Determination of disulfide bond arrangement in bombyxin-IV, an insulin superfamily peptide from the silkworm,Bombyx mori, by combination of thermolysin digestion of natural peptide and selective synthesis of disulfide bond isomers

The mode of disulfide linkages in bombyxin-IV, an insulin superfamily peptide consisting of A- and B-chains, was determined as A6–A11, A7–B10, and A20–B22. An intermolecular bond of A20–B22 was identified by sequencing and mass spectrometric analysis of the fragments generated by thermolysin digestion of natural bombyxin-IV. The mode of the remaining two bridges was determined by chemical and selective synthesis of three possible disulfide bond isomers of bombyxin-IV. A- and B-chains were synthesized by solid-phase method, and three disulfide bonds were bridged stepwise and in a fully controlled manner. Retention time on reversed-phase high-performance liquid chromatography (HPLC), thermolysin digests, and biological activity of the synthetic [A6–A11, A7–B10, A20–B22-cystine]-bombyxin-IV revealed that it was identical with the natural bombyxin-IV. Two other isomers with respect to disulfide bond arrangement, [A6–A7, A11–B10, A20–B22-cystine]- and [A6–B10, A7–A11, A20–B22-cystine]-bombyxin-IVs, were distinguishable from the natural one by use of HPLC, thermolysin digestion, and bioassay.     

Synthesis of bombyxin-IV,an insulin superfamily peptide from the silkworm,Bombyx mori,by stepwise and selective formation of three disulfide Bridges

We report the synthesis of bombyxin-IV, a disulfide-linked, heterodimeric, insulin superfamily peptide from the silkworm,Bombyx mori. The two chains (A- and B-chains) were synthesized separately by the solid-phase method using fluoren-9-ylmethoxycarbonyl (Fmoc) group as a protecting group for α-amino group. Three disulfide bonds were bridged step by step (A6–A11, A20–B22, and A7–B10) in a good yield. Synthetic bombyxin-IV was identical with natural one with regard to the retention time on a reversed-phase column and the molecular weight measured by mass spectrometry. Circular dichroism (CD) spectrum of the synthetic bombyxin-IV was very similar to that of the natural one. The specific activity of synthetic bombyxin-IV is equal to that of natural one (0.1 ng/Samia unit). These results suggest that the synthetic bombyxin-IV has the tertiary structure identical with the natural peptide. Our method developed for synthesis of bombyxin-IV would be generally applicable to the synthesis of insulin-like heterodimeric peptides.     

Synthesis of bombyxin-IV, an insulin-like heterodimeric peptide from the silkworm, Bombyx mori

Bombyxin-IV, a molecular species of bombyxin, which is a member of insulin-like heterodimeric peptides of the silkworm Bombyx mori with prothoracicotropic hormone activity, was synthesized. The A- and B-chains of bombyxin-IV containing four and two Cys residues, respectively, were first synthesized separately by solid phase chemistry using Boc protocol. Then they were coupled by stepwise removal of two different protecting groups at the cysteinyl thiols for semiselective formation of disulfide bridges to give bombyxin-IV in 8% yield. The synthetic bombyxin-IV was shown to have chromatographic and biological properties identical with those of natural bombyxin-IV.     

Two-step selective formation of three disulfide bridges in the synthesis of the C-terminal epidermal growth factor-like domain in human blood coagulation factor IX.

The 45-residue C-terminal EGF-like domain in human blood coagulation factor IX has been synthesized by a 2-step method to form selectively 3 disulfide bridges. Four out of 6 cysteines are blocked with either trityl or 4-methyl-benzyl, and the remaining 2 cysteines are blocked with acetamidomethyl (Acm). In the first step, 4 free cysteinyl thiols are released concurrently with the removal of all protecting groups except Acm and are oxidized to form 1 of the 3 possible isomers containing 2 pairs of disulfides. In the second step, iodine is used to remove the Acm groups to yield the third disulfide bridge. This approach reduces the number of possible disulfide bridging patterns from 15 to 3. To determine the optimal protecting group strategy, 3 peptides are synthesized, each with Acm blocking 1 of the 3 pairs of cysteines involved in disulfide bridges: Cys5 to Cys16 (Cys 1-3), Cys12 to Cys26 (Cys 2-4), or Cys28 to Cys41 (Cys 5-6). Only the peptide having the Cys 2-4 pair blocked with Acm forms the desired disulfide isomer (Cys 1-3/5-6) in high yield after the first step folding, as identified by proteolytic digestion in conjunction with mass spectrometric peptide mapping. Thus, the choice of which pair of cysteines to block with Acm is critically important. In the case of EGF-like peptides, it is better to place the Acm blocking groups on one of the pairs of cysteines involved in the crossing of disulfide bonds.     

Isolation and structure determination of a paralytic peptide from the hemolymph of the silkworm, Bombyx mori.

