Jürgen Engel

A new nomenclature for the laminins

The authors have adopted a new nomenclature for the laminins. They are numbered with arabic numerals in the order discovered. The previous A, B1 and B2 chains, and their isoforms, are alpha, beta and gamma, respectively, followed by an arabic numeral to identify the isoform. For example, the first laminin identified from the Engelbreth-Holm-Swarm tumor is laminin-1 with the chain composition alpha 1 beta 1 gamma 1. The genes for these chains are LAMA1, LAMB1 and LAMC1, respectively.

Shapes, domain organizations and flexibility of laminin and fibronectin, two multifunctional proteins of the extracellular matrix

Abstract Laminin from a mouse tumor basement membrane and fibronectin from human blood plasma were examined by electron microscopy using rotary shadowing and negative staining and by transmission scanning electron microscopy of unstained samples. Laminin was visualized as a rigid, asymmetric cross consisting of a long (77 nm) and three apparently identical short (36 nm) arms. The rod-like arms (diameter about 2 nm) terminated in globular units (diameter 5 to 7 nm). Additional globules were found near the terminal units in the short arms. A large pepsin-resistant fragment of laminin appeared as a rigid structure with three arms (length 26 nm, preferred angle 90 °), which presumably represented parts of the three short arms of laminin. Fibronectin could be visualized as two identical strands (length 61 nm, diameter about 2 nm), which did not reveal distinct globular units. These strands very likely comprised single peptide chains connected to each other at one end, enclosing a fixed angle of about 70 °. Electron microscopy also indicated a limited flexibility of the arms of both laminin and fibronectin, comparable to the stiffness of tropomyosin or DNA. The electron microscopic images of the shapes and dimensions of laminin, of fragments of laminin, and of fibronectin are consistent with the specific molecular weights and with the hydrodynamic properties determined in solution. The arms of fibronectin showed three distinct regions at which preferential bending occurred. These sites apparently correspond to flexible segments connecting more compact domains previously identified in biochemical studies. No sites of preferential bending were visible in the arms of laminin. Although laminin and fibronectin have some similar biological activities (binding of cells, collagen, glycosaminoglycans), the corresponding functional domains are differently arranged in the two molecules.

Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV.

Recombinant mouse nidogen and two fragments were produced in mammalian cells and purified from culture medium without resorting to denaturing conditions. The truncated products were fragments Nd‐I (positions 1–905) comprising the N‐terminal globule and rod‐like domain and Nd‐II corresponding mainly to the C‐terminal globule (position 906–1217). Recombinant nidogen was indistinguishable from authentic nidogen obtained by guanidine dissociation from tumor tissue with respect to size, N‐terminal sequence, CD spectra and immunochemical properties. They differed in protease stability and shape indicating that the N‐terminal domain of the more native, recombinant protein consists of two globules connected by a flexible segment. This established a new model for the shape of nidogen consisting of three globes of variable mass (31–56 kDa) connected by either a rod‐like or a thin segment. Recombinant nidogen formed stable complexes (Kd less than or equal to 1 nM) with laminin and collagen IV in binding assays with soluble and immobilized ligands and as shown by electron microscopy. Inhibition assays demonstrated different binding sites on nidogen for both ligands with different specificities. This was confirmed in studies with fragment Nd‐I binding to collagen IV and fragment Nd‐II binding to laminin fragment P1. In addition, recombinant nidogen but not Nd‐I was able to bridge between laminin or P1 and collagen IV. Formation of such ternary complexes implicates a similar role for nidogen in the supramolecular organization of basement membranes.

A new crystal structure, Ca2+ dependence and mutational analysis reveal molecular details of E‐cadherin homoassociation

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 Ca2+ dependency of both interactions was investigated. Low Ca2+ concentrations (50 μM) stabilized the rod‐like structure of E‐cadherin. At medium Ca2+ concentration (500 μM), two adjacent ectodomains in a pentamer formed cis‐dimers. At high Ca2+ 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 Ca2+‐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 cis‐interaction. Structural and biochemical data suggest a link between Ca2+ binding in the millimolar range and Trp2 docking, both events being essential for the trans‐association.

The Crystal Structure of a Five-Stranded Coiled Coil in COMP: A Prototype Ion Channel?

Oligomerization by the formation of α-helical bundles is common in many proteins. The crystal structure of a parallel pentameric coiled coil, constituting the oligomerization domain in the cartilage oligomeric matrix protein (COMP), was determined at 2.05 angstroms resolution. The same structure probably occurs in two other extracellular matrix proteins, thrombospondins 3 and 4. Complementary hydrophobic interactions and conserved disulfide bridges between the α helices result in a thermostable structure with unusual properties. The long hydrophobic axial pore is filled with water molecules but can also accommodate small apolar groups. An “ion trap” is formed inside the pore by a ring of conserved glutamines, which binds chloride and probably other monatomic anions. The oligomerization domain of COMP has marked similarities with proposed models of the pentameric transmembrane ion channels in phospholamban and the acetylcholine receptor.

