Meat allergy associated with galactosyl-α-(1,3)-galactose (α-Gal)-Closing diagnostic gaps by anti-α-Gal IgE immune profiling.

BACKGROUND: Glycoproteins and glycolipids of some mammalian species contain the disaccharide galactosyl-α-(1,3)-galactose (α-Gal). It is known that α-Gal is immunogenic in humans and causes glycan-specific IgG and also IgE responses with clinical relevance. α-Gal is part of the IgE-reactive monoclonal therapeutic antibody cetuximab (CTX) and is associated with delayed anaphylaxis to red meat. In this study, different α-Gal-containing analytes are examined in singleplex and multiplex assays to resolve individual sensitization patterns with IgE against α-Gal. METHODS: Three serum groups, α-Gal-associated meat allergy (MA) patients, idiopathic anaphylaxis (IA) patients with suspected MA, and non-meat-allergic healthy control individuals (HC), were analyzed via singleplex allergy diagnostics and a newly established immunoblot diagnostic system. The new dot blot detection system resolved individual IgE sensitization profiles for α-Gal-containing analytes CTX, bovine thyroglobulin (Bos d TG), and human serum albumin (HSA)-conjugated α-Gal. RESULTS: Singleplex allergy diagnostics using the α-Gal analytes CTX and Bos d TG confirms the history of MA patients in 91% and 88% of the cases, respectively. A novel dot blot-based assay system for the detection of IgE against α-Gal reveals individual IgE sensitization profiles for α-Gal-containing analytes. An α-Gal-associated IgE cross-reactivity profile (IgE against CTX, Bos d TG, and HSA-α-Gal) was identified, which is associated with MA. CONCLUSIONS: Detection of individual sensitization patterns with different α-Gal-containing analytes provides the basis for an individual allergy diagnosis for α-Gal-sensitized patients. Higher amounts of α-Gal in pork and beef innards compared to muscle meat as indicated by a higher staining intensity are a plausible explanation for the difference in allergic symptom severity.

Assessment and reporting of the clinical immunogenicity of therapeutic proteins and peptides-harmonized terminology and tactical recommendations.

Immunogenicity is a significant concern for biologic drugs as it can affect both safety and efficacy. To date, the descriptions of product immunogenicity have varied not only due to different degrees of understanding of product immunogenicity at the time of licensing but also due to an evolving lexicon that has generated some confusion in the field. In recent years, there has been growing consensus regarding the data needed to assess product immunogenicity. Harmonization of the strategy for the elucidation of product immunogenicity by drug developers, as well as the use of defined common terminology, can benefit medical practitioners, health regulatory agencies, and ultimately the patients. Clearly, understanding the incidence, kinetics and magnitude of anti-drug antibody (ADA), its neutralizing ability, cross-reactivity with endogenous molecules or other marketed biologic drugs, and related clinical impact may enhance clinical management of patients treated with biologic drugs. To that end, the authors present terms and definitions for describing and analyzing clinical immunogenicity data and suggest approaches to data presentation, emphasizing associations of ADA development with pharmacokinetics, efficacy, and safety that are necessary to assess the clinical relevance of immunogenicity.

Controlled release recombinant human interferon-α2b for treating patients with chronic hepatitis C genotype 1: a phase 2a clinical trial.

