Engineered variants of human glutamic acid decarboxylase (GAD) and autoantibody epitope recognition.

Of the two homologous forms of glutamic acid decarboxylase, GAD65 and GAD67, only GAD65 is a common target of autoimmunity. Epitope profiles of autoantibodies to GAD65 (GADA) in 140 type 1 diabetes, adult-onset diabetes mellitus (AODM), and thyroid diseases (TD) were studied. Probes were GAD65, GAD65/67 hybrids (displaying separately GAD65 residues 1-95, 96-444, and 445-585), delta GAD65 (a truncated GAD65 spanning residues 69-585), and GAD67. delta GAD65 and GAD65 detected 137 and 125 positive patients, respectively. The hybrids reacted with 113 sera and in 3 cases disclosed cryptic epitopes. Eighteen patients reacted with GAD67, indicating GAD65-GAD67 cross-reactivity. Most patients recognized both middle and C-terminal epitopes, had low reactivity against N-terminal epitopes, and seldom displayed reactivity limited to the N or C terminus. Compared with type 1 and AODM, TD patients showed a greater prevalence of multiple reactivity and higher incidence of GAD67 positivity.

Tryptophan mutants of intestinal fatty acid-binding protein: ultraviolet absorption and circular dichroism studies.

An UV absorption and CD study of intestinal fatty acid-binding protein is presented. Since there are only two Trp residues in the molecule, two single-Trp mutants were prepared to deconvolute their signals. The individual contribution of the eight Phe and four Tyr residues was not established; however, Phe global contribution is relatively free of interferences from the other chromophores and was observed directly. CD spectra showed that Phe vibronic structure was unusually sharp and seems to monitor very specific details in the three-dimensional structure. The global signal from Tyr was assigned only approximately due to band broadening and overlapping. At the upper end of the CD spectrum, strong positive (1)L(b) Trp transitions from Trp 82 and strong negative (1)L(b) Trp transitions from Trp 6 were observed. (1)L(a) transitions were overall weak, positive for Trp 82 and negative for Trp 6, nearly cancelling each other out in the final spectrum. The above assignment is of practical and fundamental interest to monitor folding, binding, and molecular dynamics down to microdomain resolution. The assignment of Trp bands allowed comparison with previous data from CRABP1, another member of the IFABP family with 28% identical residues. It was found that structural homology extends beyond sequence and tertiary fold to include optical properties of equivalent Trp residues in the structure.

Combined measurement of diabetes mellitus immunological markers: an assessment of its benefits in adult-onset patients.

The convenience of combining the measurement of antibodies to glutamic acid decarboxylase (GADA), protein tyrosine phosphatase (IA-2A), and autoantibodies to insulin (IAA) in diabetic patients was assessed. We analysed 71 type 1 and 115 adult-onset diabetic patients. The latter were grouped into three categories according to the time of evolution to insulin dependence. The main findings were as follows: (i) in type 1 diabetes, the combined analysis of GADA and IA-2A showed a sensitivity of 87.4% and was not appreciably improved by adding IAA; (ii) out of 31 adults who required insulin immediately or within the first two years of diagnosis, 41.9, 29.0, and 6.5% were positive for at least one, two or all three, and all three markers, respectively; GADA was the most prevalent (35.5%) and IA-2A the least represented (16.1%); (iii) 34 adult patients with slow evolution to insulin dependence showed a completely different profile: 5.9% were GADA positive and 23.5% were IAA positive and no double or triple positivity was observed as all patients were IA-2A negative; and (iv) 50 type 2 patients who had not required insulin treatment showed a low incidence of GADA (4%) as the only marker present. We conclude that a combined double-antigen test for GADA and IA-2A is a useful strategy for prospective screening of type 1 diabetes. However, in adults, the profile of individual markers discloses the course to insulin dependence. Therefore, it seems advisable to measure the markers separately, to allow a better classification of these patients, and help define their treatment.