Two Adjacent Trimeric Fas Ligands Are Required for Fas Signaling and Formation of a Death-Inducing Signaling Complex

ABSTRACT The membrane-bound form of Fas ligand (FasL) signals apoptosis in target cells through engagement of the death receptor Fas, whereas the proteolytically processed, soluble form of FasL does not induce cell death. However, soluble FasL can be rendered active upon cross-linking. Since the minimal extent of oligomerization of FasL that exerts cytotoxicity is unknown, we engineered hexameric proteins containing two trimers of FasL within the same molecule. This was achieved by fusing FasL to the Fc portion of immunoglobulin G1 or to the collagen domain of ACRP30/adiponectin. Trimeric FasL and hexameric FasL both bound to Fas, but only the hexameric forms were highly cytotoxic and competent to signal apoptosis via formation of a death-inducing signaling complex. Three sequential early events in Fas-mediated apoptosis could be dissected, namely, receptor binding, receptor activation, and recruitment of intracellular signaling molecules, each of which occurred independently of the subsequent one. These results demonstrate that the limited oligomerization of FasL, and most likely of some other tumor necrosis factor family ligands such as CD40L, is required for triggering of the signaling pathways.

Single amino acid substitutions in one Ca2+ binding site of uvomorulin abolish the adhesive function

We show that a synthetic peptide corresponding to the sequence of one putative Ca2+ binding motif of the cell adhesion molecule uvomorulin is able to complex Ca2+. This function is abolished if the first Asp in the peptide is replaced by Lys. Accordingly, we expressed in L cells mutant uvomorulin with a replacement of Asp to Lys or Ala. Mutant protein was resistant to Ca2+/trypsin under mild conditions but became susceptible at or near the site of replacement at higher concentrations, leaving the remaining Ca2+ binding domains protected. Remarkably, in cell aggregation assays both mutant uvomorulins failed to mediate cell adhesiveness, demonstrating that a single amino acid substitution in one Ca2+ binding site inactivates the adhesive function.

EGF‐like domains in extracellular matrix proteins: Localized signals for growth and differentiation?

Multidomain proteins of the extracellular matrix (ECM) play an important role in development and maintenance of cellular organization and in tissue repair. Several ECM proteins such as laminin, tenascin, and thrombospondin contain domains with homology to epidermal growth factor (EGF) and exhibit growth promoting activity. The mitogenic activity of laminin is restricted to a fragment which consists of about 25 repeating domains with partial homology to EGF and comprises the rod‐like inner regions of the three short arms of the four armed molecule. The mitogenic activity does not correlate with promotion of cell attachment and neurite outgrowth for which major functional sites have been found in other regions of the laminin molecule. It is suggested that EGF‐like domains in laminin, in other ECM proteins and in the extracellular portions of some membrane proteins are signals for cellular growth and differentiation. Because they are integral parts of large molecules and often of supramolecular assemblies these domains are well suited to stimulate neighboring cells in a specific and vectorial way. This concept of localized growth or differentiation signals offers an attractive mechanism for the regulation of cellular development.

The structure of cell adhesion molecule uvomorulin. Insights into the molecular mechanism of Ca2+-dependent cell adhesion.

We have determined the amino acid sequence of the Ca2+‐dependent cell adhesion molecule uvomorulin as it appears on the cell surface. The extracellular part of the molecule exhibits three internally repeated domains of 112 residues which are most likely generated by gene duplication. Each of the repeated domains contains two highly conserved units which could represent putative Ca2+‐binding sites. Secondary structure predictions suggest that the putative Ca2+‐binding units are located in external loops at the surface of the protein. The protein sequence exhibits a single membrane‐spanning region and a cytoplasmic domain. Sequence comparison reveals extensive homology to the chicken L‐CAM. Both uvomorulin and L‐CAM are identical in 65% of their entire amino acid sequence suggesting a common origin for both CAMs.

D-23129: a new anticonvulsant with a broad spectrum activity in animal models of epileptic seizures

The anticonvulsant activity of the novel drug D-23129 (N-(2-amino-4-(4-fluorobenzylamino)phenyl)carbamic acid ethyl ester) was evaluated in animal models of epileptic seizures. D-23129 was active after oral and intraperitoneal administration in rats and mice in a range of anticonvulsant tests at nontoxic doses. The compound was active against electrically induced seizures (MES, ED50 rat p.o. = 2.87 mg/kg), against seizures induced chemically by pentylenetetrazole (s.c. PTZ, ED50 mouse p.o. = 13.5 mg/kg), picrotoxin and N-methyl-D-aspartate (NMDA) and in a genetic animal model, the DBA/2 mouse. It was not active against seizures induced by bicuculline and strychnine. Motor impairment, evaluated with the rotarod test and by observation in the open field, was minimal at doses showing anticonvulsant activity. D-23129 was very effective in elevating the threshold for electrically and chemically induced seizures. Considering the dose increasing the MES threshold by 50% (TID50 mouse i.p. = 1.6 mg/kg; TID50 rat i.p. = 0.72 mg/kg) and the TD50 obtained in the rotarod test, the protective index of D-23129 is better than that of valproate and phenytoin. During 14 days chronic oral treatment with 15 mg/kg, no development of tolerance was observed. D-23129 thus presents an orally active, safe, broad spectrum anticonvulsant agent, which is structurally unrelated to anticonvulsants currently used. We expect that D-23129 will improve the treatment of refractory seizures in humans.