Better convenience and tolerability and sustained therapeutic concentrations might improve interferon (IFN) treatment for chronic hepatitis C virus (HCV) infection. In an open-label, randomized study, controlled release free (chemically unmodified) recombinant human IFN-α(2b) in poly(ether-ester) microspheres (CR-rhIFN-α(2b)), was injected at doses of 160, 320, 480 or 640 µg every 2 weeks for 12 weeks with concomitant weight-based oral ribavirin in 32 treatment-naïve patients with chronic HCV genotype 1. Treatment was well tolerated, with 31 patients (97%) successfully completing the study. Full doses of CR-rhIFN-α(2b) were administered on 96% of scheduled occasions. Flu-like symptoms were generally mild and brief. Injection site reactions developed in 13 patients (41%), and neutropenia occurred in six of eight patients receiving 640 µg. In the 320, 480 and 640 µg groups, 62-75% of patients achieved a ≥2 log(10) HCV RNA reduction by 4 weeks and 88-100% by 12 weeks. For those groups, the pooled median time to ≥2 log(10) reduction was 11 days (95% confidence interval, 7-35 days). In those groups, viral reduction below the limit of detection was accomplished in 25% of patients by 4 weeks and in 62% by 12 weeks. The 160-µg dose was less potent. After CR-rhIFN-α(2b) injection, stable plateau levels of serum IFN-α(2b) were generally reached within 72 h. Treatment-emergent neutralizing antibodies to IFN-α(2b) were observed in one patient. No antibodies to host plant proteins were detected. CR-rhIFN-α(2b) with ribavirin cotherapy was well tolerated and displayed potent early antiviral activity in patients with chronic HCV genotype 1.

[Autoantibody detection by indirect immunofluorescence on HEp-2 cells]. / Autoantikörpernachweis mittels indirekter Immunfluoreszenz an HEp-2-Zellen.

Systemic autoimmune diseases are characterized by the presence of antinuclear autoantibodies (ANA). Diluted patient sera are typically used to screen for the presence of ANA by immunfluorescence microscopy with fixed HEp-2 cells. Despite high-quality test kits, reports of different laboratories frequently present controversial results. This article recommends unified processing and interpretation of HEp-2 based screening for autoantibodies. Suggestions are made for the selection of high-quality test kits, optimized processing and diagnostic procedures. In addition to a relevant clinical diagnosis and an experienced laboratory specialist, the following procedure is highly recommended to achieve good laboratory practice: Initial HEp-2 based screening by indirect immunofluorescence, starting with a 1:80 serum dilution, and evaluation in a bright fluorescence microscope, pathological values from a titer of 1:160 upwards, internal quality checks and unified interpretation. We aim to improve diagnosis and care of patients with autoimmune diseases as a central focus of the European Autoimmunity Standardization Initiative (EASI).

Serological and clinical characterization of anti-dsDNA and anti-PM/Scl double-positive patients.

Antibodies to double-stranded desoxyribonucleic acid (dsDNA) and to the polymyositis/scleroderma (PM/Scl) complex are regarded as serological markers for systemic lupus erythematosus (SLE) and PM/Scl overlap syndrome, respectively. In a previous study, serum samples were identified that contained antibodies specific for both dsDNA and PM/Scl. Fourteen of these sera were available for more detailed investigation including the autoantibody profile as determined by several methods including an addressable laser bead assay, Crithidia luciliae indirect immunofluorescence test (CLIFT) and a PM1-Alpha ELISA. Moreover, 300 samples from connective tissue disease patients and 30 PM/Scl positive samples were screened for anti-dsDNA(+)/PM/Scl(+) specimens by CLIFT, dsDNA ELISA, and PM1-Alpha ELISA. We confirmed anti-dsDNA and anti-PM/Scl reactivity in 2/7 samples from the previous study. One sample had also anti-chromatin and anti-SS-A reactivity and the second sample was oligoreactive. In addition, 2/300 (0.7%) unselected samples from connective tissue disease patients were identified with anti-dsDNA and anti-PM/Scl reactivity. In a panel of PM1-Alpha positive samples (n = 30) collected regardless of the diagnosis of the patients, no anti-dsDNA reactivity was found. All anti-dsDNA(+)/anti-PM/Scl(+) patients identified fulfilled sufficient criteria to be classified as definite SLE and also had at least one feature of systemic sclerosis (i.e., sclerodactyly and/or Raynaud's phenomenon). Only 1/4 patients had clinical evidence of dermatomyositis. The combination of anti-dsDNA(+)/anti-PM/Scl(+) in patients suffering from connective tissue disease is less frequently found than previously described when newer assays are used. Clinically, anti-dsDNA(+)/anti-PM/Scl(+) patients may define a small subgroup of SLE patients with additional features of systemic sclerosis.

