Arthritis following recombinant outer surface protein A vaccination for Lyme disease.

As more individuals receive outer surface protein A (OspA) vaccination, adverse effects not detected during phase III clinical trials may become apparent. Although arthritis has been described following other human vaccines, we found no reports of human cases after Lyme disease vaccination. We describe 4 males (2 children, 2 adults) who developed arthritis following recombinant OspA vaccination. The potential arthritogenic effect of OspA suggested by in vitro and animal studies finds a clinical correlate in these 4 cases.     

A surface on the G protein β-subunit involved in interactions with adenylyl cyclases

Receptor activation of heterotrimeric G proteins dissociates Gα from the Gβγ complex, allowing both to regulate effectors. Little is known about the effector-interaction regions of Gβγ. We had used molecular modeling to dock a peptide encoding the region of residues 956–982 of adenylyl cyclase (AC) 2 onto Gβ to identify residues on Gβ that may interact with effectors. Based on predictions from the model, we synthesized peptides encoding sequences of residues 86–105 (Gβ86–105) and 115–135 (Gβ115–135) from Gβ. The Gβ86–105 peptide inhibited Gβγ stimulation of AC2 and blocked Gβγ inhibition of AC1 and by itself inhibited calmodulin-stimulated AC1, thus displaying partial agonist activity. Substitution of Met-101 with Asn in this peptide resulted in the loss of both the inhibitory and partial agonist activities. Most activities of the Gβ115–135 peptide were similar to those of Gβ86–105 but Gβ115–135 was less efficacious in blocking Gβγ inhibition of AC1. Substitution of Tyr-124 with Val in the Gβ115–135 peptide diminished all of its activities. These results identify the region encoded by amino acids 84–143 of Gβ as a surface that is involved in transmitting signals to effectors.     

A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate

Evidence for a two-metal ion mechanism for cleavage of the HH16 hammerhead ribozyme is provided by monitoring the rate of cleavage of the RNA substrate as a function of La³⁺ concentration in the presence of a constant concentration of Mg²⁺. We show that a bell-shaped curve of cleavage activation is obtained as La³⁺ is added in micromolar concentrations in the presence of 8 mM Mg²⁺, with a maximal rate of cleavage being attained in the presence of 3 μM La³⁺. These results show that two-metal ion binding sites on the ribozyme regulate the rate of the cleavage reaction and, on the basis of earlier estimates of the K_d values for Mg²⁺ of 3.5 mM and >50 mM, that these sites bind La³⁺ with estimated K_d values of 0.9 and >37.5 μM, respectively. Furthermore, given the very different effects of these metal ions at the two binding sites, with displacement of Mg²⁺ by La³⁺ at the stronger (relative to Mg²⁺) binding site activating catalysis and displacement of Mg²⁺ by La³⁺ at the weaker (relative to Mg²⁺) (relative to Mg²⁺) binding site inhibiting catalysis, we show that the metal ions at these two sites play very different roles. We argue that the metal ion at binding site 1 coordinates the attacking 2′-oxygen species in the reaction and lowers the pK_a of the attached proton, thereby increasing the concentration of the attacking alkoxide nucleophile in an equilibrium process. In contrast, the role of the metal ion at binding site 2 is to catalyze the reaction by absorbing the negative charge that accumulates at the leaving 5′-oxygen in the transition state. We suggest structural reasons why the Mg²⁺–La³⁺ ion combination is particularly suited to demonstrating these different roles of the two-metal ions in the ribozyme cleavage reaction.     

Crystal structure of varicella-zoster virus protease

Varicella-zoster virus (VZV), an α-herpes virus, is the causative agent of chickenpox, shingles, and postherpetic neuralgia. The three-dimensional crystal structure of the serine protease from VZV has been determined at 3.0-Å resolution. The VZV protease is essential for the life cycle of the virus and is a potential target for therapeutic intervention. The structure reveals an overall fold that is similar to that recently reported for the serine protease from cytomegalovirus (CMV), a herpes virus of the β subfamily. The VZV protease structure provides further evidence to support the finding that herpes virus proteases have a fold and active site distinct from other serine proteases. The VZV protease catalytic triad consists of a serine and two histidines. The distal histidine is proposed to properly orient the proximal histidine. The identification of an α-helical segment in the VZV protease that was mostly disordered in the CMV protease provides a better definition of the postulated active site cavity and reveals an elastase-like S′ region. Structural differences between the VZV and CMV proteases also suggest potential differences in their oligomerization states.     

