Selective protein modification by the hydroperoxide intermediate in a photoprotein,aequorin

During the course of protein modification program, we employed a recombinant aequorin, the apo-protein reconstituted with coelenterazine, and found out that the photolytic hyperperoxide modified three –S–SCH₂CHOHCHOHCH₂SH groups to –S–SCH₂CHOHCHCH–SO)H or –S–SCH₂CHOHCHCH–S(O)OH of terminal DTT connected to cysteine residues of the C¹⁴⁵, C¹⁵² and C¹⁸⁰, which turned out to locate near the chromophore.     

Amino acid sequence of the calcium-dependent photoprotein aequorin

The Ca(II)-dependent photoprotein aequorin produces the luminescence of the marine coelenterate Aequorea victoria. The complete amino acid sequence of aequorin has been determined. A complete set of nonoverlapping peptides was produced by cyanogen bromide cleavage. These peptides were aligned by using the amino-terminal sequence of the intact protein and the sequences of selected arginyl and lysyl cleavage products. Although the aequorin preparations employed in these studies were homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the presence of a minimum of 3 isotypes was demonstrated by the location of 17 sites of sequence microheterogeneity. Two amino acid variants were observed at each of 16 positions while 1 position had 3 different replacements. The protein as isolated has 189 amino acids with an unblocked amino terminus. According to the sequence reported here, the molecular weight of the apoprotein is 21 459 while that of the holoprotein is 21 914. The molecule possesses three internally homologous domains which were judged to be EF-hand Ca(II) binding domains by several different criteria. Aequorin is homologous to troponin C and to calmodulin. These findings demonstrate that aequorin is a member of the Ca(II) binding protein superfamily.     

EDTA-binding and acylation of the Ca2+-sensitive photoprotein aequorin

The rate of phosphorylation and concomitant inactivation of purified pig heart muscle pyruvate dehydrogenase complex by intrinsic kinase (EC 2.7.1.99) is markedly accelerated by the addition of coenzyme A to the incubation medium, showing a half-maximum effect at 1.8 μM. The pantetheine moiety is the effective part of the coenzyme A molecule. The free thiol group is prerequisite for the stimulatory action, acetyl-CoA, benzoyl-CoA or CoAS-SCoA being ineffectual. The thiol's specificity is evidenced by showing that dithiothreitol, 2-mercaptoethanol or glutathione up to 5 mM failed to replace coenzyme A. The possibility is considered that coenzyme A might act as a physiological modifier of pyruvate dehydrogenase kinase activity.     

Bioluminescent immunoassay of thyrotropin and thyroxine using obelin as a label

Solid-phase bioluminescent immunoassay of thyroid hormones, human thyrotropin (hTSH) and two forms of thyroxine (T4), whose determinations are vitally important for diagnostics of thyroid diseases and the efficiency of treatment, is described. The recombinant obelin, a Ca(2+)-regulated photoprotein originally derived from the luminous marine hydroid Obelia longissima, is employed as a bioluminescent label. To produce obelin conjugates with anti-hTSH, anti-T4 immunoglobulins (IgG), and T4, additional SH groups are introduced into the obelin molecule using Traut's reagent (2-iminothiolane) and then obelin possessing extra SH groups is conjugated with succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate-activated IgGs or T4. The total yield of obelin conjugates determined by luminescent activity is 60-65% after all chemical and purification procedures. The obtained conjugates are stable to lyophilization and in solution for at least 9 months at 4 degrees C, with loss of activity not exceeding 10%. The application of obelin conjugates for determination of the hTSH, total T4, and free T4 in standard, control, and patient sera displays high sensitivity and reproducibility of results. The results of bioluminescent immunoassays are closely comparable to those obtained by the radioimmunoassay method (R=0.95-0.99).     

