A novel group of Moraxella catarrhalis UspA proteins mediates cellular adhesion via CEACAMs and vitronectin.

Moraxella catarrhalis (Mx) is a common cause of otitis media and exacerbation of chronic obstructive pulmonary disease, an increasing worldwide problem. Surface proteins UspA1 and UspA2 of Mx bind to a number of human receptors and may function in pathogenesis. Genetic recombination events in the pathogen can generate hybrid proteins termed UspA2H. However, whether certain key functions (e.g. UspA1-specific CEACAM binding) can be exchanged between these adhesin families remains unknown. In this study, we have shown that Mx can incorporate the UspA1 CEACAM1-binding region not only into rare UspA1 proteins devoid of CEACAM-binding ability, but also into UspA2 which normally lack this capacity. Further, a screen of Mx isolates revealed the presence of novel UspA2 Variant proteins (UspA2V) in ∼14% of the CEACAM-binding population. We demonstrate that the expression of UspA2/2V with the CEACAM-binding domain enable Mx to bind both to cell surface CEACAMs and to integrins, the latter via vitronectin. Such properties of UspA2/2V have not been reported to date. The studies demonstrate that the UspA family is much more heterogeneous than previously believed and illustrate the in vivo potential for exchange of functional regions between UspA proteins which could convey novel adhesive functions whilst enhancing immune evasion.

Heterotetrameric forms of human phenylalanine hydroxylase: co-expression of wild-type and mutant forms in a bicistronic system.

Hybrid forms of human phenylalanine hydroxylase (hPAH) mutants have been found to present catalytic activities lower than predicted from the individual recombinant forms, indicating that interallelic complementation could be a major determinant of the metabolic phenotype of compound heterozygous phenylketonuric (PKU) patients. To provide a molecular explanation for interallelic complementation we have here developed a bicistronic expression system and a purification strategy to obtain isolated hPAH heteromeric forms. On co-expression of WT-hPAH (~50% tetramer; ~10% dimer) and the N- and C-terminally truncated form ΔN102/ΔC24-hPAH (~80% dimer) no heterodimers were recovered. Moreover, by co-expression of WT-hPAH and the N-terminally truncated form ΔN102-hPAH (~95% tetramer), heterotetramers, as a result of an assembly of two different homodimers, were isolated. The recovered (WT)/(ΔN102)-hPAH heterotetramers revealed a catalytic activity deviating significantly from that calculated by averaging the respective recombinant homotetrameric forms. The heterotetramer assembly also results in conformational changes in the WT-hPAH protomer, as detected by trypsin limited proteolysis. The finding that the presence of two homodimers with different kinetic parameters influences the properties of the resulting heterotetrameric protein indicates that the dimers exhibit interactions which are transmitted across the assembled tetramer. The bicistronic expression system developed here allowed the isolation of hybrid forms that exhibit negative interallelic complementation, and may represent a model system for studying the molecular pathogenic mechanisms of PAH gene mutations in compound heterozygous PKU patients, providing the rationale to understand the observed inconsistencies both in genotype/phenotype correlations and in the response to BH(4) supplementation.

Design of high-affinity S100-target hybrid proteins.

S100B and S100A10 are dimeric, EF-hand proteins. S100B undergoes a calcium-dependent conformational change allowing it to interact with a short contiguous sequence from the actin-capping protein CapZ (TRTK12). S100A10 does not bind calcium but is able to recruit the N-terminus of annexin A2 important for membrane fusion events, and to form larger multiprotein complexes such as that with the cation channel proteins TRPV5/6. In this work, we have designed, expressed, purified, and characterized two S100-target peptide hybrid proteins comprised of S100A10 and S100B linked in tandem to annexin A2 (residues 1-15) and CapZ (TRTK12), respectively. Different protease cleavage sites (tobacco etch virus, PreScission) were incorporated into the linkers of the hybrid proteins. In situ proteolytic cleavage monitored by (1)H-(15)N HSQC spectra showed the linker did not perturb the structures of the S100A10-annexin A2 or S100B-TRTK12 complexes. Furthermore, the analysis of the chemical shift assignments ((1)H, (15)N, and (13)C) showed that residues T102-S108 of annexin A2 formed a well-defined alpha-helix in the S100A10 hybrid while the TRTK12 region was unstructured at the N-terminus with a single turn of alpha-helix from D108-K111 in the S100B hybrid protein. The two S100 hybrid proteins provide a simple yet extremely efficient method for obtaining high yields of intact S100 target peptides. Since cleavage of the S100 hybrid protein is not necessary for structural characterization, this approach may be useful as a scaffold for larger S100 complexes.

