Engineering novel complement activity into a pulmonary surfactant protein

Complement neutralizes invading pathogens, stimulates inflammatory and adaptive immune responses, and targets non- or altered-self structures for clearance. In the classical and lectin activation pathways, it is initiated when complexes composed of separate recognition and activation subcomponents bind to a pathogen surface. Despite its apparent complexity, recognition-mediated activation has evolved independently in three separate protein families, C1q, mannose-binding lectins (MBLs), and serum ficolins. Although unrelated, all have bouquet-like architectures and associate with complement-specific serine proteases: MBLs and ficolins with MBL-associated serine protease-2 (MASP-2) and C1q with C1r and C1s. To examine the structural requirements for complement activation, we have created a number of novel recombinant rat MBLs in which the position and orientation of the MASP-binding sites have been changed. We have also engineered MASP binding into a pulmonary surfactant protein (SP-A), which has the same domain structure and architecture as MBL but lacks any intrinsic complement activity. The data reveal that complement activity is remarkably tolerant to changes in the size and orientation of the collagenous stalks of MBL, implying considerable rotational and conformational flexibility in unbound MBL. Furthermore, novel complement activity is introduced concurrently with MASP binding in SP-A but is uncontrolled and occurs even in the absence of a carbohydrate target. Thus, the active rather than the zymogen state is default in lectin·MASP complexes and must be inhibited through additional regions in circulating MBLs until triggered by pathogen recognition

Paths reunited: initiation of the classical and lectin pathways of complement activation

Understanding the structural organisation and mode of action of the initiating complex of the classical pathway of complement activation (C1) has been a central goal in complement biology since its isolation almost 50 years ago. Nevertheless, knowledge is still incomplete, especially with regard to the interactions between its subcomponents C1q, C1r and C1s that trigger activation upon binding to a microbial target. Recent studies have provided new insights into these interactions, and have revealed unexpected parallels with initiating complexes of the lectin pathway of complement: MBL–MASP and ficolin–MASP. Here, we develop and expand these concepts and delineate their implications towards the key aspects of complement activation via the classical and lectin pathways

Colloquium on Rome II: the 2007 Regulation on the Law Applicable to Non-contractual Obligations -- European and American Perspectives

Diana Wallis, vice president of the European Parliament, joined other American and European experts in private international law to explore new European Union legislation on the law applicable to torts and to compare it to approaches in U.S. law during a two-day event sponsored by the University of Georgia Dean Rusk Center - International, Comparative and Graduate Legal Studies and the Georgia Society of International and Comparative Law. The colloquium was held November 5-6, 2007 in the Larry Walker Room of Dean Rusk Hall

The crystal structure of Pneumolysin at 2.0 Å resolution reveals the molecular packing of the pre-pore complex

Pneumolysin is a cholesterol-dependent cytolysin (CDC) and virulence factor of Streptococcus pneumoniae. It kills cells by forming pores assembled from oligomeric rings in cholesterol-containing membranes. Cryo-EM has revealed the structures of the membrane-surface bound pre-pore and inserted-pore oligomers, however the molecular contacts that mediate these oligomers are unknown because high-resolution information is not available. Here we have determined the crystal structure of full-length pneumolysin at 1.98 Å resolution. In the structure, crystal contacts demonstrate the likely interactions that enable polymerisation on the cell membrane and the molecular packing of the pre-pore complex. The hemolytic activity is abrogated in mutants that disrupt these intermolecular contacts, highlighting their importance during pore formation. An additional crystal structure of the membrane-binding domain alone suggests that changes in the conformation of a tryptophan rich-loop at the base of the toxin promote monomer-monomer interactions upon membrane binding by creating new contacts. Notably, residues at the interface are conserved in other members of the CDC family, suggesting a common mechanism for pore and pre-pore assembly

Crystal structure of an inulosucrase from Halalkalicoccus jeotgali B3T-a halophilic archaeal strain

