Mucopolysaccharidosis type I (MPS I) is due to deficiency of α-l-iduronidase (IDUA) and subsequent storage of undegraded glycosaminoglycans (GAG). The severe form of the disease, known as Hurler syndrome, is characterized by mental retardation and neurodegeneration of unknown etiology. To identify potential biomarkers and unveil the neuropathology mechanism of MPS I disease, two-dimensional polyacrylamide gel electrophoresis (PAGE) and nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) were applied to compare proteome profiling of brains from MPS I and control mice (5-month old). A total of 2055 spots were compared, and 25 spots (corresponding to 50 different proteins) with a fold change ≥ 3.5 and a p value < 0.05 between MPS I and control mice were further analyzed by nanoLC-MS/MS. These altered proteins could be divided into three major groups based on Gene Ontology (GO) terms: proteins involved in metabolism, neurotransmission and cytoskeleton. Cytoskeletal proteins including ACTA1, ACTN4, TUBB4B and DNM1 were significantly downregulated. STXBP1, a regulator of synaptic vesicle fusion and docking was also downregulated, indicating impaired synaptic transmission. Additionally, proteins regulating Ca2 + and H+ homeostasis including ATP6V1B2 and RYR3 were downregulated, which may be related to disrupted autophagic and endocytotic pathways. Notably, there is no altered expression in proteins associated with cell death, ubiquitin or inflammation. These results for the first time highlight the important role of alterations in metabolism pathways, intracellular ionic homeostasis and the cytoskeleton in the neuropathology of MPS I disease. The proteins identified in this study would provide potential biomarkers for diagnostic and therapeutic studies of MPS I.

Surface exposed protein antigens of the malaria parasite Plasmodium falciparum frequently harbor multiple dimorphic amino acid positions. These are associated with parasite immune evasion and represent a major obstacle for subunit vaccine design. Here, we have analyzed the flexibility of the humoral immune response against a semiconserved sequence (YX44LFX47KEKMX52L) of the key malaria blood stage vaccine candidate merozoite surface protein-1 (MSP-1). Monoclonal antibodies (mAbs) raised against one of the six described natural sequence variants of MSP-143–53 were analyzed for cross-reactivity with the other allelic forms, which differ in one to three positions from the immunizing sequence. Enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) spectroscopy demonstrated marked differences in mAb binding avidity to the variant sequences and isothermal titration calorimetry (ITC) provided evidence for a very low affinity of some of the interactions. In immunofluorescence analysis (IFA) and Western blotting analysis, the mAbs nevertheless stained all analyzed parasite clones expressing MSP-143–53 variant sequences. When used for the evaluation of humoral immune responses in clinical malaria vaccine trials, these two commonly used methods may thus not be suitable to distinguish biologically functional high affinity antibody responses from irrelevant low-affinity cross-reactivities.

The monoclonal antibody A6 binds a conformational epitope comprising mainly the CC' surface loop on the N-terminal fibronectin type-III domain of the extracellular interferon gamma receptor (IFNgammaR). The crystal structure of an A6 Fab-IFNgammaR complex revealed an interface rich in the aromatic side chains of Trp, Tyr, and His residues. These aromatic side chains appear to interact with both polar and hydrophobic groups at the interface, a property which, in general, may be advantageous for ligand binding. To analyze these interactions in more detail, the affinities of 19 A6 alanine-scanning mutants for the IFNgammaR have been measured, using engineered A6 single chain variable region fragments, and a surface plasmon resonance biosensor. Energetically important side chains (DeltaG(mutant) - DeltaG(wt) > 2.4 kcal/mol), that form distinct hot spots in the binding interface, have been identified on both proteins. These include V(L)W92 in A6, whose benzenoid ring appears well situated for a pi-cation (or pi-amine) interaction with the side chain of receptor residue K47 and simultaneously for T-stacking onto the indole ring of W82 in the receptor. At another site, energetically important residues V(H)W52 and V(H)W53, as well as V(H)D54 and V(H)D56, surround the aliphatic side chain of the hot receptor residue K52. Taken together, the results show that side chains distributed across the interface, including many aromatic ones, make key energetic contributions to binding. In addition, the receptor CC' loop has been subjected to random mutagenesis, and receptor mutants with high affinity for A6 have been selected by phage display. Residues previously identified as important for receptor binding to A6 were conserved in the clones isolated. Some mutants, however, showed a much improved affinity for A6, due to changes at Glu55, a residue that appeared to be energetically unimportant for binding the antibody by alanine-scanning mutagenesis. An E55P receptor mutant bound A6 with a 600-fold increase in affinity (K(D) approximately 20 pM), which is one of the largest improvements in affinity from a single point mutation reported so far at any protein-protein interface.

