Search PubMed⌕ Search

Biomedical subjects

A W Koch

Publications and source records attributed to A W Koch.

7 recordsLinked to original sources
PubMed1999

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 microM) stabilized the rod-like structure of E-cadherin. At medium Ca2+ concentration (500 microM), 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.

Base Sequence↗
PubMed1999

Homophilic adhesion by cadherins.

Cadherins mediate cell-cell adhesion through homophilic interactions. High-resolution structures have greatly enhanced our understanding of this phenomenon over the past few years. Nonetheless, some of the original concepts about cadherin interactions need revision, with the new structural and additional mutagenesis data currently available. Furthermore, in vivo studies on cadherins have provided supplementary information.

Amino Acid Substitution↗
PubMed1998

Spinalin, a new glycine- and histidine-rich protein in spines of Hydra nematocysts.

Here we present the cloning, expression and immunocytochemical localization of a novel 24 kDa protein, designated spinalin, which is present in the spines and operculum of Hydra nematocysts. Spinalin cDNA clones were identified by in situ hybridization to differentiating nematocytes. Sequencing of a full-length clone revealed the presence of an N-terminal signal peptide, suggesting that the mature protein is sorted via the endoplasmic reticulum to the post-Golgi vacuole in which the nematocyst is formed. The N-terminal region of spinalin (154 residues) is very rich in glycines (48 residues) and histidines (33 residues). A central region of 35 residues contains 19 glycines, occurring mainly as pairs. For both regions a polyglycine-like structure is likely and this may be stabilized by hydrogen bond-mediated chain association. Similar sequences found in loricrins, cytokeratins and avian keratins are postulated to participate in formation of supramolecular structures. Spinalin is terminated by a basic region (6 lysines out of 15 residues) and an acidic region (9 glutamates and 9 aspartates out of 32 residues). Western blot analysis with a polyclonal antibody generated against a recombinant 19 kDa fragment of spinalin showed that spinalin is localized in nematocysts. Following dissociation of the nematocyst's capsule wall with DTT, spinalin was found in the insoluble fraction containing spines and the operculum. Immunocytochemical analysis of developing nematocysts revealed that spinalin first appears in the matrix but then is transferred through the capsule wall at the end of morphogenesis to form spines on the external surface of the inverted tubule and the operculum.

Amino Acid Sequence↗
PubMed1997

Calcium binding and homoassociation of E-cadherin domains.

Cadherins are single pass transmembrane glycoproteins which mediate calcium dependent cell-cell adhesion by homophilic interactions. To reveal the molecular details of calcium binding and homoassociation, we recombinantly expressed in Escherichia coli a domain pair consisting of the first two domains of E-cadherin (ECAD12) and the single domains 1, 2, and 5. ECAD12 encompasses the most N-terminal of the four putative calcium-binding pockets in the extracellular region of E-cadherin. Equilibrium dialysis experiments revealed that the single domains do not bind Ca2+, but ECAD12 was found to bind three calcium ions. ECAD12 dimerizes (Kd = 0.08 +/- 0.02 mM) in the presence of Ca2+ as we could demonstrate by analytical ultracentrifugation. Calcium binding to ECAD12 induces conformational changes which were monitored by electrophoretic mobility and by circular dichroism. By analyzing our equilibrium dialysis data with a single binding site model, we found an average Kd of 460 microM for the three bound Ca2+. Assuming a model for three binding sites, which slightly increased the quality of the fit, we obtained two identical Kds of 330 microM and a third much higher Kd of 2 mM. The entire extracellular region of E-cadherin, which was recombinantly expressed in mammalian cells, binds nine Ca2+ with a much lower average Kd of 30 microM. Therefore, we conclude that the four calcium binding pockets are not identical. Since binding to ECAD12 occurs at Ca2+ concentrations close to those in the extracellular space, we suggest that the N-terminal domain pair might be involved in calcium regulation of E-cadherin mediated cell-cell adhesion.

Animals↗