Several archaea harbour genes that code for fructosyltransferase (FTF) enzymes. These enzymes have not been characterized yet at structure-function level, but are of extreme interest in view of their potential role in the synthesis of novel compounds for food, nutrition and pharmaceutical applications. In this study, 3D structure of an inulin-type fructan producing enzyme, inulosucrase (InuHj), from the archaeon Halalkalicoccus jeotgali was resolved in its apo form as well as with bound substrate (sucrose) molecule and first transglycosylation product (1-kestose). This is the first crystal structure of an FTF from halophilic archaea. Its overall five-bladed β-propeller fold is conserved with previously reported FTFs, but also shows some unique features. The InuHj structure is closer to those of Gram-negative bacteria, with exceptions such as residue E266, which is conserved in FTFs of Gram-positive bacteria and has possible role in fructan polymer synthesis in these bacteria as compared to fructooligosaccharide (FOS) production by FTFs of Gram-negative bacteria. Highly negative electrostatic surface potential of InuHj, due to a large amount of acidic residues, likely contributes to its halophilicity. The complex of InuHj with 1-kestose indicates that the residues D287 in the 4B-4C loop, Y330 in 4D-5A and D361 in the unique α2 helix may interact with longer FOSs and facilitate the binding of longer FOS chains during synthesis. The outcome of this work will provide targets for future structure-function studies of FTF enzymes, particularly those from archaea

Analytical Models for Valence Fermions in Isotropic Traps

For isotropic confining Ioffe-Pritchard or TOP potentials, a valence fermion trapped with a closed core of other fermions can be described by an analytical effective one-particle model with a physical eigenspectrum. Related constructions exist for Paul and Penning traps. The analytical models arise from quantum-mechanical supersymmetry.Comment: accepted for publication in Physics Letters

High glucose disrupts oligosaccharide recognition function via competitive inhibition : a potential mechanism for immune dysregulation in diabetes mellitus

Diabetic complications include infection and cardiovascular disease. Within the immune system, host-pathogen and regulatory host-host interactions operate through binding of oligosaccharides by C-type lectin. A number of C-type lectins recognise oligosaccharides rich in mannose and fucose – sugars with similar structures to glucose. This raises the possibility that high glucose conditions in diabetes affect protein-oligosaccharide interactions via competitive inhibition. Mannose binding lectin, soluble DC-SIGN & DC-SIGNR, and surfactant protein D, were tested for carbohydrate binding in the presence of glucose concentrations typical of diabetes, via surface plasmon resonance and affinity chromatography. Complement activation assays were performed in high glucose. DC-SIGN and DC-SIGNR expression in adipose tissues was examined via immunohistochemistry. High glucose inhibited C-type lectin binding to high-mannose glycoprotein and binding of DC-SIGN to fucosylated ligand (blood group B) was abrogated in high glucose. Complement activation via the lectin pathway was inhibited in high glucose and also in high trehalose - a nonreducing sugar with glucoside stereochemistry. DC-SIGN staining was seen on cells with DC morphology within omental and subcutaneous adipose tissues. We conclude that high glucose disrupts C-type lectin function, potentially illuminating new perspectives on susceptibility to infectious and inflammatory disease in diabetes. Mechanisms involve competitive inhibition of carbohydrate-binding within sets of defined proteins, in contrast to broadly indiscriminate, irreversible glycation of proteins

Characterization of Microfibrillar-associated Protein 4 (MFAP4) as a Tropoelastin- and Fibrillin-binding Protein Involved in Elastic Fiber Formation

MFAP4 (microfibrillar-associated protein 4) is an extracellular glycoprotein found in elastic fibers without a clearly defined role in elastic fiber assembly. In the present study, we characterized molecular interactions between MFAP4 and elastic fiber components. We established that MFAP4 primarily assembles into trimeric and hexameric structures of homodimers. Binding analysis revealed that MFAP4 specifically binds tropoelastin and fibrillin-1 and -2, as well as the elastin cross-linking amino acid desmosine, and that it co-localizes with fibrillin-1-positive fibers in vivo. Site-directed mutagenesis disclosed residues Phe(241) and Ser(203) in MFAP4 as being crucial for type I collagen, elastin, and tropoelastin binding. Furthermore, we found that MFAP4 actively promotes tropoelastin self-assembly. In conclusion, our data identify MFAP4 as a new ligand of microfibrils and tropoelastin involved in proper elastic fiber organization