Bioluminescence immunoassay for angiotensin II using aequorin as a label

Angiotensin II is a biologically active component of the renin-angiotensin system. High levels of angiotensin II may be responsible for hypertension and heart failure because they increase systemic vascular resistance, arterial pressure, and sodium and fluid retention. Therefore, it is important to monitor angiotensin II levels for the treatment of hypertension and heart diseases. The goal of this work was to develop a bioluminescence immunoassay using aequorin as a label to measure angiotensin II levels in human plasma. This method utilizes a genetically engineered fusion protein between angiotensin II and aequorin. For that, the C terminus of angiotensin II was fused to the N terminus of apoaequorin using molecular biology techniques. A heterogeneous immunoassay was then developed for the determination of angiotensin II. A detection limit of 1 pg/mL was obtained with the optimized assay, allowing for the determination of angiotensin II at physiological levels in human plasma.     

Bioluminescence immunoassay for cortisol using recombinant aequorin as a label

The analysis of hormones in saliva is a powerful tool in the assessment of a patient's endocrine function, since it allows multiple noninvasive samplings. Since salivary levels of most hormones are 10 to 50 times lower than plasma levels, accurate and highly sensitive assays are needed for saliva measurements. Herein, we describe the development of a solid-phase competitive immunoassay for cortisol in saliva, in which a mutant of the photoprotein aequorin has been used as a label. We have chemically conjugated cortisol to aequorin at different molar ratios. The various cortisol-aequorin conjugates were characterized in terms of bioluminescent activity and affinity for the anti-cortisol antibody. The conjugate that gave the best analytical performance was used for the development of the immunoassay and the analysis of cortisol in saliva samples. The conjugates were stable for at least 6 months when stored at 4 degrees C. The method fulfilled all the standard requirements of precision and accuracy. The optimized immunoassay gave a detection limit of 300 fmol/tube, corresponding to 3 nmol/L, with a linear dynamic range of 10-1000 nmol/L. Therefore, cortisol can be detected down to 0.1 ng in 100 microl of saliva sample using this assay, without any sample pretreatment. This detection limit is almost one order of magnitude lower than the physiological levels of salivary cortisol, which are reported to be 10-25 nmol/L. This allows the quantification of salivary cortisol to be performed in the linear range of the calibration curve, which is most reliable for quantification purposes.     

Bioluminescence of the Ca-binding photoprotein, aequorin, after histidine modification

Modification studies of the 5 histidine residues in aequorin employing site-directed mutagenesis and diethyl pyrocarbonate suggested that His¹⁶⁹ may be the site of binding of molecular oxygen in aequorin. The modification of this residue led to complete loss of activity, whereas modification of the remaining 4 histidine residues yielded mutant aequorins with varying bioluminescence activities.     

Thermostable mutants of the photoprotein aequorin obtained by in vitro evolution

Aequorin is a photoprotein that emits light upon binding calcium. Aequorin mutants showing increased intensity or slow decay of bioluminescence were isolated by in vitro evolution combining DNA shuffling and functional screening in bacteria. Luminescence decay mutants were isolated at the first round of screening and carried mutations located in EF-hand calcium binding sites or their vicinity. During in vitro evolution, the luminescence intensity of the population of mutants increased with the frequency of effective mutations whereas the frequency of other amino acid substitutions remained roughly stable. Luminescence intensity mutations neighbored the His-16 or His-169 coelenterazine binding residues or were located in the first EF-hand. None of the selected mutants exhibited an increase in photon yield when examined in a cell-free assay. However, we observed that two mutants, Q168R and L170I, exhibited an increase of the photoprotein lifetime at 37 degrees C that may underlie their high luminescence intensity in bacteria. Further analysis of Q168R and L170I mutations showed that they increased aequorin thermostability. Conversely, examination of luminescence decay mutants revealed that the F149S substitution decreased aequorin thermostability. Finally, screening of a library of random Gln-168 and Leu-170 mutants confirmed the involvement of both positions in thermostability and indicated that optimal thermostability was conferred by Q168R and L170I mutations selected through in vitro evolution. Our results suggest that Phe-149 and Gln-168 residues participate in stabilization of the coelenterazine peroxide and the triggering of photon emission by linking the third EF-hand to Trp-129 and His-169 coelenterazine binding residues.     

