Mapping the collagen-binding site in the von Willebrand factor-A3 domain

The multimeric glycoprotein von Willebrand factor (VWF) mediates platelet adhesion to collagen at sites of vascular damage. The binding site for collagen types I and III is located in the VWF-A3 domain. Recently, we showed that His(1023), located near the edge between the "front" and "bottom" faces of A3, is critical for collagen binding (Romijn, R. A., Bouma, B., Wuyster, W., Gros, P., Kroon, J., Sixma, J. J., and Huizinga, E. G. (2001) J. Biol. Chem. 276, 9985-9991). To map the binding site in detail, we introduced 22 point mutations in the front and bottom faces of A3. The mutants were expressed as multimeric VWF, and binding to collagen type III was evaluated in a solid-state binding assay and by surface plasmon resonance. Mutation of residues Asp(979), Ser(1020), and His(1023) nearly abolished collagen binding, whereas mutation of residues Ile(975), Thr(977), Val(997), and Glu(1001) reduced binding affinity about 10-fold. Together, these residues define a flat and rather hydrophobic collagen-binding site located at the front face of the A3 domain. The collagen-binding site of VWF-A3 is distinctly different from that of the homologous integrin alpha(2) I domain, which has a hydrophilic binding site located at the top face of the domain. Based on the surface characteristics of the collagen-binding site of A3, we propose that it interacts with collagen sequences containing positively charged and hydrophobic residues. Docking of a collagen triple helix on the binding site suggests a range of possible engagements and predicts that at most eight consecutive residues in a collagen triple helix interact with A3.     

LRP/amyloid beta-peptide interaction mediates differential brain efflux of Abeta isoforms

LRP (low-density lipoprotein receptor-related protein) is linked to Alzheimer's disease (AD). Here, we report amyloid beta-peptide Abeta40 binds to immobilized LRP clusters II and IV with high affinity (Kd = 0.6-1.2 nM) compared to Abeta42 and mutant Abeta, and LRP-mediated Abeta brain capillary binding, endocytosis, and transcytosis across the mouse blood-brain barrier are substantially reduced by the high beta sheet content in Abeta and deletion of the receptor-associated protein gene. Despite low Abeta production in the brain, transgenic mice expressing low LRP-clearance mutant Abeta develop robust Abeta cerebral accumulations much earlier than Tg-2576 Abeta-overproducing mice. While Abeta does not affect LRP internalization and synthesis, it promotes proteasome-dependent LRP degradation in endothelium at concentrations > 1 microM, consistent with reduced brain capillary LRP levels in Abeta-accumulating transgenic mice, AD, and patients with cerebrovascular beta-amyloidosis. Thus, low-affinity LRP/Abeta interaction and/or Abeta-induced LRP loss at the BBB mediate brain accumulation of neurotoxic Abeta.     

Guidelines to come to minimized tensile strength loss upon cellulase application

Application of cellulase technology in the textile production process often results in a certain loss of tensile strength along with the desired performance. In this paper guidelines are given how to come to minimization or even prevention of tensile strength loss. Part of the considerations is based on the hypothesis given in the accompanying paper (Lenting and Warmoeskerken, 2001, J. Biotechnol.) concerning the mechanism of interaction between cellulase action and applied shear force. Recommendations given concern the enzyme choice, process parameters and enzyme targeting.     

The role of glyceraldehyde 3-phosphate dehydrogenase (GapA-1) in <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> adherence to human cells

Background  

Glyceraldehyde 3-phosphate dehydrogenases (GAPDHs) are cytoplasmic glycolytic enzymes, which although lacking identifiable secretion signals, have also been found localized to the surface of several bacteria (and some eukaryotic organisms); where in some cases they have been shown to contribute to the colonization and invasion of host tissues. Neisseria meningitidis is an obligate human nasopharyngeal commensal which can cause life-threatening infections including septicaemia and meningitis. N. meningitidis has two genes, gapA-1 and gapA-2, encoding GAPDH enzymes. GapA-1 has previously been shown to be up-regulated on bacterial contact with host epithelial cells and is accessible to antibodies on the surface of capsule-permeabilized meningococcal cells. The aims of this study were: 1) to determine whether GapA-1 was expressed across different strains of N. meningitidis; 2) to determine whether GapA-1 surface accessibility to antibodies was dependant on the presence of capsule; 3) to determine whether GapA-1 can influence the interaction of meningococci and host cells, particularly in the key stages of adhesion and invasion.     