Head-to-tail and side-by-side oligomerization of human carbonic anhydrase II: a small angle X-ray scattering study.

Solvent-induced directional aggregation of human carbonic anhydrase II (hCA) was studied by small angle X-ray scattering and fluorescence and fourth-derivative ultraviolet absorption spectroscopy. We propose that hCA at 5 mg ml(-1) in pure water forms head-to-tail oligomers built up, on average, by four to five monomers. At higher protein concentrations, the oligomers associate pair-wise and side-by-side. Spectroscopic evidence suggests that the subunits forming the aggregates are tightly folded, but with a structure that differs, at least locally, from the native state. A more complex aggregation pattern was observed under solvent conditions that favor the removal of zinc from the enzyme-active site, conditions under which the subunits are significantly less compact than in water. hCA may provide a useful model to investigate the effects of additives and genetic manipulation on protein aggregation.

Export and folding of signal-sequenceless Bacillus licheniformis beta-lactamase in Escherichia coli.

Two genetically engineered variants of the Bacillus licheniformis beta-lactamase gene were expressed in Escherichia coli. One variant coded for the exo-small mature enzyme without the signal peptide. The other coded for the exo-large mature enzyme preceded by 10, mostly polar, residues from an incomplete heterologous signal. As observed following the extraction by a lysozyme-EDTA treatment, the signal-less variant was exported to the periplasm with nearly 20% efficiency, whereas the variant with the N-terminal extension was translocated to a lesser degree; interestingly, nearly all of the former and half of the latter were extracted by osmotic shock, which may be of importance for our understanding of cellular compartments. The fact that a signal-less protein is translocated with substantial yields raises questions about the essential role of signal peptides for protein export. As folding and export are related processes, we investigated the folding in vitro of the two variants. No differences were found between them. In the absence of denaturant, they are completely folded, fully active and have a large DeltaG of unfolding. Under partially denaturing conditions they populate several partially folded states. The absence of significant amounts of a non-native state under native conditions makes a thermodynamic partitioning between folding and export less likely. In addition, kinetic measurements indicated that these B. licheniformis lactamases fold much faster than E. coli beta-lactamase. This behavior suggests that they are exported by a kinetically controlled process, mediated by one or more still unidentified interactions that slow folding and allow a folding intermediate to enter the export pathway.

Replacement of methionine-161 with threonine eliminates a major by-product of human glutamate decarboxylase 65-kDa variant expression in Escherichia coli.

Most insulin-dependent diabetes mellitus patients gen-erate conformational autoantibodies to the islet-cell 65-kDa variant of human glutamate decarboxylase (GAD65), and several immunochemical tests for the early detection of type-1 diabetes rely on GAD65 antibody (GADA) assessment using properly folded recombinant GAD65 as the antigen. In addition, preventive therapies based on tolerization by GAD65 administration may be available in the near future. Therefore, there exists a strong interest in a facile and economically sound expression procedure for this antigen. Several attempts to produce, in native form, wild-type GAD65 in Escherichia coli have failed. However, this difficulty was recently surmounted in our laboratory by expressing GAD65 as a fusion protein with thioredoxin [Papouchado, Valdez, Ghiringhelli, Poskus and Ermácora (1997) Eur. J. Biochem. 246, 350-359]. In this work, a new GAD65 hybrid gene was prepared by joining engineered cDNA obtained from human and rat tissues. The new gene was modified additionally to finally code for human GAD65 with a single amino-acid substitution: Met-161-->Thr. This change impeded the co-expression of a 48-kDa by-product from an internal translation site. Also, a second 58-kDa by-product was identified as a GAD65 C-terminal proteolytic fragment that co-purifies with thioredoxin-M161T GAD65. The new GAD65 variant was expressed and easily purified, yielding an antigen that performed equally or better than wild-type GAD65 in the reference radiobinding assay for GADA. The procedure provides an inexpensive source of large amounts of fully active and immunochemically competent GAD65.

Engineering a compact non-native state of intestinal fatty acid-binding protein.