SUMMARY Immunopotentiating reconstituted influenza virosomes (IRIV) were used as a delivery system for the synthetic peptide-based malaria vaccine SPf66. The reduced SPf66 peptide molecules containing terminal cysteine residues were covalently attached to phosphatidylethanolamine with the heterobifunctional crosslinker γ-maleimidobutyric acid N-hydroxysuccinimide ester. The SPf66-phosphatidylethanolamine was incorporated into IRIV and BALB/c mice were immunized twice by intramuscular injection with peptide-loaded virosomes. Titres of elicited anti-SPf66 IgG were determined by ELISA. These titres were significantly higher and the required doses of antigen were lower, when mice had been preimmunized with a commercial whole virus influenza vaccine. After preimmunization with the influenza vaccine, SPf66-IRIV elicited far more consistently anti-SPf66 antibody responses than SPf(66)n adsorbed to alum. MoAb produced by four B cell hybridoma clones derived from a SPf66-IRIV-immunized mouse cross-reacted with Plasmodium falciparum blood stage parasites in immunofluorescence assays. All four MoAbs were specific for the merozoite surface protein-1 (MSP-1)-derived 83.1 portion of SPf66. Sequencing of their functionally rearranged κ light chain variable region genes demonstrated that the four hybridomas were generated from clonally related splenic B cells. Biomolecular interaction analyses (BIA) together with these sequencing data provided evidence for the selection of somatically mutated affinity-matured B cells upon repeated immunization with SPf66-IRIV. The results indicate that IRIV are a suitable delivery system for synthetic peptide vaccines and thus have a great potential for the design of molecularly defined combined vaccines targeted against multiple antigens and development stages of one parasite, as well as against multiple pathogens.

A complex formed between the extracellular human interferon gamma receptor α-chain (hIFNγR) and the Fab fragment of the neutralizing antibody A6 has been studied by site-directed mutagenesis. Five complementarity determining regions of the A6 antibody interact primarily with the CC′ surface loop of the receptor, from Lys47 to Trp56, although contact is also made with residues in the neighbouring F strand, in particular with Trp82. The relative contribution that individual side-chains make to complex stabilization was assessed with 21 receptors mutants, whose affinity for A6 was monitored using a surface plasmon resonance biosensor, as well as by solution-phase competition ELISA. The results reveal two lysine side-chains (Lys47 and Lys52), an asparagine side-chain (Asn53), and two aromatic side-chains (Tyr49 and Trp82) in the receptor that are important for recognition by A6. The role of aromatic side-chains in antibody-antigen recognition is of particular interest, not least in this case because 13 aromatic groups (six Tyr, six Trp and one His) are present at the interface (four in VL, six in VH and three in the receptor), and several are proximal to the charged and polar side-chains of Lys47, Lys52 and Asn53 in the receptor. The results highlight the possibility for aromatic rings to participate in networks of co-operative interactions with not only hydrophobic, but also charged and hydrogen bond donor and acceptor groups, properties that are well suited for creating binding sites for protein epitopes, regardless of the distribution of polar and non-polar surface residues. These findings may contribute, therefore, to an understanding of how surface groups on proteins are captured by the often aromatic-rich hypervariable loops of antibodies, and may be of value for the design of molecules with novel recognition properties.

A straightforward and effective method of stabilizing a P-hairpin conformation in a cyclic protein loop mimetic is described, which exploits the templating effect of a heterochiral D-Pro-L-Pro dipeptide unit. A twelve-residue P-hairpin loop was grafted from the extracellular interferon gamma receptor onto the heterochiral D-Pro-L-Pro dipeptide template to afford a fourteen-residue cyclic peptide. The residues directly attached to the D-Pro-L-Pro template are shown by NMR spectroscopy to structurally mimic corresponding residues in adjacent antiparallel beta-strands in the receptor. MD Simulations with and without time-averaged distance restraints support this view and indicate that the tip of the loop is more flexible, as inferred also for the receptor protein from crystallographic data. The templating effect of the heterochiral diproline unit also promotes efficient backbone cyclization of the fourteen-residue linear peptide precursor, suggesting that a wide variety of related protein loop mimetics incorporating the D-Pro-L-Pro template might be readily accessible.

