dimerization domain of M2BP, because it has been formerly shown that recombinant domain 1 is monomeric. M2BP-3,4 showed a concentration-dependent self-association, and aggregates of different size and shape were shown by electron microscopy. In contrast to this irregular aggregation of M2BP-3,4, it has been formerly shown that a fragment comprising domains 2-4 still has the ability to form ringlike structures, although the rings are protein-filled, and thus domain 2 appears to be indispensable for ring formation. Solid phase assays showed that M2BP-3,4 contains binding sites for galectin-3, nidogen, and collagens V and VI, whereas M2BP-1,2 is inactive in binding. Both fragments showed no cell adhesive activity in contrast to native M2BP, suggesting that a concerted binding action and/or multivalent interactions of rings are necessary for cell attachment. Mac-2-binding protein (M2BP)1 is a secreted glycoprotein present in the extracellular matrix of several tissues (1) and in extracellular fluids such as serum and milk (2). Elevated levels are observed in some tumors and viral infections (3, 4). In vitro, human M2BP induces production of interleukin-1, interleukin-6, and other cytokines by blood monocytes and stimulates natural killer cell and lymphokine-activated killer cell activity (5, 6). Mouse cyclophilin C-associated protein is 69% identical to M2BP, and it seems likely that the two proteins are functional homologues. Gene-targeted cyclophilin C-associated protein-deficient mice are viable but show an up-regulation of the endotoxin and proinflammatory response (7).M2BP is extensively glycosylated and interacts with galectin-3 (former name Mac-2), and it also interacts with other extracellular proteins such as collagens IV, V, and VI, fibronectin, and nidogen (1, 2, 8, 9). M2BP binds to galectin-3 on the cell surface and induces homotypic cell aggregation (10). ␤ 1 -Integrin-mediated cell adhesion to M2BP was also demonstrated (1).Tissue extracted and recombinant M2BP for...