Biliverdin-binding Proteins Research Articles

Binding and sequestration of poison frog alkaloids by a plasma globulin.

Alkaloids are important bioactive molecules throughout the natural world, and in many animals they serve as a source of chemical defense against predation. Dendrobatid poison frogs bioaccumulate alkaloids from their diet to make themselves toxic or unpalatable to predators. Despite the proposed roles of plasma proteins as mediators of alkaloid trafficking and bioavailability, the responsible proteins have not been identified. We use chemical approaches to show that a ~50 kDa plasma protein is the principal alkaloid-binding molecule in blood of poison frogs. Proteomic and biochemical studies establish this plasma protein to be a liver-derived alkaloid-binding globulin (ABG) that is a member of the serine-protease inhibitor (serpin) family. In addition to alkaloid-binding activity, ABG sequesters and regulates the bioavailability of 'free' plasma alkaloids in vitro. Unexpectedly, ABG is not related to saxiphilin, albumin, or other known vitamin carriers, but instead exhibits sequence and structural homology to mammalian hormone carriers and amphibian biliverdin-binding proteins. ABG represents a new small molecule binding functionality in serpin proteins, a novel mechanism of plasma alkaloid transport in poison frogs, and more broadly points toward serpins acting as tunable scaffolds for small molecule binding and transport across different organisms.

A genetically encoded far-red fluorescent calcium ion biosensor derived from a biliverdin-binding protein.

Far-red and near-infrared (NIR) genetically encoded calcium ion (Ca2+ ) indicators (GECIs) are powerful tools for in vivo and multiplexed imaging of neural activity and cell signaling. Inspired by a previous report to engineer a far-red fluorescent protein (FP) from a biliverdin (BV)-binding NIR FP, we have developed a far-red fluorescent GECI, designated iBB-GECO1, from a previously reported NIR GECI. iBB-GECO1 exhibits a relatively high molecular brightness, an inverse response to Ca2+ with ΔF/Fmin = -13, and a near-optimal dissociation constant (Kd ) for Ca2+ of 105 nM. We demonstrate the utility of iBB-GECO1 for four-color multiplexed imaging in MIN6 cells and five-color imaging in HEK293T cells. Like other BV-binding GECIs, iBB-GECO1 did not give robust signals during in vivo imaging of neural activity in mice, but did provide promising results that will guide future engineering efforts. SIGNIFICANCE: Genetically encoded calcium ion (Ca2+ ) indicators (GECIs) compatible with common far-red laser lines (~630-640 nm) on commercial microscopes are of critical importance for their widespread application to deep-tissue multiplexed imaging of neural activity. In this study, we engineered a far-red excitable fluorescent GECI, designated iBB-GECO1, that exhibits a range of preferable specifications such as high brightness, large fluorescence response to Ca2+ , and compatibility with multiplexed imaging in mammalian cells.

Multiple origins of green coloration in frogs mediated by a novel biliverdin-binding serpin

Many vertebrates have distinctive blue-green bones and other tissues due to unusually high biliverdin concentrations-a phenomenon called chlorosis. Despite its prevalence, the biochemical basis, biology, and evolution of chlorosis are poorly understood. In this study, we show that the occurrence of high biliverdin in anurans (frogs and toads) has evolved multiple times during their evolutionary history, and relies on the same mechanism-the presence of a class of serpin family proteins that bind biliverdin. Using a diverse combination of techniques, we purified these serpins from several species of nonmodel treefrogs and developed a pipeline that allowed us to assemble their complete amino acid and nucleotide sequences. The described proteins, hereafter named biliverdin-binding serpins (BBS), have absorption spectra that mimic those of phytochromes and bacteriophytochromes. Our models showed that physiological concentration of BBSs fine-tune the color of the animals, providing the physiological basis for crypsis in green foliage even under near-infrared light. Additionally, we found that these BBSs are most similar to human glycoprotein alpha-1-antitrypsin, but with a remarkable functional diversification. Our results present molecular and functional evidence of recurrent evolution of chlorosis, describe a biliverdin-binding protein in vertebrates, and introduce a function for a member of the serpin superfamily, the largest and most ubiquitous group of protease inhibitors.