The last three C-terminal residues (129-131) of intestinal fatty acid-binding protein (IFABP) participate in four main-chain hydrogen bonds and two electrostatic interactions to sequentially distant backbone and side-chain atoms. To assess if these interactions are involved in the final adjustment of the tertiary structure during folding, we engineered an IFABP variant truncated at residue 128. An additional mutation, Trp-6-->Phe, was introduced to simplify the conformational analysis by optical methods. Although the changes were limited to a small region of the protein surface, they resulted in an IFABP with altered secondary and tertiary structure. Truncated IFABP retains some cooperativity, is monomeric, highly compact, and has the molecular dimensions and shape of the native protein. Our results indicated that residues 129-131 are part of a crucial conformational determinant in which several long-range interactions, essential for the acquisition of the native state, are established. This work suggests that carefully controlled truncation can populate equilibrium non-native states under physiological conditions. These non-native states hold a great promise as experimental models for protein folding.

Highly-sensitive and specific enzyme-linked immunosorbent assays for GAD65 autoantibodies using a thioredoxin-GAD65 fusion antigen.

Autoantibodies against glutamic acid decarboxylase (GAD65) are present in the sera of most patients with recently diagnosed insulin-dependent diabetes mellitus (IDDM). These antibodies appear years before the clinical symptoms, and they are considered to be early markers of the disease. To detect GAD65 autoantibodies (GADA), we developed new enzyme-linked immunosorbent assays (ELISA) with a fusion protein thioredoxin-GAD65 (Trx-GAD65) produced in E. coli as the antigen. These assays were compared with the reference radiobinding assay (RBA). Since most GADA are directed against native epitopes, and adsorption of GAD65 to plastic may cause disruption of its native conformation, the new assays rely on the following immobilization procedures: (a) capture ELISA (c-ELISA) with Trx-GAD65 (protocol A) or biotin-Trx-GAD (protocol B) indirectly immobilized by a non-adsorptive process; (b) ELISA with antigen-antibody preincubation in solution (p-ELISA) in which GADA were reacted first with Trx-GAD65 (protocol C) or biotin-Trx-GAD (protocol D) and the free antigen was determined by conventional ELISA. The results obtained with 42 newly diagnosed IDDM patients and 30 normal individuals were as follows: RBA had 79% sensitivity (percentage of IDDM patients detected) and 97% specificity (100% minus the percentage of false positives). c-ELISA showed low sensitivity (36 and 50%, respectively for protocols A and B), and high specificity (100 and 97%, respectively). p-ELISA were highly-sensitive (74 and 79%, respectively) and specific (97 and 93% for protocols C and D, respectively). Thus, protocols C and D had a performance similar to the reference method. The results reported here provide the basis for simple, highly-sensitive, specific, and widely-applicable tests for GADA that eliminate many of the drawbacks of the radioactive methods.

Time differential perturbed angular correlation of 111In-EDTA linked to the single free cysteine of bovine serum albumin.

By means of a facile chemical modification of the bovine serum albumin molecule, it was possible to measure its hydrodynamic radius with high accuracy (approximately 3%) using the TDPAC technique. The new approach presented here allows a wide use of the TDPAC technique to perform high precision studies of backbone dynamics of almost any protein.

Expression of properly folded human glutamate decarboxylase 65 as a fusion protein in Escherichia coli.