[Endocrine orbitopathy and significance of autoantibodies against 1D protein]. / Endokrinní orbitopatie a význam autoprotilátek proti 1D proteinu.

BACKGROUND: Endocrine ophthalmopathy is a chronic eye disease, characterized by inflammation in parabulbar and retrobulbar space, occurring usually in Graves' thyrotoxicosis. Although the pathogenesis of the disease has not been clarified until now, it is accepted that this disease is of an autoimmune nature, where the targets of the autoimmune reaction are the antigens shared by thyroid and orbit-tissue. The autoantibodies against recombinant 1D protein are highly specific and sensitive for the diagnosis of endocrine orbitopathy. METHODS AND RESULTS: The aim of our study was to establish, whether the autoantibodies against 1D protein are found predominantly in patients with clinically expressed endocrine orbitopathy. We evaluated in 30 patients with clinically expressed endocrine orbitopathy the thickness of the three retrobulbar eye muscles, damaged by endocrine orbitopathy, determined the parameters of thyroid hormones and anti-TSH receptor autoantibodies. In all patients the detection of circulating autoantibodies against recombinant 1D protein was performed. Autoantibodies against recombinant 1D protein were found in all patients with clinically expressed endocrine orbitopathy. CONCLUSIONS: Immunoreactivity did not depend on the duration or severity of the eye disease, neither on patients' age. We did not find any correlation between the thickness of eye muscles and the titre of anti-TSH receptor autoantibodies, levels of ssTSH and free thyroxine and also any correlation between the thickness of eye muscles and the disease duration.

Autologous whole blood injections to patients with chronic urticaria and a positive autologous serum skin test: a placebo-controlled trial.

BACKGROUND: Patients with chronic urticaria (CU) frequently exhibit positive skin test reactions to autologous serum (ASST). Therapies aimed at inducing tolerance to circulating histamine-releasing factors in ASST+ CU patients, e.g. by treatment with autologous whole blood (AWB), have not yet been tested. OBJECTIVE: To test whether ASST+ CU patients can benefit from repeated low-dose intramuscular injections of AWB. METHODS: We characterized CU severity and duration, anti-Fc(epsilon)RI and anti-IgE expression, use of antihistamines, and quality of life in 56 CU patients (ASST+: 35, ASST-: 21) and assessed the therapeutic effects of 8 weekly AWB injections in a randomized, placebo-controlled, single-blind, parallel-group trial. RESULTS: Numbers, size, intensity, and/or duration of CU symptoms, quality of life, as well as expression of anti-Fc(epsilon)RI or anti-IgE were similar in ASST+ and ASST- CU patients. However, CU in ASST+ patients was of longer duration and required markedly more antihistaminic medication. Interestingly, ASST+ patients, but not ASST- patients, showed significantly (1) reduced CU activity, (2) decreased use of antihistamines, and (3) improved quality of life after AWB treatment. Placebo treatment was ineffective in both groups, but differences of AWB and placebo treatment responses did not achieve statistical significance in either group, most likely due to the limited number of patients treated. CONCLUSION: Our findings suggest that ASST+ CU is clinically different from other CU subforms and that ASST+ CU patients can benefit from AWB therapy.

HLA antigen expression in autoimmune endocrinopathies.