Rational Construction of Compact de Novo-Designed Biliverdin-Binding Proteins.

We report the rational construction of de novo-designed biliverdin-binding proteins by first principles of protein design, informed by energy minimization modeling in Rosetta. The self-assembling tetrahelical bundles bind biliverdin IXa (BV) cofactor autocatalytically in vitro, like photosensory proteins that bind BV (and related bilins or linear tetrapyrroles) despite lacking sequence and structural homology to the natural counterparts. Upon identification of a suitable site for ligation of the cofactor to the protein scaffold, stepwise placement of residues stabilized BV within the hydrophobic core. Rosetta modeling was used in the absence of a high-resolution structure to inform the structure-function relationships of the cofactor binding pocket. Holoprotein formation stabilized BV, resulting in increased far-red BV fluorescence. Via removal of segments extraneous to cofactor stabilization or bundle stability, the initial 15 kDa de novo-designed fluorescence-activating protein was truncated without any change to its optical properties, down to a miniature 10 kDa “mini”, in which the protein scaffold extends only a half-heptad repeat beyond the hypothetical position of the bilin D-ring. This work demonstrates how highly compact holoprotein fluorochromes can be rationally constructed using de novo protein design technology and natural cofactors.

CDNA sequences of two biliproteins, BP1 and BP2, from the cabbage white butterfly, Pieris rapae and their tissue- and stage-specific accumulation

Two similar full-length cDNAs of biliprotein were isolated and shown to encode the two isoelectric forms of biliverdin-binding proteins (BPs): BP1 and BP2 in Pieris rapae. Sequence analysis of two cDNA clones shows that both BPs contain a 567-bp open reading frame which predicts a 189-amino acid protein and a 15-amino acid signal peptide. The calculated isoelectric points are pI=7.25 (BP1) and 6.74 (BP2), respectively. Comparison of two sequences of BP1 and BP2 reveals 12 base differences in the open reading frame, of which three nucleotide changes lead to two amino acid substitutions. The 5′-UTR from the two clones shows no difference, but an additional 45-bp fragment is inserted in the 3′-UTR of BP2 making its message a little longer than that of BP1. Northern blot analysis confirmed that the BP mRNAs are expressed from the late 4th instar to the adult stage with exception of prepupae and newly ecdysed pupae. While the BP1 transcript was prevalent in the larval stage, the BP2 transcript was abundant in the whole body only after the pupal stage in P. rapae. Both BPs were detected in a stage-specific pattern in the epidermis, testis, hindgut, wing, brain, and egg, with a lesser amount in the fat body. Two-dimensional gel electrophoresis and Western blot analyses revealed that BP1 was dominant in tissues from larvae, BP2 was dominant in tissues from pupal stages, and both BPs appeared in tissues from the adult stage, though BP2 was predominant.

Purification and characterization of biliverdin-binding vitellogenin from the hemolymph of the common cutworm, Spodoptera litura.

Biliverdin-binding vitellogenin (Vg) was purified from adult female hemolymph of the common cutworm, Spodoptera litura, by using gel filtration and ion exchange chromatographies. The molecular mass of the protein was 490 kDa and it was composed of two 188-kDa subunits. Three internal amino acid sequences obtained by digestion of the protein with lysylendopeptidase showed high similarity to those of Bombyx mori Vg, supporting the purified blue protein to be vitellogenin. latroscan analyses demonstrated the presence of biliverdin in Vg that occupied 2.4% of total lipid components. Among the lipids of Vg (9.5 micrograms total lipids per 100 micrograms protein), diacylglycerol was the most predominant, followed by phospholipid, hydrocarbons, and then triacylglycerol, while in biliverdin-binding proteins (BPs) purified from larval hemolymph (3.1 micrograms total lipids per 100 micrograms protein), phospholipid was the most abundant lipid followed by diacylglycerol; hydrocarbons and triacylglycerol were minor components. Vg was first detected in the hemolymph of female pupae one day before eclosion, but injection of 5 micrograms of methoprene into a 3-day-old pupa induced Vg in the hemolymph 4 days earlier than in the control. Methoprene also induced a faster decline in BP-A and BP-B titers in the hemolymph with a corresponding increase of the Vg titer. These results suggest that juvenile hormone (JH) induces not only vitellogenesis but also the uptake of these proteins by stimulating the metamorphosis of fat body during the pupal stage.