Immobilization of proteins to a solid phase leads to denaturation of the adsorbed molecules which may subsequently affect biological interactions. However, for many applications maintenance of the native structure is desired. Therefore, an indirect immobilization system was developed, based on binding of the albumin binding domain (ABP) of streptococcal protein G to rat serum albumin (RSA) precoated on a solid phase (RSA-microtiter plates). Escherichia coli vectors were adapted for production of recombinant protein fused to ABP and the 6 X His-tag. The expressed ABP tag was found to form homodimers. Plasmon resonance was used to study the interaction between an ABP fusion protein and immobilized RSA. Apparent on- and off-rates were calculated using a model for a bivalent analyte (k(a1) = 3.37 x 10(4) M(-1) s(-1), k(d1) = 1.23 x 10(-4) s(-1)). Thus, the stability of the ABP-RSA interaction can be explained by a slow off-rate. This was confirmed by chase experiments in an ELISA format. The ABP-RSA interaction remained stable after addition of different albumins. This immobilization system was used for the development of an ELISA to detect antibodies against Borna disease virus protein p40. The use of RSA-microtiter plates for indirect immobilization of ABP fusion protein was shown to be superior to direct adsorption on plastic. To obtain maximal antibody binding ten times less antigen was needed for indirect immobilization compared to direct adsorption. The binding capacity of the RSA-microtiter plates was determined to be about 0.8 pmol of monomeric ABP protein.

The extracellular interferon γ receptor α-chain comprises two immunoglobulin-like domains, each with fibronectin type-III topology, which are responsible for binding interferon γ at the cell surface. The epitopes on the human receptor recognized by three neutralizing antibodies, A6, γR38 and γR99, have been mapped by homolog scanning mutagenesis. In this way, a loop connecting β-strands C and C′ in the N-terminal domain was identified as a key component of the epitopes bound by A6 and γR38, whereas γR99 binds to the C-terminal domain in a region including strands A and B and part of the large C′E loop. The epitope for A6 was confirmed in a crystal structure of a complex between a recombinant N-terminal receptor domain and the Fab fragment from A6, determined by X-ray diffraction to 2.8 Å resolution. The antibody-antigen interface buries 1662 Å2 of protein surface, including 22 antibody residues from five complementarity determining regions, primarily through interactions with the CC′ surface loop of the receptor. The floor of the antigen binding cavity is formed mainly by residues from CDR L3 and CDR H3 while a surrounding ridge is formed by residues from all other CDRs except L2. Many potential polar interactions, as well as 13 aromatic side-chains, four in VL, six in VH and three in the receptor, are situated at the interface. The surface of the receptor contacted by A6 overlaps to a large extent with that contacted by interferon-γ, in the ligand-receptor complex. However, the conformation of this epitope is very different in the two complexes, demonstrating that conformational mobility in a surface loop on this cytokine receptor permits steric and electrostatic complementarity to two quite differently shaped binding sites.

Three single-chain antibody fragments that recognize the extracellular human interferon gamma receptor alpha-chain (IFN gamma R), and inhibit the binding of human IFN gamma, have been produced in Escherichia coli. These fragments are derived from murine anti-receptor monoclonal antibodies, and comprise the variable heavy (VH) domain linked to the variable light (VL) chain through a 15 amino acid linker [(GGGGS)3]. Using surface plasmon resonance technology (BIAcore), the soluble proteins were shown to retain a high affinity for recombinant IFN gamma R, and by radioimmunoassay to possess a high inhibitory activity towards IFN gamma-binding to human Raji cells. The antibody fragments most likely recognize epitopes that overlap the cytokine binding site on the receptor surface. Attempts to dissect further the antibodies to isolated VH- and VL-chains and to synthetic linear and cyclic peptides derived from the individual complementarity determining regions failed to afford fragments with significant IFN gamma R binding affinity. Nevertheless, these native-like variable region fragments and petidomimetics derived from them are of interest in the design of novel IFN gamma R antagonists.