The HLA allelic frequency was determined in three groups of autoimmune endocrinopathies: A) 30 patients with autoimmune thyroiditis, B) 20 patients with polyglandular activation of autoimmunity, and C) 10 patients with the autoimmune polyglandular syndrome type II. The groups were defined by the clinical state and serological parameters. Healthy blood donors of Caucasian population from the US database of HLA frequencies served as the controls. In group A, a higher occurrence of HLA-A24 (21.7 %) was found as compared to group B (5.0 %) and to the controls (8.5 %), of HLA-B27 (15.0 %) and of HLA-DR-11 (20 %) as compared to the controls (4.2 % and 8.5 %). In group B, a higher occurrence of HLA-A3 (25.0 %) was found as compared to group A (10 %) and to the controls (11.8 %), and of HLA-B8 (22.5 %) as compared to group A (8.3 %) and to the controls (8.6 %). In this group the occurrence of HLA-DR3 (30.0 %) was higher as compared to group A (10.0 %) and to the controls (9.8 %) and of HLA-B8 (30.0 %) as compared to group A (8.3 %) and to the controls (8.6 %). Genetic markers indicate a similarity of groups B and C. Patients in these groups could be at different stages of the same disease, however, some distinctions between them lead us to consider the possibility whether different epigenetic factors could extend the difference between these groups in the course of clinical development.

Clinical evaluation of 5-S-cysteinyldopa testing using a new and optimized detection system as a tumour marker for malignant melanoma.

5-S-cysteinyldopa (5-S-CD) has been described as a tumour marker for the detection of human metastatic melanoma. We investigated the clinical utility of a new and optimized method to detect 5-S-CD by analysing 207 plasma samples derived from 138 patients with clinical stage I/II ( = 60), III ( = 32) or IV ( = 46) melanoma. Control groups consisted of 27 patients with non-melanoma skin diseases and 30 healthy volunteers. 5-S-CD plasma levels were determined using a new analytical technique based on a fully automated solid phase extraction coupled online to a novel high performance liquid chromatography method. In all the samples from the healthy control subjects 5-S-CD plasma concentrations were below 2.0 microg/l. Increased 5-S-CD-levels (>/=2.0 microg/l) were found in 52%, 67% and 81% of the plasma samples from patients with stages I/II, III and IV malignant melanoma, respectively. The mean values of 5-S-CD were found to rise with increasing tumour stage. Among 27 samples from patients with non-melanoma skin disease, slightly elevated 5-S-CD levels between 2.3 and 2.6 microg/l were found in only four samples from patients with multiple dysplastic naevi. In conclusion, our improved analytical technique provides a high sensitivity in all stages of the disease and represents a useful technique for monitoring melanoma patients.

Cicatricial pemphigoid: IgA and IgG autoantibodies target epitopes on both intra- and extracellular domains of bullous pemphigoid antigen 180.

BACKGROUND: Cicatricial pemphigoid (CP) is an autoimmune subepidermal blistering disease where autoantibodies target various components of the dermal-epidermal junction, including the bullous pemphigoid antigen 180 (BP180). OBJECTIVE: We determined the exact specificity of circulating IgG and IgA autoantibodies to BP180 in a large number of CP patients. METHODS: Twenty-six consecutive CP sera were analysed by Western blotting using a panel of cell-derived and recombinant proteins covering the entire BP180 molecule. RESULTS: Circulating autoantibodies were detected in all CP sera. Seven sera reacting with laminin-5 were excluded from further analyses; the remaining 19 sera recognized BP180, including six sera (32%) that showed only IgA reactivity to this protein. With the combined use of the soluble BP180 ectodomain (LAD-1) and recombinant BP180 NC16A, 16 of these 19 CP sera (84%) targeted BP180. IgG reactivity was preferentially found against NC16A, whereas IgA antibodies predominantly recognized LAD-1. Thirty-two per cent of the BP180-reative sera revealed reactivity with the intracellular domain of this protein. CONCLUSIONS: Our findings demonstrate that autoantibodies in CP target epitopes on both extra- and intracellular domains of BP180 and highlight the importance of testing for both IgG and IgA reactivity in these patients' sera.

Mapping of epitopes on the BP180 ectodomain targeted by IgA and IgG autoantibodies in patients with the lamina lucida-type of linear IgA disease.