Effects of a juvenile hormone analog, methoprene, on the hemolymph titers of biliverdin-binding proteins in the common cutworm, Spodoptera litura (Lepidoptera: Noctuidae).

Effects of methoprene (a juvenile hormone analog) on the larval development and hemolymph titers of biliverdin-binding proteins (BPs) in Spodoptera litura were studied. BP-A first appeared in the hemolymph of the fifth (penultimate) instar larvae, decreased during the molting to the sixth instar and prominently increased during the sixth (last) instar. On the other hand, the titer of BP-B, a predominant component in preceding stadia, greatly increased during the fifth instar, drastically decreased before the ecdysis to sixth instar, and slightly increased during the sixth (last) instar. Methoprene (0.1–5 μg/insect) topically applied on day 0 of the fifth instar caused little effect on the duration of the fifth instar and on the titers of BPs, while the application to day 0 sixth instars extended the last instar dose-dependently and changed the BP titers as follows: BP-A showed a similar increase as in untreated control, but continued to increase further during the prolonged feeding period; BP-B titer showed a increase within one day of the application. Double applications of methoprene at one day interval induced a stepwise increase of the BP-B titer at each application, but not of BP-A. These results suggest that BP-B synthesis is triggered by juvenile hormone, which also regulates the synthesis of BP-A indirectly by blocking larval-pupal transformation.

Purification and characterization of biliverdin-binding protein from larval hemolymph of the swallowtail butterfly, Papilio xuthus L.

The biliverdin-binding protein from the larval hemolymph of the swallowtail butterfly, Papilio xuthus L., was purified and characterized. The crude biliverdin-binding protein, obtained by ammonium sulfate fractionation, was purified in two steps, the first one by gel filtration chromatography and the second one by ion-exchange chromatography. The molecular mass of the purified protein was analyzed by SDS-polyacrylamide gel electrophoresis and estimated to be 21 kDa. The Namino terminal sequence of P. xuthus biliverdin-binding protein analyzed up to the 19th residue showed that 42% of the amino acid sequence are sequence similarity to the bilin-binding protein from Pieris brassicae. These results suggest that the P. xuthus biliverdin-binding protein belongs to the insecticyanin-type.

Purification and properties of two blue biliproteins from the larval hemolymph and integument of Rhodinia fugax (Lepidoptera: Saturniidae)

Blue biliproteins (BPs) are found in the hemolymph and integument of the fifth instar larvae of the saturniid silkworm, Rhodinia fugax. An efficient method of isolating BPs from the hemolymph, epidermis and cuticle using hydrophobic interaction chromatography and ion-exchange chromatography was devised. The BPs from the hemolymph, epidermis and cuticle have molecular weights of approximately 24 000, 48 000 and 23 000 Da by gel-filtration, respectively. Using matrix-assisted laser desorption ionization–time of flight/mass spectrometry (MALDI–TOF/MS), the respective molecular masses were determined to be 22 641, 22 908 and 22 737 Da. Based on these results, BP molecules from the hemolymph and cuticle are assumed to be monomers, whereas the epidermal BP is a dimer. The amino acid composition and N-terminal amino acid sequences of the BPs from the hemolymph and cuticle (BP-I) are very similar, but the BP from the epidermis (BP-II) is quite different. The N-terminal amino acid sequences of these BPs share approximately 50% identity with the biliproteins from other lepidopteran insects. The blue color of BP is due to the presence of bile pigments, which are non-covalently bound to the apoprotein. The absorbance spectrum of BP-I from the hemolymph revealed maxima at 280 and 669 nm, while that of BP-II showed maxima at 280, 385 and 663 nm. The pigment dimethyl esters were extracted from BP-I and BP-II with acidic methanol and dichloromethane. The results of these analyses suggest that the blue pigments of BP-I and BP-II are different; BP-I contains a phorcabilin-like pigment while BP-II contains biliverdin IX γ. In an immunoblot analysis, anti-BP-I antibodies, produced against hemolymph BP-I, reacted with immunoreactive proteins in the hemolymph and cuticle of R. fugax. These anti-BP-I antibodies did not react with BP-II and only cross-reacted weakly with Samia cynthia ricini biliverdin-binding protein (BBP)-II.

