Annette K. Shrive

Three dimensional structure of human C-reactive protein.

The structure of the classical acute phase reactant human C-reactive protein provides evidence that phosphocholine binding is mediated through calcium and a hydrophobic pocket centred on Phe 66. The residue Glu 81 is suitably positioned to interact with the choline group. A cleft on the pentameric face opposite to that containing the calcium site may have an important functional role. The structure provides insights into the molecular mechanisms by which this highly conserved plasma protein, for which no polymorphism or deficiency state is known, may exert its biological role.

Topology and Structure of the C1q-Binding Site on C-Reactive Protein

The host defense functions of human C-reactive protein (CRP) depend to a great extent on its ability to activate the classical complement pathway. The aim of this study was to define the topology and structure of the CRP site that binds C1q, the recognition protein of the classical pathway. We have previously reported that residue Asp112 of CRP plays a major role in the formation of the C1q-binding site, while the neighboring Lys114 hinders C1q binding. The three-dimensional structure of CRP shows the presence of a deep, extended cleft in each protomer on the face of the pentamer opposite that containing the phosphocholine-binding sites. Asp112 is part of this marked cleft that is deep at its origin but becomes wider and shallower close to the inner edge of the protomer and the central pore of the pentamer. The shallow end of the pocket is bounded by the 112–114 loop, residues 86–92 (the inner loop), the C terminus of the protomer, and the C terminus of the pentraxin α-helix 169–176, particularly Tyr175. Mutational analysis of residues participating in the formation of this pocket demonstrates that Asp112 and Tyr175 are important contact residues for C1q binding, that Glu88 influences the conformational change in C1q necessary for complement activation, and that Asn158 and His38 probably contribute to the correct geometry of the binding site. Thus, it appears that the pocket at the open end of the cleft is the C1q-binding site of CRP.

The two types of 3-dehydroquinase have distinct structures but catalyze the same overall reaction.

The structures of enzymes catalyzing the reactions in central metabolic pathways are generally well conserved as are their catalytic mechanisms. The two types of 3-dehydroquinate dehydratase (DHQase) are therefore most unusual since they are unrelated at the sequence level and they utilize completely different mechanisms to catalyze the same overall reaction. The type I enzymes catalyze a cis-dehydration of 3-dehydroquinate via a covalent imine intermediate, while the type II enzymes catalyze a trans-dehydration via an enolate intermediate. Here we report the three-dimensional structures of a representative member of each type of biosynthetic DHQase. Both enzymes function as part of the shikimate pathway, which is essential in microorganisms and plants for the biosynthesis of aromatic compounds including folate, ubiquinone and the aromatic amino acids. An explanation for the presence of two different enzymes catalyzing the same reaction is presented. The absence of the shikimate pathway in animals makes it an attractive target for antimicrobial agents. The availability of these two structures opens the way for the design of highly specific enzyme inhibitors with potential importance as selective therapeutic agents.

High resolution structural insights into ligand binding and immune cell recognition by human lung surfactant protein D

Lung surfactant protein D (SP-D) can directly interact with carbohydrate residues on pulmonary pathogens and allergens, stimulate immune cells, and manipulate cytokine and chemokine profiles during the immune response in the lungs. Therapeutic administration of rfhSP-D, a recombinant homotrimeric fragment of human SP-D comprising the alpha-helical coiled-coil neck plus three CRDs, protects mice against lung allergy and infection caused by the fungal pathogen Aspergillus fumigatus. The high resolution crystal structures of maltose-bound rfhSP-D to 1.4A, and of rfhSP-D to 1.6A, define the fine detail of the mode and nature of carbohydrate recognition and provide insights into how a small fragment of human SP-D can bind to allergens/antigens or whole pathogens, and at the same time recruit and engage effector cells and molecules of humoral immunity. A previously unreported calcium ion, located on the trimeric axis in a pore at the bottom of the funnel formed by the three CRDs and close to the neck-CRD interface, is coordinated by a triad of glutamate residues which are, to some extent, neutralised by their interactions with a triad of exposed lysine residues in the funnel. The spatial relationship between the neck and the CRDs is maintained internally by these lysine residues, and externally by a glutamine, which forms a pair of hydrogen-bonds within an external cleft at each neck-CRD interface. Structural links between the central pore and the cleft suggest a possible effector mechanism for immune cell surface receptor binding in the presence of bound, extended natural lipopolysaccharide and phospholipid ligands. The structural requirements for such an effector mechanism, involving both the trimeric framework for multivalent ligand binding and recognition sites formed from more than one subunit, are present in both native hSP-D and rfhSP-D, providing a possible explanation for the significant biological activity of rfhSP-D.