Autoantibodies to the islet-cell 65-kDa variant of glutamate decarboxylase (GAD65) are found in most insulin-dependent diabetes mellitus (IDDM) patients many years before the appearance of clinical symptoms of the disease. As IDDM-preventive therapies may be available in the future, an international effort is taking place to develop widely applicable anti-GAD immunochemical tests. These tests would help to detect individuals at risk before the full installation of the disease and to enroll them in prevention programs. Autoantibodies to GAD65 are mostly directed to conformational epitopes, and the enzyme is a complex molecule with a prosthetic group and 15 cysteine residues. Thus, the conformational integrity of GAD65 is essential for an appropriate anti-GAD assay. Isolation of large amounts of GAD65 from pancreas or other tissues is impractical, and no successful production of properly folded GAD65 has been reported in bacteria. Native recombinant GAD65 for immunochemical tests is usually obtained from eukaryotic expression systems. Since the large-scale production of a recombinant protein in an eukaryotic system is expensive and technically difficult, we investigated the expression of GAD65 in Escherichia coli as an alternative. A number of DNA constructs intended to export the enzyme to the periplasmic space or to improve its cytoplasmic solubility were designed and tested. Our results provide a solution to the two main problems associated with the expression of GAD65 in E. coli: misfolding, leading to the formation of inclusion bodies; and the presence of alternative initiation sites for translation that causes the preferential production of truncated variants of GAD65. We describe here the production of properly folded, fully active, and immunochemically competent GAD65 as an N-terminal fusion protein with thioredoxin. An account of the reactivity of the produced protein with sera of six IDDM patients is also presented.

Detection of glutamic acid decarboxylase (GAD) autoantibodies by indirect immunofluorescence using CHO cells expressing recombinant human GAD65.

The reaction between human glutamic acid decarboxylase (GAD65) expressed in CHO cells and GAD antibodies was studied by indirect immunofluorescence (IIF). The monoclonal antibodies GAD1 and GAD6, which recognize conformational and continuous GAD epitopes respectively, yielded distinct staining patterns. Twelve of 26 sera from newly-diagnosed insulin-dependent diabetes mellitus (IDDM) patients displayed a variety of anti GAD specific IIF images encompassing the two extremes observed with the monoclonal antibodies. None of 21 normal sera tested positive in this assay. As a control, the sera were tested by a reference immunoprecipitation (IP) assay using in vitro produced, folded 35S-GAD65. Only one of the patient sera reacted by IP using heat- and detergent-denatured 35S-GAD65 indicating that most of the auto-antibodies recognized only a folded antigen. Eleven patient sera were both IIF and IP anti-GAD-positive. The IIF reactivity of these sera was blocked by soluble GAD from brain extracts. One serum was positive only by IIF, and its reactivity was not blocked by soluble GAD. Eight sera were positive only by IP. Our results established differences in anti GAD antibodies in terms of their capacity to recognize human GAD65 in the context of transformed CHO cells compared with conventional IP assays. These differences should be considered in future attempts to improve the available assays for the detection of IDDM autoantibodies.

Purification and partial characterization of a fatty acid-binding protein from the yeast, Yarrowia lipolytica.

A low-molecular-mass fatty acid-binding protein was isolated from the cytosol of the yeast Yarrowia lipolytica. Purification was achieved by a two-step procedure involving size-exclusion and cation-exchange chromatography. The isolated protein exists as a monomer of 15 kDa, is basic and has a blocked N-terminus. Internal amino acid sequencing suggests that this protein may belong to a novel class of fatty acid-binding proteins.

Mapping the structure of a non-native state of staphylococcal nuclease.

Non-native states of proteins populated at extremes of pH, or by mutation or truncation of the protein sequence, are thought to be equilibrium models for kinetic intermediates on the folding pathway. While the global physical properties of these molecules have been well characterized, analysis of their structure by NMR spectroscopy has proven difficult. Here we report the use of a new chemical cleavage technique, based on reactive oxygen species, to map the backbone fold of a truncated form of staphylococcal nuclease in a non-native state. The fragment adopts a native-like fold, however the technique also reveals regions of non-native structure.

Mapping staphylococcal nuclease conformation using an EDTA-Fe derivative attached to genetically engineered cysteine residues.