Binding of outer surface protein A and human lymphocyte function-associated antigen 1 peptides to HLA-DR molecules associated with antibiotic treatment-resistant Lyme arthritis

OBJECTIVE: To assess the binding of outer surface protein A (OspA) and human lymphocyte function-associated antigen 1 (hLFA-1) peptides to 5 major histocompatibility complex (MHC) molecules. METHODS: Peptide binding to the MHC molecules was determined by in vitro binding assays, and binding was correlated with the frequencies of the 5 MHC molecules in patients with treatment-resistant Lyme arthritis. RESULTS: The HLA-DRB1*0401 molecule bound both OspA(163-175) and hLFA-1alpha(L330-342) well. Although the magnitude of the binding was less, the DRB1*0404 molecule also showed binding of both peptides. The DRB1*0101 molecule bound OspA(163-175) well, but hLFA-1alpha(L330-342) only weakly; the DRB1*0801 or *1101 molecule bound both peptides weakly, if at all. The magnitude of OspA(163-175) binding correlated well with the frequencies of the DRB1 alleles in patients with treatment-resistant arthritis, but the binding of hLFA-1alpha(L330-342) showed only an association with the DRB*04 alleles. CONCLUSION: These correlations support the hypothesis that OspA(163-175) is the critical epitope in triggering antibiotic treatment-resistant Lyme arthritis. However, the inability of the DRB*0101 molecule to bind hLFA-1alpha(L330-342) suggests that this peptide may not be a relevant autoantigen, at least in DRB1*0101-positive patients.     

Therapeutic passive vaccination against chronic Lyme disease in mice

Passive and active immunization against outer surface protein A (OspA) has been successful in protecting laboratory animals against subsequent infection with Borrelia burgdorferi. Antibodies (Abs) to OspA convey full protection, but only when they are present at the time of infection. Abs inactivate spirochetes within the tick and block their transmission to mammals, but do not affect established infection because of the loss of OspA in the vertebrate host. Our initial finding that the presence of high serum titers of anti-OspC Abs (5 to 10 μg/ml) correlates with spontaneous resolution of disease and infection in experimentally challenged immunocompetent mice suggested that therapeutic vaccination with OspC may be feasible. We now show that polyclonal and monospecific mouse immune sera to recombinant OspC, but not to OspA, of B. burgdorferi resolve chronic arthritis and carditis and clear disseminated spirochetes in experimentally infected C.B.-17 severe combined immunodeficient mice in a dose-dependent manner. This was verified by macroscopical and microscopical examination of affected tissues and recultivation of spirochetes from ear biopsies. Complete resolution of disease and infection was achieved, independent of whether OspC-specific immune sera (10 μg OspC-specific Abs) were repeatedly given (4× in 3- to 4-day intervals) before the onset (day 10 postinfection) or at the time of fully established arthritis and carditis (days 19 or 60 postinfection). The results indicate that in mice spirochetes constitutively express OspC and are readily susceptible to protective OspC-specific Abs throughout the infection. Thus, an OspC-based vaccine appears to be a candidate for therapy of Lyme disease.     

Serologic diagnosis of Lyme disease using recombinant outer surface proteins A and B and flagellin.

Recombinant (r) outer surface proteins (Osp) A and B, flagellin, and an immunogenic region of flagellin (41-G) from Borrelia burgdorferi were evaluated using immunoblot and ELISA for usefulness as substrates in diagnostic testing for Lyme disease. Antibodies to rOspA, rOspB, and r-flagellin were detected by immunoblot and ELISA using the recombinant proteins. Patients with late disease responded to rOspA, rOspB, and r-flagellin, or only to r-flagellin, whereas patients with early disease showed no response or responded only to r-flagellin. Patients who developed antibodies to r-flagellin also responded to 41-G. The data suggest that recombinant B. burgdorferi antigens can serve as substrates for immunoblot and ELISA, which may be helpful in diagnosing Lyme disease.     