Calcium binding sites in tomato bushy stunt virus visualized by laue crystallography

We have collected Laue diffraction data from crystals of tomato bushy stunt virus using the full white X-ray spectrum from the wiggler magnet of the Synchrotron Radiation Source at Daresbury, U.K. A single 24 second exposure of a crystal soaked in EDTA yielded a data set that was 90% complete between 6 and 3.5 A resolution. A large proportion of the data could be measured using an overlap deconvolution routine to separate spatially overlapping reflections in the dense Laue photograph. Reflections with I greater than 2 sigma I (40% of the data set) were subjected to wavelength normalization. A difference Fourier map between these reflections and a monochromatic native set showed, after icosahedral averaging, the three pairs of Ca2+ binding sites related by quasi-symmetry and the movement of a liganding loop in the protein at the A/C subunit interface. The extent and quality of the data obtained from a single Laue photograph of this virus were thus sufficient to detect clearly such small structural alterations. In a second experiment, a Laue photograph was taken from a crystal that was soaked first in EDTA and then in GdCl3. A difference Fourier map between this Laue data set and the Laue data set from the EDTA-soaked crystal showed clearly the Gd3+ sites in the capsid, demonstrating that the Laue technique is a reliable and efficient means for data collection with virus crystals.

Isolation and characterisation of pentraxin-like serum proteins from the common carp Cyprinus carpio

There is increasing economic and ecological interest in the development of assays for the early detection of infection, disease activity and environmental stress in marine and freshwater animals. In humans the serum pentraxin C-reactive protein (CRP) is universally used as a clinical indicator of inflammation and underlying infection. As a first step towards assessing the potential of an immunoassay for CRP in fish, we have isolated and characterised common carp Cyprinus carpio CRP and a highly specific and sensitive anti-carp CRP polyclonal antibody has been raised. The results show levels of CRP in healthy fish similar to those found in healthy humans. A protein of unknown function, which displays the characteristic calcium-dependent phosphate monoester binding exhibited by CRP and some similarity to the known fish pentraxin sequences, has also been identified.

Inflammatory interactions in fish exposed to pollutants and parasites: a role for apoptosis and C reactive protein

Although previous studies have highlighted the inflammatory responses of fish infected with parasites and exposed to pollutants, very little is known about how these two stressors interact within the fish. In this review, which also contains original data, the effect of these two parameters on the fish inflammatory response is assessed and, in particular, the role of apoptosis and the acute phase protein, C reactive protein, is evaluated. In Cyprinus carpio exposed to 0.5 mg NH4+ l(-1) or 0.1 mg Cd2+ l(-1) and experimentally infected with the blood fluke, Sanguinicola inermis, the pollutant type and the order in which the fish experiences the parasite and toxicant, significantly affects the ultrastructural appearance and cellular content of the pronephros and thymus. This is reflected in the intensity of infection where the pollutant appears to have less effect on an established infection. Both stressors, pollutant and infection, may mediate their effects via the endocrine system. Studies have revealed that cortisol at 100 ng ml(-1) is able to induce apoptosis in pronephric cells of carp and that an increase in apoptosis is associated with an increase in phagocytosis in this immune organ. In addition, C reactive protein, which is used as a biomarker of the inflammatory response in humans and other mammals, is evaluated as a possible indicator of physiological states in fish exposed to pathogens and pollutants.