Six single cysteine variants of staphylococcal nuclease were reacted with the iron complex of (EDTA-2-aminoethyl) 2-pyridyl disulfide (EPD-Fe) [Ermácora, M. R., Delfino, J. M., Cuenoud, B., Schepartz, A., & Fox, R. O. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6383-6387] and used to assess the ability of this cleavage reagent to faithfully report on the structure of nonnative protein states. The act of mutation and modification did not significantly alter the protein's global structure, as measured by CD and enzymatic activity, and only modestly affected its stability. The reaction was conformation dependent and generated specific cleavage products that mapped tertiary interactions present in the folded state. Several parameters relevant to the cleavage reaction and its use as a conformational probe were analyzed. Proximity and solvent accessibility are the most important parameters in determining the cleavage pattern and can be used to predict cleavage sites in the native protein. The cleavage reaction can be performed in the presence of high denaturant concentration, in the presence of SDS, and under a wide range of pH values; thus it can readily be applied to the study of equilibrium folding intermediates. Mass spectrometric analysis combined with N-terminal sequencing identified cleavage products consistent with a single cleavage event per protein molecule and revealed one cleavage mechanism which was not previously considered for protein oxidative degradation, although it was reported for hydroxyl radical induced cleavage of small peptides. Identification of the cleavage sites obtained from each variant allowed a nearest-neighbor mapping of the secondary structural elements of nuclease. Quantitation of specific cleavage products was used to monitor the disruption of the interaction between helices H2 and H3 in equilibrium unfolding experiments. The resulting unfolding curve revealed a local conformational heterogeneity at low denaturant concentration which was not observed when the same transition was monitored by the change in fluorescence of a single nuclease tryptophan. Interestingly, the midpoint of the transition and the second half of the unfolding curve were the same, as monitored by the two probes. This indicates that the lifetime of the reactive oxygen species generated by the cleavage reagent is short compared to the unfolding equilibrium rate constants and that the cleavage technique identifies a native-like folding intermediate not detected by fluorescence. The experiments presented herein demonstrate that EPD-Fe-mediated protein cleavage is an appropriate technique for the study of nonnative protein structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Oxidative polypeptide cleavage mediated by EDTA-Fe covalently linked to cysteine residues.

Chemical cleavage with reactive oxygen species generated by EPD-Fe, a protein-tethered EDTA-Fe reagent, has been proposed as a method to map the structure of nonnative equilibrium protein folding intermediates [Ermácora, M. R., Delfino, J. M., Cuenoud, B., Schepartz, A., & Fox, R. O. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6383-6387]. The chemical structure of protein cleavage products and the mechanism of backbone scission for this class of reagents have been unclear. Here, we report the nature of EPD-Fe-mediated backbone cleavage of a small model peptide. The EPD-Fe reagent was attached to a partially alpha-helical peptide, alpha 1BA1a (Ac-AEAEEAAKKAKEACKA-NH2), through a mixed disulfide. Backbone cleavage was initiated by addition of the iron reductant ascorbate. Chemical analysis of the novel cleavage products revealed an oxidative cleavage mechanism, probably initiated by diffusible hydroxyl radicals. The EPD-Fe-mediated cleavage technique appears to be suitable for the analysis of nonnative protein states such as the molten globule.

Mapping interactions between the catalytic domain of resolvase and its DNA substrate using cysteine-coupled EDTA-iron.

Single cysteine-substituted mutants of gamma delta resolvase have been covalently modified using a novel sulfhydryl-specific EDTA derivative, EDTA-2-aminoethyl 2-pyridyl disulfide (EPD). Iron, chelated by the coupled EDTA and in the presence of reducing agent, generates reactive oxygen species that result in localized cleavage of the DNA to which resolvase is bound. The procedure provides valuable information on two fronts. First, it allows the identification of regions or surfaces of the protein that are in close proximity to DNA even though they may not be part of the DNA-binding domain. Second, it allows identification of the portions of DNA that are closest to each EDTA-derivatized cysteine, since the DNA cleavages observed are highly localized and their efficiency drops rapidly as a function of the distance between the EDTA-Fe complex and the deoxyribose target. We have used the procedure to investigate the interaction of gamma delta resolvase with the three DNA binding sites that constitute its recombination substrate, res. The data indicate that the two N-terminal domains of a resolvase dimer interact symmetrically with site I, which contains the recombination cross-over point, but asymmetrically with the accessory sites, II and III. The patterns of DNA cleavage obtained with several different EDTA-coupled mutants have enabled us to propose a model for the interaction between resolvase and site I.