Chemical modification of proteases for wool cuticle scale removal

Over the last few decades several enzymatic processes to improve properties of wool fabrics like felting tendency, shrink resistance, dyeing ability and handling characteristics have been described. Previous investigations into the use of proteases to hydrolyse the cuticles at the surface of wool fibres, resulted in high strength and weight losses. Therefore restriction of the enzyme activity to the wool surface or control of enzyme diffusion to the cortex cells is required.

To change the diffusion behaviour of proteases in wool fibres, the soluble polymer PEG was covalently attached to a protease from Bacillus lentus. Modified enzymes with different molecular weights were compared. These modified enzymes retained up to 80% of their activity in the standard assay while hydrolysis of wool fibres was successfully restricted to cuticles, resulting in a 90% decrease in weight losses compared to non-modified enzymes.     

The complete mitochondrial DNA sequence of the shark Mustelus manazo: evaluating rooting contradictions to living bony vertebrates

A remarkable example of a misleading mitochondrial protein tree is presented, involving ray-finned fishes, coelacanths, lungfishes, and tetrapods, with sea lampreys as an outgroup. In previous molecular phylogenetic studies on the origin of tetrapods, ray-finned fishes have been assumed as an outgroup to the tetrapod/lungfish/coelacanth clade, an assumption supported by morphological evidence. Standard methods of molecular phylogenetics applied to the protein-encoding genes of mitochondria, however, give a bizarre tree in which lamprey groups with lungfish and, therefore, ray-finned fishes are not the outgroup to a tetrapod/lungfish/coelacanth clade. All of the dozens of published phylogenetic methods, including every possible modification to maximum likelihood known to us (such as inclusion of site heterogeneity and exclusion of potentially misleading hydrophobic amino acids), fail to place the ray-finned fishes in a biologically acceptable position. A likely cause of this failure may be the use of an inappropriate outgroup. Accordingly, we have determined the complete mitochondrial DNA sequence from the shark, Mustelus manazo, which we have used as an alternative and more proximal outgroup than the lamprey. Using sharks as the outgroup, lungfish appear to be the closest living relative of tetrapods, although the possibility of a lungfish/coelacanth clade being the sister group of tetrapods cannot be excluded.      

Surface loop 199-204 in blood coagulation factor IX is a cofactor-dependent site involved in macromolecular substrate interaction.

In factor IX residues 199-204 encompass one of six surface loops bordering its substrate-binding groove. To investigate the contribution of this loop to human factor IX function, a series of chimeric factor IX variants was constructed, in which residues 199-204 were replaced by the corresponding sequence of factor VII, factor X, or prothrombin. The immunopurified and activated chimeras were indistinguishable from normal factor IXa in hydrolyzing a small synthetic substrate, indicating that this region is not involved in the interaction with substrate residues on the N-terminal side of the scissile bond. In contrast, replacement of loop 199-204 resulted in a 5-25-fold reduction in reactivity toward the macromolecular substrate factor X. This reduction was due to a combination of increased K(m) and reduced k(cat). In the presence of factor VIIIa the impaired reactivity toward factor X was largely restored for all factor IXa variants, resulting in a more pronounced stimulation by factor VIIIa compared with normal factor IXa (3 to 5 x 10(4)-fold versus 5 x 10(3)-fold). Inhibition by antithrombin was only slightly affected for the factor IXa variant with the prothrombin loop sequence, whereas factor IXa variants containing the analogous residues of factor VII or factor X were virtually insensitive to antithrombin inhibition. In the presence of heparin, however, all chimeric factor IXa variants formed complexes with antithrombin. Thus the cofactors heparin and factor VIIIa have in common that they both alleviate the deleterious effects of mutations in the factor IX loop 199-204. Collectively, our data demonstrate that loop 199-204 plays an important role in the interaction of factor IXa with macromolecular substrates.