The crystal structure of nitrophorin 4 at 1.5 A resolution: transport of nitric oxide by a lipocalin-based heme protein.

Nitrophorins are nitric oxide (NO) transport proteins from the saliva of blood-feeding insects, which act as vasodilators and anti-platelet agents. Rhodnius prolixus, an insect that carries the trypanosome that causes Chagas' disease, releases four NO-loaded nitrophorins during blood feeding, whereupon the ligand is released into the bloodstream or surrounding tissue of the host. Histamine, a signaling molecule released by the host upon tissue damage, is tightly bound by the nitrophorins; this may facilitate the release of NO and reduce inflammation in the host. Recombinant nitrophorin 4 (NP4) was expressed in Escherichia coli, reconstituted with heme, and found to bind NO and histamine in a manner similar to that of the natural protein. The crystal structure of NP4 revealed a lipocalin-like eight-stranded beta barrel, with heme inserted into one end of the barrel. His59 ligates the proximal site on the heme, a solvent molecule (NH3) ligates the distal site, and three additional solvent molecules occupy the distal pocket. Buried in the protein interior are Glu55 and three solvent molecules. A detailed comparison with other lipocalins suggests that NP4 is closely related to the biliverdin-binding proteins from insects. The nitrophorins have a unique hemoprotein structure and are completely unlike the globins, the only other hemoproteins designed to transport dissolved gases. Compared with the recently described structure of NP1, the NP4 structure is considerably higher resolution, confirms the unusual placement of ionizable groups in the protein interior, and clarifies the solvent arrangement in the distal pocket. It also provides a striking example of structural homology where sequence homology is minimal.

Purification and characterization of two insecticyanin-type proteins from the larval hemolymph of the Eri-silkworm, Samia cynthia ricini

Two different biliverdin-binding proteins, designated BBP-I and BBP-II, were purified from the larval hemolymph of the Eri-silkworm, Samia cynthia ricini. These proteins were readily isolated from the hemolymph of fifth instar larvae using two chromatographic steps, hydrophobic interaction chromatography and ion exchange chromatography. Both BBPs were easily separated by Q-Sepharose HP column chromatography. BBP-I has an apparent molecular weight of 24 kDa, as determined by gel-filtration and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Native BBP-II had a molecular weight of 48 kDa estimated by gel-filtration. SDS–PAGE revealed a single band with a molecular weight of 26 kDa. Moreover, the molecular weights of BBP-I and BBP-II were determined to be 20,468 and 22,708 by MALDI-TOF/MS (matrix-assisted laser desorption ionization-time of flight/mass spectrometry), respectively. On this basis, BBP-I and BBP-II molecules are assumed to be a monomer and a dimer, respectively. The blue color of BBPs collected from the hemolymph is attributed to the presence of biliverdin IX γ, which is non-covalently and stoichiometrically bound to the apoprotein, based on absorbance maxima at 359 and 695 nm in methanol:HCl (95:5, v/v). One molecule of BBP-I contains one molecule of biliverdin IX γ, whereas BBP-II contains two molecules of biliverdin IX γ. The amino acid compositions of BBP-I and BBP-II are different, although the N-terminal sequences of both BBPs have a 48% identity. These BBPs were found in the hemolymph of fourth and fifth instar larvae. The newly molted fifth instar larvae had the highest concentration of BBP-I in the hemolymph. This gradually decreased during larval development. In contrast to BBP-I, the level of BBP-II was low, and increased slightly at the same developmental stage in S. cynthia ricini larvae.