Crosstalk between the protein surface and hydrophobic core in a core-swapped fibronectin type III domain.

Two homologous fibronectin type III (fnIII) domains, FNfn10 (the 10th fnIII domain of human fibronectin) and TNfn3 (the third fnIII domain of human tenascin), have essentially the same backbone structure, although they share only approximately 24% sequence identity. While they share a similar folding mechanism with a common core of key residues in the folding transition state, they differ in many other physical properties. We use a chimeric protein, FNoTNc, to investigate the molecular basis for these differences. FNoTNc is a core-swapped protein, containing the "outside" (surface and loops) of FNfn10 and the hydrophobic core of TNfn3. Remarkably, FNoTNc retains the structure of the parent proteins despite the extent of redesign, allowing us to gain insight into which components of each parent protein are responsible for different aspects of its behaviour. Naively, one would expect properties that appear to depend principally on the core to be similar to TNfn3, for example, the response to mutations, folding kinetics and side-chain dynamics, while properties apparently determined by differences in the surface and loops, such as backbone dynamics, would be more like FNfn10. While this is broadly true, it is clear that there are also unexpected crosstalk effects between the core and the surface. For example, the anomalous response of FNfn10 to mutation is not solely a property of the core as we had previously suggested.

Use of chimeric antibodies as positive controls in an enzyme-linked immunosorbent assay for diagnosis of scrub typhus (infection by Orientia tsutsugamushi).

The use of human sera collected from individuals of known infected and noninfected status is necessary for the validation of diagnostic assays and for the determination of cutoff values. However, the routine inclusion of pooled human sera from infected individuals for use as positive controls in commercial assay kits has many disadvantages. Sufficient quantities of sera can be difficult to obtain, and there are ethical and safety issues to be considered. Additionally, each batch of control material requires standardization, as each will differ in antibody titer. We have genetically engineered chimeric immunoglobulin G (IgG), IgM, and IgA antibodies consisting of mouse-derived variable regions and human constant regions derived from peripheral blood lymphocytes. The chimeric nature of these antibodies allows the desired antigen specificity created through mouse immunization and hybridoma technology while retaining a human constant region required for recognition by the enzyme-conjugated antihuman signal antibody. We have investigated the potential use of chimeric IgG with specificity for the major surface antigen of Orientia tsutsugamushi as an alternative positive control for inclusion in a commercial enzyme-linked immunosorbent assay kit for the diagnosis of rickettsia scrub typhus (caused by infection with O. tsutsugamushi). Chimeric IgG was expressed in stably transfected CHO cells, allowing production of unlimited quantities. The purified protein was found to have a much greater specificity for the scrub typhus antigen than the serum-derived controls. The methods described could be applied to other assay kits for the detection of antibodies against infectious agents.

Tripartite chimeras comprising functional domains derived from the cytosolic NADPH oxidase components p47phox, p67phox, and Rac1 elicit activator-independent superoxide production by phagocyte membranes: an essential role for anionic membrane phospholipids.

The superoxide-generating NADPH oxidase is converted to an active state by the assembly of a membrane-localized cytochrome b(559) with three cytosolic components: p47(phox), p67(phox), and GTPase Rac1 or Rac2. Assembly involves two sets of protein-protein interactions: among cytosolic components and among cytosolic components and cytochrome b(559) within its lipid habitat. We circumvented the need for interactions among cytosolic components by constructing a recombinant tripartite chimera (trimera) consisting of the Phox homology (PX) and Src homology 3 (SH3) domains of p47(phox), the tetratricopeptide repeat and activation domains of p67(phox), and full-length Rac1. Upon addition to phagocyte membrane, the trimera was capable of oxidase activation in vitro in the presence of an anionic amphiphile. The trimera had a higher affinity (lower EC(50)) for and formed a more stable complex (longer half-life) with cytochrome b(559) compared with the combined individual components, full-length or truncated. Supplementation of membrane with anionic but not neutral phospholipids made activation by the trimera amphiphile-independent. Mutagenesis, truncations, and domain replacements revealed that oxidase activation by the trimera was dependent on the following interactions: 1) interaction with anionic membrane phospholipids via the poly-basic stretch at the C terminus of the Rac1 segment; 2) interaction with p22(phox) via Trp(193) in the N-terminal SH3 domain of the p47(phox) segment, supplementing the electrostatic attraction; and 3) an intrachimeric bond among the p67(phox) and Rac1 segments complementary to their physical fusion. The PX domain of the p47(phox) segment and the insert domain of the Rac1 segment made only minor contributions to oxidase assembly.