Binding of outer surface protein A and human lymphocyte function–associated antigen 1 peptides to HLA–DR molecules associated with antibiotic treatment–resistant Lyme arthritis

Objective

To assess the binding of outer surface protein A (OspA) and human lymphocyte function–associated antigen 1 (hLFA‐1) peptides to 5 major histocompatibility complex (MHC) molecules.

Methods

Peptide binding to the MHC molecules was determined by in vitro binding assays, and binding was correlated with the frequencies of the 5 MHC molecules in patients with treatment‐resistant Lyme arthritis.

Results

The HLA–DRB1*0401 molecule bound both OspA_163–175 and hLFA‐1α_L330–342 well. Although the magnitude of the binding was less, the DRB1*0404 molecule also showed binding of both peptides. The DRB1*0101 molecule bound OspA_163–175 well, but hLFA‐1α_L330–342 only weakly; the DRB1*0801 or *1101 molecule bound both peptides weakly, if at all. The magnitude of OspA_163–175 binding correlated well with the frequencies of the DRB1 alleles in patients with treatment‐resistant arthritis, but the binding of hLFA‐1α_L330–342 showed only an association with the DRB*04 alleles.

Conclusion

These correlations support the hypothesis that OspA_163–175 is the critical epitope in triggering antibiotic treatment–resistant Lyme arthritis. However, the inability of the DRB*0101 molecule to bind hLFA‐1α_L330–342 suggests that this peptide may not be a relevant autoantigen, at least in DRB1*0101‐positive patients.

     

Geographic clustering of an outer surface protein A mutant of Borrelia burgdorferi. Possible implications of multiple variants for Lyme disease persistence

DNA sequences encoding full-length outer surface protein (Osp) A were amplified from four joint fluid samples over 4.5 months from a patient with chronic Lyme arthritis, with a variant from wild type only found in sample 3. Rather than a mutation in vivo, these findings suggested a mixed infection in which BORRELIA: containing the wild-type and mutant ospA were waxing and waning in the patient's joint. If so, we reasoned that the mutant should be present in the community. We therefore took the novel epitope resulting from the mutation, expressed as a fusion protein in Escherichia coli, and performed Western blots on 80 high-titred stored sera; however, all except that of our index patient were negative. We then collected 36 stored sera from patients with Lyme disease residing within 10 miles of where the index patient had lived. An additional two sera from this circumscribed area were positive (P = 0.038). These findings show that results from single samples can be misleading, and suggest that the OspAs expressed in force late in Lyme arthritis are the same ones introduced initially into the host. Moreover, they allow a speculative mechanism for disease persistence not previously considered, in which antigenically distinct B. burgdorferi variant proteins present themselves serially to the immune system.     

Mapmodulin: A possible modulator of the interaction of microtubule-associated proteins with microtubules

We have purified and characterized a 31-kDa protein named mapmodulin that binds to the microtubule-associated proteins (MAPs) MAP2, MAP4, and tau. Mapmodulin binds free MAPs in strong preference to microtubule-associated MAPs, and appears to do so via the MAP’s tubulin-binding domain. Mapmodulin inhibits the initial rate of MAP2 binding to microtubules, a property that may allow mapmodulin to displace MAPs from the path of organelles translocating along microtubules. In support of this possibility, mapmodulin stimulates the microtubule- and dynein-dependent localization of Golgi complexes in semi-intact CHO cells. To our knowledge, mapmodulin represents the first example of a protein that can bind and potentially regulate multiple MAP proteins.     

Mutated mitogen-activated protein kinase: A tumor rejection antigen of mouse sarcoma

The molecular basis of the polymorphic tumor rejection antigens of chemically induced sarcomas of inbred mice remains a mystery, despite the discovery of these antigens over 40 years ago and their critical importance to the foundation of tumor immunology. In an analysis of a panel of BALB/c 3-methylcholanthrene-induced tumors, we identified one tumor, CMS5, that elicited a strong cytotoxic T cell response with exquisite specificity for CMS5. A stable cloned line of T cells with this specificity (C18) was used to screen a CMS5 cDNA expression library. The gene encoding the C18-defined antigen was identified as a mutated form of a mouse mitogen-activated protein kinase, ERK2, and a peptide incorporating the resulting amino acid substitution (lysine to glutamine) was efficiently recognized by C18. Vaccination with this peptide elicited specific resistance to CMS5 challenge. Extensive efforts to isolate antigen-loss variants of CMS5 were unsuccessful, suggesting that the mutated mitogen-activated protein kinase is essential for maintenance of the malignant phenotype.     

Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: structural basis for recognition of B-cell receptors and superantigen activity

Staphylococcus aureus produces a virulence factor, protein A (SpA), that contains five homologous Ig-binding domains. The interactions of SpA with the Fab region of membrane-anchored Igs can stimulate a large fraction of B cells, contributing to lymphocyte clonal selection. To understand the molecular basis for this activity, we have solved the crystal structure of the complex between domain D of SpA and the Fab fragment of a human IgM antibody to 2.7-A resolution. In the complex, helices II and III of domain D interact with the variable region of the Fab heavy chain (V(H)) through framework residues, without the involvement of the hypervariable regions implicated in antigen recognition. The contact residues are highly conserved in human V(H)3 antibodies but not in other families. The contact residues from domain D also are conserved among all SpA Ig-binding domains, suggesting that each could bind in a similar manner. Features of this interaction parallel those reported for staphylococcal enterotoxins that are superantigens for many T cells. The structural homology between Ig V(H) regions and the T-cell receptor V(beta) regions facilitates their comparison, and both types of interactions involve lymphocyte receptor surface remote from the antigen binding site. However, T-cell superantigens reportedly interact through hydrogen bonds with T-cell receptor V(beta) backbone atoms in a primary sequence-independent manner, whereas SpA relies on a sequence-restricted conformational binding with residue side chains, suggesting that this common bacterial pathogen has adopted distinct molecular recognition strategies for affecting large sets of B and T lymphocytes.     

Prostate stem cell antigen: A cell surface marker overexpressed in prostate cancer

The identification of cell surface antigens is critical to the development of new diagnostic and therapeutic modalities for the management of prostate cancer. Prostate stem cell antigen (PSCA) is a prostate-specific gene with 30% homology to stem cell antigen 2, a member of the Thy-1/Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cell surface antigens. PSCA encodes a 123-aa protein with an amino-terminal signal sequence, a carboxyl-terminal GPI-anchoring sequence, and multiple N-glycosylation sites. PSCA mRNA expression is prostate-specific in normal male tissues and is highly up-regulated in both androgen-dependent and -independent prostate cancer xenografts. In situ mRNA analysis localizes PSCA expression in normal prostate to the basal cell epithelium, the putative stem cell compartment of the prostate. There is moderate to strong PSCA expression in 111 of 126 (88%) prostate cancer specimens examined by in situ analysis, including high-grade prostatic intraepithelial neoplasia and androgen-dependent and androgen-independent tumors. Flow cytometric analysis demonstrates that PSCA is expressed predominantly on the cell surface and is anchored by a GPI linkage. Fluorescent in situ hybridization analysis localizes the PSCA gene to chromosome 8q24.2, a region of allelic gain in more than 80% of prostate cancers. A mouse homologue with 70% amino acid identity and similar genomic organization to human PSCA has also been identified. These results support PSCA as a target for prostate cancer diagnosis and therapy.     

Mso1p: A yeast protein that functions in secretion and interacts physically and genetically with Sec1p

The yeast Sec1p protein functions in the docking of secretory transport vesicles to the plasma membrane. We previously have cloned two yeast genes encoding syntaxins, SSO1 and SSO2, as suppressors of the temperature-sensitive sec1–1 mutation. We now describe a third suppressor of sec1–1, which we call MSO1. Unlike SSO1 and SSO2, MSO1 is specific for sec1 and does not suppress mutations in any other SEC genes. MSO1 encodes a small hydrophilic protein that is enriched in a microsomal membrane fraction. Cells that lack MSO1 are viable, but they accumulate secretory vesicles in the bud, indicating that the terminal step in secretion is partially impaired. Moreover, loss of MSO1 shows synthetic lethality with mutations in SEC1, SEC2, and SEC4, and other synthetic phenotypes with mutations in several other late-acting SEC genes. We further found that Mso1p interacts with Sec1p both in vitro and in the two-hybrid system. These findings suggest that Mso1p is a component of the secretory vesicle docking complex whose function is closely associated with that of Sec1p.     