Blue fluorescent protein from the calcium-sensitive photoprotein aequorin is a heat resistant enzyme, catalyzing the oxidation of coelenterazine

Blue fluorescent protein from the calcium-sensitive photoprotein aequorin (BFP-aq) was prepared and determined to be a heat resistant enzyme, catalyzing the luminescent oxidation of coelenterazine (luciferin) with molecular oxygen as a general luciferase. After treatment with excess ethylenediaminetetraacetic acid to remove Ca2+ from BFP-aq, the blue fluorescence shifted to a greenish fluorescence. This greenish fluorescent protein (gFP-aq) was identified as a non-covalent complex of apoaequorin with coelenteramide (oxyluciferin) in a molar ratio of 1:1. By incubation with coelenterazine in the absence of reducing reagents, gFP-aq was converted to aequorin at 25 degrees C. BFP-aq and gFP-aq possessing both fluorescence and luminescence activities may work as novel reporter proteins.     

Preparation and initial characterization of crystals of the photoprotein aequorin from Aequorea victoria

Crystals of recombinant aequorin, the photoprotein from the jellyfish Aequorea victoria, have been grown from solutions containing sodium phosphate. The crystals grow as thin plates which diffract to beyond 2.2 Å resolution. The crystals are orthorhombic, space group P2₁2₁2 ₁; the axes are a = 89.1(1), b = 88.4(1), and c = 52.7(1) Å. The asymmetric unit contains two molecules. Crystals exposed to calcium ion solutions emit a steady glow and slowly deteriorate, confirming that the crystals consist of a charged, competent photoprotein. This represents the first successful preparation of single crystals of a photoprotein suitable for diffraction analysis. © 1993 Wiley-Liss, Inc.     

Blue fluorescent protein from the calcium-sensitive photoprotein aequorin: catalytic properties for the oxidation of coelenterazine as an oxygenase

Blue fluorescent protein from the calcium-binding photoprotein aequorin (BFP-aq) is a complex of Ca2+ -bound apoaequorin and coelenteramide, and shows luminescence activity like a luciferase, catalyzing the oxidation of coelenterazine with molecular oxygen. To understand the catalytic properties of BFP-aq, various fluorescent proteins (FP-aq) have been prepared from semi-synthetic aequorin and characterized in comparison with BFP-aq. FP-aq has luciferase activity and could be regenerated into native aequorin by incubation with coelenterazine. The results from substrate specificity studies of FP-aq using various coelenterazine analogues have suggested that the oxidation of coelenterazine by BFP-aq in the luciferase reaction and the regeneration process to aequorin might involve the same catalytic site of BFP-aq.     

Galactose-dependent expression of the recombinant Ca2(+)-binding photoprotein aequorin in yeast

Aequorin is a Ca2(+)-binding protein that emits light upon reacting with Ca2+ and has been used as a probe for monitoring changes in the intracellular free Ca2+ concentration, [Ca2+]i. The protein consists of three components: apoaequorin (apoprotein), molecular oxygen and a chromophore. The present study was designed to conditionally express the apoaequorin cDNA of the jellyfish Aequorea victoria under the control of the GAL1 promoter in the yeast Saccharomyces cerevisiae and to investigate whether apoaequorin can be accumulated in high enough concentration in the cells to detect a Ca2+ signal in vitro. The results showed that the cells accumulated sufficient amounts of recombinant apoaequorin when incubated in the galactose-based medium and that the protein was active and not toxic to the cells, suggesting that the recombinant apoaequorin may be applicable to monitoring changes in [Ca2+]i in intact yeast cells.     

One-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography

A hexahistidine tag was fused to the N-terminus of apoaequorin. A suitable vector encoding the fusion protein was constructed and used for transformation of Escherichia coli JM109 cells. Apoaequorin was overexpressed under the control of tac promoter. It was found, however, that most of the protein existed in the form of inclusion bodies. Inclusion bodies were solubilized with urea, followed by purification and refolding of (His)(6)-apoaequorin in a single chromatographic step by immobilized metal-ion affinity chromatography using Ni(2+)-nitrilotriacetic acid agarose. The purity, as determined by SDS-PAGE analysis, was greater than 80%. The yield was 0.7-1 mg apoaequorin from a 50 ml bacterial culture. The kinetics of light emission of purified aequorin upon addition of Ca(2+) was typical of the commercial aequorin. The luminescence of the purified aequorin was a linear function of its concentration extending over six orders of magnitude. As low as 0.5 attomoles purified aequorin gave a signal-to-noise ratio of 1.8.     