Characterisation of receptors for IGF-I and insulin; evidence for hybrid insulin/IGF-I receptor in human coronary artery endothelial cells.

OBJECTIVE: Coronary artery disease is a prevalent cause of morbidity and mortality in diabetes. Little is known about insulin-like growth factor-I receptors (IGF-IR) and insulin receptors (IR) in human coronary endothelium. Our aim was to characterize IGF-IR and IR in human coronary artery endothelial cells (HCAEC). DESIGN: Cultured human coronary artery endothelial cells were used. Gene expression was measured by quantitative real-time RT-PCR analysis and receptor affinity by ligand binding. Receptor protein, phosphorylation of IGF-IR and IR beta-subunit as well as the presence of hybrid insulin receptor/Insulin-like growth factor-I receptor (Hybrid IR/IGF-IR) was analyzed by immunoprecipitation and Western blot. Postreceptor effects of insulin and IGF-I were assed by (3)H-thymidine incorporation. RESULTS: The gene expression of IGF-IR was several folds higher than that of IR. and insulin receptor isoform A (IR-A) was 20-fold more expressed than insulin receptor isoform B (IR-B) in HCAEC. The specific binding of (125)I-IGF-I was higher than that of (125)I-insulin. Insulin and the new long acting insulin analog, glargine, interacted with the IGF-IR with over thousand and 100-fold less potency than IGF-I itself, whereas IGF-II had 6 times lower potency than IGF-I. Phosphorylation of the IGF-IR beta-subunit was obtained by concentrations of 10(-10)-10(-8)M IGF-I, 10(-6)M of insulin, inconsistently by 10(-8)M insulin and not at all by 10(-10)-10(-9)M insulin. The IR beta-subunit was phosphorylated by insulin and IGF-I at concentrations of 10(-9)-10(-8)M. When immunoprecipitating with specific monoclonal anti-IR or anti-IGF-IR alpha-subunit antibodies we found bands situated in slightly different positions suggesting the presence of Hybrid IR/IGF-IR. IGF-I, IGF-II and insulin (10(-9)-10(-7)M) had no significant effect on (3)H-thymidine incorporation into DNA. CONCLUSIONS: Human coronary endothelial cells express more IGF-IR than IR, mainly IR-A, and also Hybrid IR/IGF-IR. Both IGF-I and insulin phosphorylate their receptors, but only IGF-I seems to phosphorylate Hybrid IR/IGF-IR. Our study provides experimental evidence for a possible role of IGF-IR, IR and Hybrid IR/IGF-IR in human coronary artery endothelial cells.

Functionally diverging molecular quasi-species evolve by crossing two enzymes.

Molecular evolution is frequently portrayed by structural relationships, but delineation of separate functional species is more elusive. We have generated enzyme variants by stochastic recombinations of DNA encoding two homologous detoxication enzymes, human glutathione transferases M1-1 and M2-2, and explored their catalytic versatilities. Sampled mutants were screened for activities with eight alternative substrates, and the activity fingerprints were subjected to principal component analysis. This phenotype characterization clearly identified at least three distributions of substrate selectivity, where one was orthogonal to those of the parent-like distributions. This approach to evolutionary data mining serves to identify emerging molecular quasi-species and indicates potential trajectories available for further protein evolution.

Purification and activity of the Rho ADP-ribosylating binary C2/C3 toxin.

C3 exoenzyme from Clostridium limosum, specifically ADP-ribosylates and inactivates Rho GTPases, but not or much less than Rac and Cdc42. To bypass the poor cell accessibility of the exoenzyme, a chimeric fusion toxin was constructed consisting of C3 exoenzyme and the N-terminal adaptor domain of the enzyme component C2I of the actin-ADP-ribosylating Clostridium botulinum C2 toxin. This fusion toxin C2IN-C3 is transported into cells by interaction with the binding and translocation component (C2II) of C2 toxin. Purification and activity of the chimeric toxin is reported.