Linear IgA disease (LAD) is an autoimmune subepidermal blistering skin disease characterized by the linear deposition of IgA at the dermoepidermal junction. Serum from patients with LAD most commonly contains autoantibodies that are directed against the hemidesmosomal transmembrane glycoprotein BP180 (type XVII collagen). Various antigenic sites on the extracellular domain of this anchoring filament protein have been shown to be targeted by autoantibodies in different autoimmune bullous skin diseases, including bullous pemphigoid and cicatricial pemphigoid (CP). However, little is known about epitopes on BP180 recognized by autoantibodies in LAD. In this study, we used three recombinant GST fusion proteins, together roughly covering the entire BP180 ectodomain, to characterize the autoimmune response in serum from patients with LAD. Interestingly, we found both IgA and IgG reactivity to all three portions of the BP180 ectodomain. The strongest reactivity was observed with the C-terminal portion of BP180. This is also the major region recognized by autoantibodies in patients with CP. This finding correlates with the observation that there may be significant overlap of the clinical and immunopathological findings in LAD and CP.

Patients with bullous pemphigoid and linear IgA disease show a dual IgA and IgG autoimmune response to BP180.

Bullous pemphigoid (BP) and linear IgA disease (LAD) are autoimmune subepidermal blistering skin diseases associated with autoantibodies against the transmembrane hemidesmosomal protein BP180/type XVII collagen. It has been demonstrated previously that BP is characterized predominantly by IgG autoantibodies, while autoantibodies in LAD mainly belong to the IgA isotype. The aim of the present study was to investigate the hypothesis that there is a significant overlap in the autoantibody isotype profiles associated with these two diseases. Several new recombinant forms of BP180 were generated in the baculovirus expression system, including the full-length protein. IgG autoantibodies to BP 180 were detectable in 39 of 40 (98%) of BP sera; interestingly, 88% of BP sera also contained IgA anti-BP180 autoantibodies. Similarly, anti-BP180 reactivity in LAD sera (n=22) was also attributed to both an IgA (68%) and an IgG (76%) autoantibody response. IgA and IgG autoantibodies to the intracellular portion of BP180 were found in 14% and 28% of BP sera, respectively, and in 8% of LAD sera (same percentage for both isotypes). Our findings clearly demonstrate that both BP and LAD patients have a dual IgA and IgG autoimmune response to BP180 which is directed not only to the ectodomain, but also to the intracellular portion of this protein.

p53 autoantibodies in patients with autoimmune diseases: a quantitative approach.

With few exceptions, autoantibodies directed against the gene product of the tumor suppressor gene p53 are only detected in cancer patients. From 73 patients with various autoimmune diseases, we obtained 17 sera with elevated autoantibodies against the p53 protein comprising patients with SLE, Graves' disease, and immune vasculitis including Wegener's granulomatosis. The overall prevalence (23%) of p53 autoantibodies was comparable to that in various cancers; differences, however, were obvious with respect to the magnitude of antibody levels. Only 5% of seropositive colorectal cancer patients had levels within the critical range (150-180 U/ml) but nearly half (41%) of seropositive autoimmune disease patients were that low. None of the autoimmune disease patients exceeded 300 U/ml serum compared to more than 60% of seropositive colorectal cancer patients with higher levels. This remarkable difference in magnitude underlines the necessity of quantification of p53 autoantibodies over a mere qualitative determination. Patients with autoimmune diseases face an increased risk for malignancies. It still remains to be established whether p53 seropositivity in autoimmune diseases adds to the rare exceptions of p53AAb in non-malignant diseases or is indicative for a yet occult cancer.

Serological reactivity of recombinant 1D autoantigen and its expression in human thyroid and eye muscle tissue: a possible autoantigenic link in Graves' patients.