Dietary β-glucan stimulate complement and C-reactive protein acute phase responses in common carp (Cyprinus carpio) during an Aeromonas salmonicida infection

The effect of β-glucans as feed additive on the profile of C-reactive protein (CRP) and complement acute phase responses was studied in common carp Cyprinus carpio after exposition to a bacterial infection with Aeromonas salmonicida. Carp were orally administered with β-glucan (MacroGard®) for 14 days with a daily β-glucan intake of 6 mg per kg body weight. Fish were then intraperitoneally injected with either PBS or 1 × 10⁸ bacteria per fish and sampled at time 0, 6, 12, 24, 48, 72, 96 and 120 h post-injection (p.i.) for serum and head kidney, liver and mid-gut tissues. CRP levels and complement activity were determined in the serum samples whilst the gene expression profiles of CRP and complement related genes (crp1, crp2, c1r/s, bf/c2, c3 and masp2) were analysed in the tissues by quantitative PCR. Results obtained showed that oral administration of β-glucan for 14 days significantly increased serum CRP levels up to 2 fold and serum alternative complement activity (ACP) up to 35 fold. The bacterial infection on its own (i.e. not combined with a β-glucan feeding) did have significant effects on complement response whilst CRP was not detectably induced during the carp acute phase reaction. However, the combination of the infection and the β-glucan feeding did show significant effects on both CRP and complement profiles with higher serum CRP levels and serum ACP activity in the β-glucan fed fish than in the control fed fish. In addition, a distinct organ and time dependent expression profile pattern was detected for all the selected genes: a peak of gene expression first occurred in the head kidney tissue (6 h p.i. or 12 h p.i.), then an up-regulation in the liver several hours later (24 h p.i.) and finally up- or down-regulations in the mid-gut at 24 h p.i. and 72 h p.i. In conclusion, the results of this study suggest that MacroGard® stimulated CRP and complement responses to A. salmonicida infection in common carp.

The three-dimensional structure of calcium-depleted human C-reactive protein from perfectly twinned crystals.

C-reactive protein is a member of the pentraxin family of oligomeric serum proteins which has been conserved through evolution, homologues having been found in every species in which they have been sought. Human C-reactive protein (hCRP) is the classical acute-phase reactant produced in large amounts in response to tissue damage and inflammation and is used almost universally as a clinical indicator of infection and inflammation. The role of hCRP in host defence and the calcium-dependent ligand-binding specificity of hCRP for phosphocholine moieties have long been recognized. In order to clarify the structural rearrangements associated with calcium binding, the reported affinity of calcium-depleted hCRP for polycations and other ligands, and the role of calcium in protection against denaturation and proteolysis, the structure of calcium-depleted hCRP has been determined by X-ray crystallography. Crystals of calcium-depleted hCRP are invariably twinned and those suitable for analysis are merohedral type II twins of point group 4 single crystals. The structure has been solved by molecular replacement using the calcium-bound hCRP structure [Shrive et al. (1996), Nature Struct. Biol. 3, 346-354]. It reveals two independent pentamers which form a face-to-face decamer across a dyad near-parallel to the twinning twofold axis. Cycles of intensity deconvolution, density modification (tenfold NCS) and model building, eventually including refinement, give a final R factor of 0.19 (R(free) = 0.20). Despite poor definition in some areas arising from the limited resolution of the data and from the twinning and disorder, the structure reveals the probable mode of twinning and the conformational changes, localized in one of the calcium-binding loops, which accompany calcium binding.

Serum CRP-like protein profile in common carp Cyprinus carpio challenged with Aeromonas hydrophila and Escherichia coli lipopolysaccharide.

The potential of C-reactive protein (CRP)-like proteins to be used as a biomarker of health status in cultured carp obtained from various European fish lines has been assessed. Varying CRP-like protein levels in the serum of carp were monitored using an indirect competitive enzyme-linked immunosorbent assay. CRP-like protein basal levels in normal fish varied between carp lines, ranging on average from 2.9+/-0.15 to 12.57+/-1.19 microg ml(-1). Serum levels of CRP-like protein in carp were observed to increase several fold in fish infected with the pathogen Aeromonas hydrophila. However, carp injected with Escherichia coli lipopolysaccharide (LPS) serotype 0111:B4 did not exhibit an increase in CRP-like proteins levels.