Jean van den Elsen

Adhesion mechanism of human beta 2-glycoprotein I to phospholipids based on its crystal structure

Human β2‐glycoprotein I is a heavily glycosylated five‐domain plasma membrane‐adhesion protein, which has been implicated in blood coagulation and clearance of apoptotic bodies from the circulation. It is also the key antigen in the autoimmune disease anti‐phospholipid syndrome. The crystal structure of β2‐glycoprotein I isolated from human plasma reveals an elongated fish‐hook‐like arrangement of the globular short consensus repeat domains. Half of the C‐terminal fifth domain deviates strongly from the standard fold, as observed in domains one to four. This aberrant half forms a specific phospholipid‐binding site. A large patch of 14 positively charged residues provides electrostatic interactions with anionic phospholipid headgroups and an exposed membrane‐insertion loop yields specificity for lipid layers. The observed spatial arrangement of the five domains suggests a functional partitioning of protein adhesion and membrane adhesion over the N‐ and C‐terminal domains, respectively, separated by glycosylated bridging domains. Coordinates are in the Protein Data Bank (accession No. 1QUB).

Structure of Golgi α-mannosidase II: a target for inhibition of growth and metastasis of cancer cells

Golgi α‐mannosidase II, a key enzyme in N‐glycan processing, is a target in the development of anti‐ cancer therapies. The crystal structure of Drosophila Golgi α‐mannosidase II in the absence and presence of the anti‐cancer agent swainsonine and the inhibitor deoxymannojirimycin reveals a novel protein fold with an active site zinc intricately involved both in the substrate specificity of the enzyme and directly in the catalytic mechanism. Identification of a putative GlcNAc binding pocket in the vicinity of the active site cavity provides a model for the binding of the GlcNAcMan5GlcNAc2 substrate and the consecutive hydrolysis of the α1,6‐ and α1,3‐linked mannose residues. The enzyme–inhibitor interactions observed provide insight into the catalytic mechanism, opening the door to the design of novel inhibitors of α‐mannosidase II.

Interaction of Human Complement with Sbi, a Staphylococcal Immunoglobulin-binding Protein INDICATIONS OF A NOVEL MECHANISM OF COMPLEMENT EVASION BY STAPHYLOCOCCUS AUREUS

Staphylococcal immunoglobulin-binding protein, Sbi, is a 436-residue protein produced by many strains of Staphylococcus aureus. It was previously characterized as being cell surface-associated and having binding capacity for human IgG and β2-glycoprotein I. Here we show using small angle x-ray scattering that the proposed extracellular region of Sbi (Sbi-E) is an elongated molecule consisting of four globular domains, two immunoglobulin-binding domains (I and II) and two novel domains (III and IV). We further show that together domains III and IV (Sbi-III-IV), as well as domain IV on its own (Sbi-IV), bind complement component C3 via contacts involving both the C3dg fragment and the C3a anaphylatoxin domain. Preincubation of human serum with either Sbi-E or Sbi-III-IV is inhibitory to all complement pathways, whereas domain IV specifically inhibits the alternative pathway. Monitoring C3 activation in serum incubated with Sbi fragments reveals that Sbi-E and Sbi-III-IV both activate the alternative pathway, leading to consumption of C3. By contrast, inhibition of this pathway by Sbi-IV does not involve C3 consumption. The observation that Sbi-E activates the alternative pathway is counterintuitive to intact Sbi being cell wall-associated, as recruiting complement to the surface of S. aureus would be deleterious to the bacterium. Upon re-examination of this issue, we found that Sbi was not associated with the cell wall fraction, but rather was found in the growth medium, consistent with it being an excreted protein. As such, our data suggest that Sbi helps mediate bacterial evasion of complement via a novel mechanism, namely futile fluid-phase consumption.