Thyroid-associated ophthalmopathy (TAO) is a potentially severe autoimmune disease, in and around the orbit, usually accompanied by Graves' disease. It was the goal of this study to develop a serological indicator for TAO and to characterize its expression in human thyroid and eye muscle tissue. Thus, we have recloned the full-length 1D-complementary DNA and assessed its expression levels in 90 healthy and diseased human thyroids. Only Graves' patients suffering from TAO (n = 29) displayed a significant, 2.1-fold increase of 1D expression levels (P = 0.029), compared with normal controls (n = 9), as assessed using the Mann-Whitney U-test for paired, nonnormally distributed samples. In contrast, a decrease of 1D expression (to 40% of control normal values) was confined to thyroid autonomy (n = 19, P = 0.032). In all other diseased human thyroids, including Graves' thyroids from patients not suffering from clinically overt TAO (n = 9), 1D expression levels were not different from the healthy controls. 1D gene expression was demonstrated in both healthy (n = 10) and diseased (n = 10) eye muscle tissues. Furthermore, a recombinant protein derived from baculovirus-infected Sf9 insect cells was purified under both nondenaturing and denaturing conditions. While under nondenaturing conditions, the molecular mass of recombinant 1D was determined to be 85 kDa; denaturing isolation yielded the expected 64-kDa protein. Autoantibodies against denatured 1D protein were not detectable in sera of diseased or healthy subjects. Immunoreactivity against the 85-kDa, nondenatured protein, evaluated in a panel of 222 different human sera, showed that 82% of Graves' patients suffering from TAO had autoantibodies against recombinant 1D, whereas only 5% of the healthy controls were positive for antibodies against 1D. Taken together, our results demonstrate a high disease sensitivity and specificity of recombinant, nondenatured 1D, to distinguish Graves' disease with or without TAO from other forms of thyroid and/or eye disease. Prospective studies will have to show whether autoantibodies against 1D can also be used as a prognosticator of TAO.

Members of the fatty acid binding protein family are differentiation factors for the mammary gland.

Mammary gland development is controlled by systemic hormones and by growth factors that might complement or mediate hormonal action. Peptides that locally signal growth cessation and stimulate differentiation of the developing epithelium have not been described. Here, we report that recombinant and wild-type forms of mammary-derived growth inhibitor (MDGI) and heart-fatty acid binding protein (FABP), which belong to the FABP family, specifically inhibit growth of normal mouse mammary epithelial cells (MEC), while growth of stromal cells is not suppressed. In mammary gland organ culture, inhibition of ductal growth is associated with the appearance of bulbous alveolar end buds and formation of fully developed lobuloalveolar structures. In parallel, MDGI stimulates its own expression and promotes milk protein synthesis. Selective inhibition of endogenous MDGI expression in MEC by antisense phosphorothioate oligonucleotides suppresses appearance of alveolar end buds and lowers the beta-casein level in organ cultures. Furthermore, MDGI suppresses the mitogenic effects of epidermal growth factor, and epidermal growth factor antagonizes the activities of MDGI. Finally, the regulatory properties of MDGI can be fully mimicked by an 11-amino acid sequence, represented in the COOH terminus of MDGI and a subfamily of structurally related FABPs. This peptide does not bind fatty acids. To our knowledge, this is the first report about a growth inhibitor promoting mammary gland differentiation.

Structural studies on human muscle fatty acid binding protein at 1.4 A resolution: binding interactions with three C18 fatty acids.

BACKGROUND: Muscle fatty acid binding protein (M-FABP) is one of a family of cytosolic lipid-binding proteins involved in fatty acid processing. In order to investigate the precise interactions between M-FABP and its ligands and to understand the structural basis of differential binding affinity, we have compared the structures of M-FABP in complex with three C18 fatty acids. RESULTS: We describe the crystal structures of M-FABP in complex with n-octadecanoate (stearate), trans-delta 9-octadecenoate (elaidate) and cis-delta 9-octadecenoate (oleate). These structures were refined using least-squares positional and anisotropic temperature factor refinement to final R-factors of 11.4%, 12.1% and 13.2% respectively for all the data between 8.0 A and 1.4 A resolution. CONCLUSIONS: Stearate, elaidate and oleate each adopt highly similar U-shaped conformations when they bind to M-FABP within a large interior binding cavity, which also contains 13 ordered water molecules. The atomic structure of the protein is virtually identical, regardless of the nature of the bound ligand. The fatty acid is thought to enter the interior cavity of the protein via a portal in its surface while interior solvent is released through a secondary opening. The ligand affinity can be correlated with the conformational energy and the solubility of the bound ligand.