Developmental changes of storage proteins and biliverdin-binding proteins in the haemolymph and fat body of the common cutworm, Spodoptera litura

The concentrations of three storage proteins (SL-1,SL-2 and SL-3, hexamers of 70–80 kDa subunits) and two biliverdin-binding proteins (BP-A and BP-B, dimers of 165 kDa) in the haemolymph and fat body during larval and pupal development of Spodoptera litura were determined by immunodiffusion tests using polyclonal antisera. SL-1 and SL-2 (methionine-rich) first appeared in the haemolymph of one-day-old sixth (final) instar larvae, prominently increased in the haemolymph during the later feeding period and were almost totally sequestered by the fat body after gut purge. SL-3 (arylphorin) was first detected in the haemolymph during the molting period to the final larval ecdysis, increased in concentration throughout the entire feeding period of the final larval instar and was partly sequestered by the fat body several hours later than the other storage proteins. BP-A showed nearly the same pattern in the haemolymph as SL-3: BP-B increased during feeding period and decreased during molting period and attained a maximum level during the penultimate larval instar, however its concentration decreased considerably and remained low in the final larval instar. BP-A was partly and BP-B was almost totally sequestered by the fat body 8 h after sequestration of SL-1 and SL-2, rendering the fat body blue in colour. These facts suggest an additional function of biliverdin-binding proteins as amino acid storage proteins and the results show a differential uptake mechanism for these proteins by the fat body.

Insecticyanin of Agrius convolvuli: purification and characterization of the biliverdin-binding protein from the larval hemolymph.

Blue biliprotein, insecticyanin (INS), has been purified from the hemolymph of the sweet potato hornworm, Agrius convolvuli. The protein was efficiently isolated from the hemolymph of fifth instar larvae using three successive column chromatographic techniques: hydrophobic interaction chromatography, ion exchange chromatography and gel-filtration. The purified INS showed a native molecular weight of approximately 59,000 by gel-filtration. SDS-PAGE revealed a single band with Mr of approximately 26,000. Moreover, the molecular mass of INS was 21,213 by MALDI-TOF/MS. Thus, the native A. convolvuli INS molecule was assumed to be a trimer in solution. The blue coloration of A. convolvuli INS from the hemolymph was attributed to the presence of biliverdin IX gamma, due to the absorbance maxima at 360 and 695 nm, which was non-covalently bound with the apoprotein. Amino acid composition and N-terminal sequence of A. convolvuli INS is similar to M. sexta INS. A. convolvuli INS represents one of the biliverdin-binding proteins in lepidopteran insects.

Purification and characterization of four biliverdin-binding proteins from larval haemolymph of the common cutworm, Spodoptera litura

Four biliverdin-binding proteins (BPs) were purified from the larval haemolymph of Spodoptera litura by a combination of KBr gradient ultracentrifugation and chromatofocusing. In the order of highest isoelectric point, we named the proteins BP-1, BP-2, BP-3 and BP-4. All BPs were composed of subunits of 165 kDa and their native molecular weights were estimated as 390 kDa by gel filtration. In addition to biliverdin, they contained more than 3% lipids (cholesterol, cholesterol ester, triacylglycerol, diacylglycerol, monoacylglycerol and phospholipid) and ca 3% carbohydrates ( d-fucose, d-mannose, d-glucose, d-galactose and N-acetyl- d-glucosamine). The amino acid composition of BP-4 differed slightly from the other BPs. In Ouchterlony's immunodiffusion tests, antiserum against BP-2 showed cross-reactivities with BP-1, BP-2 and BP-3, and antiserum against BP-4 only reacted with BP-4. An immunoblotting test revealed strong reactivities between BP-2 antiserum and BP-1, BP-2 and BP-3, and weak cross-reactivity with BP-4. Antiserum against BP-4 only showed strong reactivity with its own antigen, and weak cross-reactivities with BP-1, BP-2 and BP-3.