Crystal structure of human calcineurin complexed with cyclosporin A and human cyclophilin

Calcineurin (Cn), a Ca(2+)/calmodulin-dependent Ser/Thr protein phosphatase, is an important participant in signaling pathways that activate T cells. It is the target of the immunosuppressive drugs cyclosporin A (CsA) and FK506. These drugs bind proteins known as cyclophilin (Cyp) and FK506-binding protein, respectively, and the drug-protein complexes in turn inhibit Cn. We report the crystal structure of a Cyp/CsA/Cn ternary complex, determined to a resolution of 3.1 A. Residues 3-9 of CsA, particularly N-methyl leucines 4 and 6, and Trp-121 of Cyp form a composite surface for interaction with Cn. The hydrophobic interface buries two hydrogen bonds. The structure accounts clearly for the effects of mutations in Cn on CsA-resistance and for the way modifications of CsA alter immunosuppressive activity.     

Crystal structure of truncated human apolipoprotein A-I suggests a lipid-bound conformation

The structure of truncated human apolipoprotein A-I (apo A-I), the major protein component of high density lipoprotein, has been determined at 4-Å resolution. The crystals comprise residues 44–243 (exon 4) of apo A-I, a fragment that binds to lipid similarly to intact apo A-I and that retains the lipid-bound conformation even in the absence of lipid. The molecule consists almost entirely of a pseudo-continuous, amphipathic α-helix that is punctuated by kinks at regularly spaced proline residues; it adopts a shape similar to a horseshoe of dimensions 125 × 80 × 40 Å. Four molecules in the asymmetric unit associate via their hydrophobic faces to form an antiparallel four-helix bundle with an elliptical ring shape. Based on this structure, we propose a model for the structure of apo A-I bound to high density lipoprotein.     

Crystal structure of murine cyclophilin C complexed with immunosuppressive drug cyclosporin A.

Cyclophilin is a cellular receptor for the immunosuppressive drug cyclosporin A (CsA). Cyclophilin C (CyPC) is highly expressed in murine kidney, making it a potential mediator of the nephrotoxic effects of CsA. The structure of murine CyPC complexed with CsA has been solved and refined to an R factor of 0.197 at a 1.64-A resolution. Superposition of the CyPC-CsA structure with the unligated cyclophilin A (CyPA) revealed significant migration of three loops: Gln-179 to Thr-189, Asp-47 to Lys-49, and Met-170 to Ile-176. The proximity of the loop Gln-179 to Thr-189 to the CsA binding site may account for the unique binding of a 77-kDa glycoprotein, CyPC binding protein (CyCAP), to CyPC. The binding of CsA to CyPC is similar to that of CsA to human T-cell cyclophilin A (CyPA). However, the conformation of CsA when bound to CyPC is significantly different from that when bound to CyPA. These differences may reflect conformational variation of CsA when bound to different proteins. Alternatively, the previous CyPA-CsA structure at low resolution may not provide sufficient details for a comparison with the CyPC-CsA structure.     

Evidence that the lipid moiety of oxidized low density lipoprotein plays a role in its interaction with macrophage receptors

The binding of oxidatively damaged red blood cells (OxRBCs) to resident mouse peritoneal macrophages correlates with an increase in phosphatidylserine on the external leaflet of the plasma membrane. Liposomes rich in phosphatidylserine can inhibit this binding and also the binding of certain apoptotic cells. We have shown previously that oxidized low density lipoproteins (OxLDL) also can inhibit the binding of OxRBCs to resident mouse peritoneal macrophages. The present studies show that microemulsions prepared from the lipids extracted from OxLDL are very effective in inhibiting the binding of OxRBCs and also, to a lesser extent, of apoptotic thymocytes to macrophages. OxRBC binding was also inhibited by cholesterol phospholipid liposomes containing oxidized 1-stearoyl-2-linoleoyl-phosphatidylcholine. The binding and uptake of ¹²⁵I-labeled OxLDL were also strongly inhibited by microemulsions of the lipids extracted from OxLDL and by cholesterol phospholipid liposomes containing oxidized 1-palmitoyl-2-arachidonoyl-phosphatidylcholine. Earlier studies have shown that the delipidated protein moiety of OxLDL can competitively inhibit macrophage binding of intact OxLDL, implicating the protein moiety as an effective receptor-binding domain of OxLDL with respect to some macrophage scavenger receptors. The present studies suggest that the lipid moiety of OxLDL may also play a role.