The Staphylococcus aureus Protein Sbi Acts as a Complement Inhibitor and Forms a Tripartite Complex with Host Complement Factor H and C3b

The Gram-positive bacterium Staphylococcus aureus, similar to other pathogens, binds human complement regulators Factor H and Factor H related protein 1 (FHR-1) from human serum. Here we identify the secreted protein Sbi (Staphylococcus aureus binder of IgG) as a ligand that interacts with Factor H by a—to our knowledge—new type of interaction. Factor H binds to Sbi in combination with C3b or C3d, and forms tripartite Sbi∶C3∶Factor H complexes. Apparently, the type of C3 influences the stability of the complex; surface plasmon resonance studies revealed a higher stability of C3d complexed to Sbi, as compared to C3b or C3. As part of this tripartite complex, Factor H is functionally active and displays complement regulatory activity. Sbi, by recruiting Factor H and C3b, acts as a potent complement inhibitor, and inhibits alternative pathway-mediated lyses of rabbit erythrocytes by human serum and sera of other species. Thus, Sbi is a multifunctional bacterial protein, which binds host complement components Factor H and C3 as well as IgG and β2-glycoprotein I and interferes with innate immune recognition.

Predicting the virulence of MRSA from its genome sequence

Microbial virulence is a complex and often multifactorial phenotype, intricately linked to a pathogens evolutionary trajectory. Toxicity, the ability to destroy host cell membranes, and adhesion, the ability to adhere to human tissues, are the major virulence factors of many bacterial pathogens, including Staphylococcus aureus. Here, we assayed the toxicity and adhesiveness of 90 MRSA (methicillin resistant S. aureus) isolates and found that while there was remarkably little variation in adhesion, toxicity varied by over an order of magnitude between isolates, suggesting different evolutionary selection pressures acting on these two traits. We performed a genome-wide association study (GWAS) and identified a large number of loci, as well as a putative network of epistatically interacting loci, that significantly associated with toxicity. Despite this apparent complexity in toxicity regulation, a predictive model based on a set of significant single nucleotide polymorphisms (SNPs) and insertion and deletions events (indels) showed a high degree of accuracy in predicting an isolates toxicity solely from the genetic signature at these sites. Our results thus highlight the potential of using sequence data to determine clinically relevant parameters and have further implications for understanding the microbial virulence of this opportunistic pathogen.

X-ray Crystal Structure of the C4d Fragment of Human Complement Component C4

C4 fulfills a vital role in the propagation of the classical and lectin pathways of the complement system. Although there are no reports to date of a C4 functional activity that is mediated solely by the C4d region, evidence clearly points to it having a vital role in a number of the properties of native C4 and its major activation fragment, C4b. Contained within the C4d region are the thioester-forming residues, the four isotype-specific residues controlling the C4A/C4B transacylation preferences, a binding site for nascent C3b important in assembling the classical pathway C5 convertase and determinants for the Chido/Rodgers (Ch/Rg) blood group antigens. In view of its functional importance, we undertook to determine the three-dimensional structure of C4d by X-ray crystallography. Here we report the 2.3A resolution structure of C4Ad, the C4d fragment derived from the human C4A isotype. Although the approximately 30% sequence identity between C4Ad and the corresponding fragment of C3 might be expected to establish a general fold similarity between the two molecules, C4Ad in fact displays a fold that is essentially superimposable on the structure of C3d. By contrast, the electrostatic characteristics of the various faces of the C4Ad molecule show marked differences from the corresponding faces of C3d, likely reflecting the differences in function between C3 and C4. Residues previously predicted to form the major Ch/Rg epitopes were proximately located and accessible on the concave surface of C4Ad. In addition to providing further insights on the current models for the covalent binding reaction, the C4Ad structure allows one to rationalize why C4d is not a ligand for complement receptor 2. Finally the structure allows for the visualization of the face of the molecule containing the binding site for C3b utilized in the assembly of classical pathway C5 convertase.

A crystal structure of the complex between human complement receptor 2 and its ligand C3d.