Escherichia coli-derived rat intestinal fatty acid binding protein with bound myristate at 1.5 A resolution and I-FABPArg106-->Gln with bound oleate at 1.74 A resolution.

Rat intestinal fatty acid binding protein (I-FABP) is a 131-residue protein composed of two short alpha-helices (alpha I and alpha II) and 10 anti-parallel beta-strands organized into two nearly orthogonal beta-sheets. The structure of crystalline I-FABP with bound tetradecanoate (myristate) has been refined to a resolution of 1.5 A and compared to the 1.2 A structure of apo-I-FABP, the 1.9 A structure of I-FABP:hexadecanoate (palmitate) and the 1.75 A structure of I-FABP:9Z-octadecanoate (oleate) to determine how this model fatty acid receptor accommodates changes in the length of its fatty acid ligand. Myristate is located in the interior of the protein. A highly ordered, electrostatic network containing 7 hydrogen (H)-bonds links the OE1 and OE2 atoms of myristate's carboxylate group, the indole nitrogen of Trp82, NH1, and NH2 of Arg106, NE2, and OE1 of Gln115, and 2 interior ordered waters. The hydrocarbon chain of the bound fatty acid is slightly bent. Its convex face lies in a crevice, forming van der Waals contacts with the side chains of several hydrophobic and aromatic residues. Its concave face is exposed to an array of 8 interior ordered waters whose positions are stabilized by H-bond interactions with other waters, H-bond interactions with the side chains of polar/ionizable residues, and van der Waals contacts with the surface of the fatty acid. Addition of 2 or 4 methylenes to myristate produces remarkably little change in the positions of I-FABP's main chain and side chain atoms and interior ordered waters. The principal alterations are in the conformation of a surface opening (portal) connecting external and internal solvent and in the position of the benzene side chain of Phe55. Changes in the conformation of the portal reflect movement of two of its components: the backbone of alpha II and a type I turn (Ala73, Asp74) connecting two beta-strands. The positions of the main chain atoms of Ala73 and Asp74 appear to be determined by their ability to form van der Waals contacts with the omega-terminus of the fatty acid. The side chain of Phe55 appears to function as an adjustable aromatic lid, located over the portal, whose position is dependent on an ability to form van der Waals contacts with a fatty acid's omega-terminus.(ABSTRACT TRUNCATED AT 400 WORDS)

Solution structure of bovine heart fatty acid-binding protein (H-FABPc).

Fatty acid-binding protein (FABP) from bovine heart, a 15 kDa cytoplasmic protein has been investigated by multi-dimensional homonuclear and heteronuclear NMR-spectroscopy. Perdeuterated palmitic acid has been used as fatty acid ligand. The tertiary structure has been determined from distance geometry calculations with the variable target functions algorithm (DIANA) utilizing 1027 interproton distance constraints, which were obtained from 1H-homonuclear NOESY spectra. Overlapping NOE crosspeaks were assigned by heteronuclear multidimensional NMR-experiments with a 15N-labelled sample. The tertiary structure resembles a beta-barrel (beta-clam) consisting of ten anti-parallel beta-strands and a short helix-turn-helix motif. The beta-strands are arranged in two nearly orthogonal beta-sheets composed of 5 strands each. The solution structure is compared with the x-ray crystal structure of bovine heart and rat intestinal FABPs.