Purification and characterization of a biliverdin-binding protein from the molting fluid of the eri silkworm, Samia cynthia ricini (Lepidoptera: Saturniidae)

1. 1. The biliverdin-binding protein (BBP) was found in the molting fluid of the saturniid silkmoth, Samia cynthia ricini at the time of the larval-pupal ecdysis. 2. 2. It was purified from the molting fluid using hydrophobic interaction chromatography, gel-filtration and high-performance liquid chromatography (HPLC). 3. 3. The highly purified BBP showed a M r of approximately 49,000 by gel permeation of HPLC analysis using a TSKgel G3000SW column. 4. 4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a single band of M r approximately 24,000, indicating that the native BBP is dimer. 5. 5. The blue coloration of BBP from the molting fluid was due to the presence of biliverdin type pigments, as judged from its absorption spectra. 6. 6. These results suggested that BBP from Samia molting fluid may be a new type of insecticyanin (INS).

Purification and characterization of biliverdin-associated cyanoprotein from eggs and hemolymph of the bean bug, Riptortus clavatus (Heteroptera: Alydidae)

Biliverdin binding protein, cyanoprotein (CP), was purified from egg extracts of the bean bug, Riptortus clavatus. The preparation was shown to be homogeneous by DEAE-chromatography and polyacrylamide gel electrophoresis (PAGE). The native molecular weight (MW) of egg CP (CP egg) was estimated to be 320,000 by PAGE and 335,000 by gel filtration. The apoprotein consisted of identical subunits with MW of 76,000. CP egg had a high content of aromatic amino acids (17% mol ratio) with absorbance peaks at 370 and 620 nm which are characteristic of biliverdins. Native CP egg was associated non-covalently with 16 molecules of biliverdin. CP egg contained about 13.0% neutral sugar (mainly mannose) associated covalently with the apoprotein, no lipid was detected. In the hemolymph of the bugs, four CP bands (CP1, 2, 3 and 4) were detected by native PAGE. These bands were immunologically related and showed properties similar to CP egg in biochemical analysis. Only CP1 was identical to CP egg. CPs specific to the hemolymph, CP2, 3 and 4, were different from CP egg in mobility by PAGE, pI and peptide mapping profiles.

The reaction mechanism of bovine kidney biliverdin reductase

The steady-state kinetics of biliverdin reductase can be studied in detail at pH 9 as under these conditions the K m for biliverdin is high enough to obtain reliable measurements of the initial rate in the absence of any biliverdin binding proteins. The initial rate kinetics and the product-inhibition studies are consistent with an ordered sequential mechanism provided the biliverdin concentration was below 20 μM. Above this concentration significant flux occurs through a substrate inhibition pathway involving an enzyme-NAD(P)-biliverdin complex. Chloride is shown to cause a significant activation of the enzyme under certain conditions and this is shown to result from an inability of biliverdin to bind to an enzyme-NAD-chloride complex.

Purification and characterization of a biliverdin-associated protein from the hemolymph of Manduca sexta.

A biliverdin binding protein, insecticyanin, has been isolated from the hemolymph of the fourth instar tobacco hornworm Manduca sexta. The protein has been purified to apparent homogeneity by conventional chromatography with a cumulative yield of 40-50%. The protein (Mw 71 600) is composed of three subunits (Mr 23 000). Each subunit binds one biliverdin molecule. Proton magnetic resonance spectroscopy and absorption spectroscopy demonstrate that the bilin is the biliverdin IX gamma isomer.