The topology of this molecular interface provides a foundation for the design of therapeutics against autoreactive B cells. The interaction of complement receptor 2 (CR2)—which is present on B cells and follicular dendritic cells—with its antigen-bound ligand C3d results in an enhanced antibody response, thus providing an important link between the innate and adaptive immune systems. Although a cocrystal structure of a complex between C3d and the ligand-binding domains of CR2 has been published, several aspects of this structure, including the position in C3d of the binding interface, remained controversial because of disagreement with biochemical data. We now report a cocrystal structure of a CR2(SCR1-2):C3d complex at 3.2 angstrom resolution in which the interaction interfaces differ markedly from the previously published structure and are consistent with the biochemical data. It is likely that, in the previous structure, the interaction was influenced by the presence of zinc acetate additive in the crystallization buffer, leading to a nonphysiological complex. Detailed knowledge of the binding interface now at hand gives the potential to exploit the interaction in vaccine design or in therapeutics directed against autoreactive B cells.

Analysis of protein glycation using phenylboronate acrylamide gel electrophoresis

The incorporation of the specialized carbohydrate affinity ligand methacrylamido phenylboronic acid in polyacrylamide gels for SDS‐PAGE analysis has been successful for the separation of carbohydrates and has here been adapted for the analysis of post‐translationally modified proteins. While conventional SDS‐PAGE analysis cannot distinguish between glycated and unglycated proteins, methacrylamido phenylboronate acrylamide gel electrophoresis (mP‐AGE) in low loading shows dramatic retention of δ‐gluconolactone modified proteins, while the mobility of the unmodified proteins remains unchanged. With gels containing 1% methacrylamido phenylboronate, mP‐AGE analysis of gluconoylated recombinant protein Sbi results in the retention of the modified protein at a position expected for a protein that has quadrupled its expected molecular size. Subsequently, mP‐AGE was tested on HSA, a protein that is known to undergo glycation under physiological conditions. mP‐AGE could distinguish between various carbohydrate‐protein adducts, using in vitro glycated HSA, and discriminate early from late glycation states of the protein. Enzymatically glycosylated proteins show no altered retention in the phenylboronate‐incorporated gels, rendering this method highly selective for glycated proteins. We reveal that a trident interaction between phenylboronate and the Amadori cis 1,2 diol and amine group provides the molecular basis of this specificity. These results epitomize mP‐AGE as an important new proteomics tool for the detection, separation, visualization and identification of protein glycation. This method will aid the design of inhibitors of unwanted carbohydrate modifications in recombinant protein production, ageing, diabetes, cardiovascular diseases and Alzheimers disease.

The Development of Boronic Acids as Sensors and Separation Tools

Synthetic receptors for diols that incorporate boronic acid motifs have been developed as new sensors and separation tools. Utilizing the reversible interactions of diols with boronic acids to form boronic esters under new binding regimes has provided new hydrogel constructs that have found use as dye-displacement sensors and electrophoretic separation tools; similarly, molecular boronic-acid-containing chemosensors were constructed that offer applications in the sensing of diols. This review provides a somewhat-personal perspective of developments in boronic-acid-mediated sensing and separation, placed in the context of the seminal works of others in the area, as well as offering a concise summary of the contributions of the co-authors in the area.

Staphylococcus aureus proteins Sbi and Efb recruit human plasmin to degrade complement C3 and C3b.

Upon host infection, the human pathogenic microbe Staphylococcus aureus (S. aureus) immediately faces innate immune reactions such as the activated complement system. Here, a novel innate immune evasion strategy of S. aureus is described. The staphylococcal proteins surface immunoglobulin-binding protein (Sbi) and extracellular fibrinogen-binding protein (Efb) bind C3/C3b simultaneously with plasminogen. Bound plasminogen is converted by bacterial activator staphylokinase or by host-specific urokinase-type plasminogen activator to plasmin, which in turn leads to degradation of complement C3 and C3b. Efb and to a lesser extend Sbi enhance plasmin cleavage of C3/C3b, an effect which is explained by a conformational change in C3/C3b induced by Sbi and Efb. Furthermore, bound plasmin also degrades C3a, which exerts anaphylatoxic and antimicrobial activities. Thus, S. aureus Sbi and Efb comprise platforms to recruit plasmin(ogen) together with C3 and its activation product C3b for efficient degradation of these complement components in the local microbial environment and to protect S. aureus from host innate immune reactions.