Inhibitory action of palmitic acid on the growth of Saccharomyces cerevisiae.

High concentrations of long-chain fatty acids have been found to be harmful to mammalian cells and prokaryotic organisms. This effect was investigated in Saccharomyces cerevisiae. Addition of 3 mmol/L palmitate to a yeast extract-peptone medium caused a significant inhibition of cell growth during the first 2 d of incubation, followed by renewed growth and palmitate utilization. Inhibition was also observed with palmitate concentrations down to 0.1 mmol/L. As inferred from catalase activity determinations, this effect was found to correlate with the absence of peroxisome proliferation. Finally, no inhibition was observed in exponential-phase cultures or in the presence of 0.1 g/L glucose, this suggesting that the physiological state of the cell may determine whether its growth will be inhibited by fatty acids.

Rat brain fatty acid-binding protein during development.

Cytosolic fatty acid-binding proteins (FABPs) have been described in rat and bovine whole brain. In the present study we investigated the distribution of FABP among white matter and gray matter as well as its changes during development. Fatty acid binding activity was similar in white and gray matter up to 40 days of age. In white matter it showed an age dependent increase thereafter, while in gray matter it remained constant throughout. Gel filtration (Sephadex G-75) of white matter cytosol of adult female rats resolved the fatty acid-binding activity in two peaks: A (Vo) and B (12-14 KDa; FABP). The specific binding activity in the FABP fraction was 10.4 pmol/micrograms of protein. The activity in peak A showed an age-dependent increase which paralleled myelin deposition. In contrast, the activity in the FABP fraction (peak B) remained undetectable up to 40 days of age, increasing thereafter. The differential distribution of cellular brain proteins with the capacity to bind fatty acids in gray matter and white matter suggests that this activity could be related to glial cells or to cell related structures such as myelin.

Conformation-dependent cleavage of staphylococcal nuclease with a disulfide-linked iron chelate.

We report the synthesis and evaluation of (EDTA-2-aminoethyl) 2-pyridyl disulfide. By using this easily prepared cysteine-specific hydrophilic reagent, an ethylenediaminetriacetic acid-Fe3+ complex (EDTA-Fe) was covalently attached to a single genetically engineered cysteine residue in staphylococcal nuclease. Upon addition of the iron reductant ascorbate, the nuclease-EDTA-Fe conjugate underwent a protein self-cleavage reaction mediated by reactive oxygen species. Sequence analysis of the products indicated that cleavage occurs close in tertiary structure to the EDTA-Fe attachment site. In the presence of denaturants, the cleavage pattern changes and the reaction is limited to residues proximal in sequence to the cysteine attachment site. These results indicate that intramolecular protein cleavage reactions mediated by EDTA-Fe can be used to evaluate changes in protein conformation. The reagent described should be a useful tool in the structural mapping of nonnative protein states populated at equilibrium, such as the molten globule, that are frequently refractory to conventional structure analysis.

Study on fatty acid binding by proteins in yeast. Dissimilar results in Saccharomyces cerevisiae and Yarrowia lipolytica.

1. The presence of soluble proteins with fatty acid binding activity was investigated in cell-free extracts from Saccharomyces cerevisiae and Yarrowia lipolytica cultures. 2. No significant fatty acid binding by proteins was detected in S. cerevisiae, even when grown on a fatty acid-rich medium, thus indicating that such proteins are not essential to fatty acid metabolism. 3. An inducible fatty acid binding protein (K0.5 = 3-4 microM) was found in Y. lipolytica which had grown on a minimal medium with palmitate as the sole source of carbon and energy. 4. The relative molecular mass of this protein was 100,000 as inferred from Sephacryl S-200 gel filtration.