Measurement of thromboxane A2-induced elevation of ionized calcium in collagen-stimulated platelets with the photoprotein, aequorin

Using aequorin-loaded rat platelets stimulated with collagen, we found two phases of Ca2+ mobilization, one coinciding with a shape change and the other with aggregation, which have not yet been detected in quin2-loaded platelets. U46619, a stable analogue of prostaglandin H2, induced only a shape change and a concomitant rapid rise in the cytoplasmic ionized calcium concentration ([Cai2+]). However, upon addition of U46619 to platelets previously stimulated with collagen in the presence of indomethacin, a rapid increase in [Cai2+] and a shape change occurred, and, after about 1 min, second increase in [Cai2+] and aggregation occurred. The actions of U46619 were inhibited by an antagonist for the thromboxane A2 (TXA2) receptor. These results suggest that the collagen-induced shape change is initiated by TXA2-induced Ca2+ mobilization, and aggregation is induced by the secondary Ca2+ mobilization induced by TXA2 and the occupation of the receptor by collagen.     

Cysteine-free mutant of aequorin as a photolabel in immunoassay development

The bioluminescent protein aequorin is a sensitive label that has been employed in a number of analytical applications. A mutant of aequorin with enhanced stability produced recombinantly in our laboratory has been employed as a label in the development of an immunoassay for digoxin. Digoxin is a cardiac glycoside used in the treatment of congestive heart failure. This drug has a very narrow therapeutic range of 0.8-2.0 ng/mL (1.0-2.5 nmol/L), thus requiring therapeutic drug monitoring. In this study, a derivative of digoxigenin was chemically conjugated to the mutant aequorin, and the resulting protein-digoxigenin derivative conjugates were characterized in terms of their luminescence properties. A solid-phase immunoassay for digoxin was then developed. The detection limit of the assay for digoxin was 1 x 10(-12) M. To demonstrate the use of this mutant aequorin as a label in biological sample analysis without any need for pretreatment of the samples, the assay was tested in serum spiked with digoxin. Interference from digoxin analogues was also evaluated to determine the specificity of the assay.     

Application of a fusion protein, metapyrocatechase/protein A, to an enzyme immunoassay

A fusion protein of metapyrocatechase and protein A was genetically produced for demonstration of effective conjugation of an enzyme with a binding protein employed in enzyme immunoassay. Plasmid pMPRA3, constructed by inserting the protein A gene into a plasmid pMK12 vector derived directly from the structural gene of metapyrocatechase, was expressed in Escherichia coli. The resulting fusion protein was shown to have promising properties for use in enzyme immunoassays due to the specific binding of the protein A moiety to the Fc portion of immunoglobulin G and to the high amplification of enzyme. Bovine serum albumin, a model antigen, was successfully determined in the concentration range from 1 x 10(-3) to 1 x 10(-7) g/ml.     

Streptavidin-aequorin fusion protein for bioluminescent immunoassay

The fusion protein of streptavidin to aequorin (STA-AQ) was highly purified from inclusion bodies in Escherichia coli cells and applied to a bioluminescent sandwich immunoassay. α-Fetoprotein (AFP), which is a serological marker of liver cancer, was used as a model analyte to test STA-AQ in an immunoassay. The measurable range of AFP by the sandwich immunoassay, using the complex of STA-AQ and the biotinylated anti-AFP antibody, was 0.02-200 ng/mL with an average coefficient of variation of 4.9%. The detection sensitivity with the complex of STA-AQ and the biotinylated anti-AFP antibody was similar to that with the complex of biotinylated aequorin, streptavidin and the biotinylated anti-AFP antibody. STA-AQ would be a useful reporter protein for immunoassays.