A peptide with paralytic activity in larvae of the silkworm, Bombyx mori, was isolated from its hemolymph. Purification procedures consisted of extraction with 50% acetone, Vydac C4 reversed-phase cartridge elution and 4 steps of reversed-phase HPLC. Injection of the purified peptide into 4th instar B. mori larvae caused rapid and rigid paralysis for 2 min at a dose of 3.4 ng/larva. This paralytic peptide consists of 23 amino acid residues containing 2 cysteines with an intra-disulfide bond. The complete amino acid sequence is: H-Glu-AsnPhe-Val-Gly-Gly-Cys-Ala-Thr-Gly-Phe-Lys-Arg-Thr-Ala-Asp-G ly-Arg-Cys-Lys-Pro-Thr-Phe-OH. The relationship between structure and the biologic activity of synthetic analogs indicated that the entire amino acid sequence and the intra-disulfide bond were necessary for biological activity.     

Effect of disulfide bond on the conformation and anticoagulant activity of an Arg-Gly-Asp motif displayed on a mutant insulin protein framework

Summary Protein engineering techniques were employed to graft the known anticoagulant Arg-Gly-Asp (RGD) motifcontaining sequences onto the surface of a mutant, inactive insulin framework. To probe the effect of a disulfide bond on the resultant anticoagulant activity, a native RGD-containing sequence from disintegrin dendroaspin, CFTPRGDMPGPYC, as well as a modified sequence, SFTPRGDMPGPYS were each examined. The peptide was placed between the C-terminal of the B chain and the N-terminal of the A chain and connected with B27 and A1 residues of the inactive insulin that lacks the characteristic intramolecular A6–11 disulfide bond within the A chain. The two RGD-containing insulin genes were over-expressed inE. coli, and purified and designated as RGD-Cys-Ins and RGD-Ser-Ins, respectively. Their amino acid compositions and mass data were in good agreement with those of expected. The RGD-Cys-Ins showed inhibition of platelet aggregation with an IC₅₀ of 3 μM, while the latter was 3.5-fold less active. Thein vivo assay also indicated that the RGD-Cys-Ins had a higher activity in prolonging the bleeding time in mice than RGD-Ser-Ins. Both RGD-Cys-Ins and RGD-Ser-Ins retained about 25% of the proinsulin immunoactivity and had almost no insulin receptor binding activity. The results indicate the necessity for the RGD motif to be conformationally constrained for it to elicit a greater anticoagulant activity.     

Effect of disulfide bond on the conformation and anticoagulant activity of an Arg-Gly-Asp motif displayed on a mutant insulin protein framework

Protein engineering techniques were employed to graft the known anticoagulant Arg-Gly-Asp (RGD) motif-containing sequences onto the surface of a mutant, inactive insulin framework. To probe the effect of a disulfide bond on the resultant anticoagulant activity, a native RGD-containing sequence from disintegrin dendroaspin, CFTPRGDMPGPYC, as well as a modified sequence, SFTPRGDMPGPYS, were each examined. The peptide was placed between the C-terminal of the B chain and the N-terminal of the A chainand connected with B27 and A1 residues of the inactive insulin that lacks the characteristic intramolecular A6-11 disulfide bondwithin the A chain. The two RGD-containing insulin genes were over-expressed in E. coli, and purified and designatedas RGD-Cys-Ins and RGD-Ser-Ins, respectively. Their amino acid compositions and mass data were in good agreement with those ofexpected. The RGD-Cys-Ins showed inhibition of platelet aggregation with an IC₅₀ of 3 M, while the latter was3.5-fold less active. The in vivo assay also indicatedthat the RGD-Cys-Ins had a higher activity in prolonging the bleeding time in mice than RGD-Ser-Ins. Both RGD-Cys-Ins and RGD-Ser-Ins retained about 25% of the proinsulin immunoactivity and had almost no insulin receptor binding activity. The results indicate the necessity for the RGD motif to be conformationally constrained for it to elicit a greater anticoagulant activity.     

Analysis of precocious metamorphosis induced by application of an imidazole derivative to early third instar larvae of the silkworm,Bombyx mori

When an imidazole derivative (KK-42) was applied to day 1 third instar larvae of the silkworm, Bombyx mori, 100% underwent precocious metamorphosis at the end of the fourth instar. Thus, the fourth instar becomes the last instar in these KK-42–treated larvae. The endocrine systems underlying the precocious metamorphosis were analyzed in the present study. Hydroprene application during the prolonged third instar after KK-42 treatment can prevent precocious metamorphosis, and the results showed dose-dependent and stage-specific effects. From analysis of the developmental changes in ecdysteroid levels in both KK-42–treated larvae and KK-42– and hydroprene-treated larvae, we conclude that changes in JH levels during the third larval instar can modify the secretion pattern of prothoracic glands and that during the next larval instar, very low ecdysteroid levels during the early stages of the presumptive last (fourth) larval instar are directly related to precocious metamorphosis. Arch. Insect Biochem. Physiol. 36:349–361, 1997. © 1997 Wiley-Liss, Inc.     

Construction and characterization of bacterial artificial chromosome libraries from the silkworm, Bombyx mori.

Two bacterial artificial chromosome (BAC) libraries were constructed using nuclear DNA from posterior silkglands of the silkworm (Bombyx mori) strains p50 and C108. The libraries contain a total of 36,864 clones, or approximately 9 genome equivalents. The average insert sizes in the libraries were 134.5 kb and 120.8 kb, respectively. PCR-based screening was performed on the p50 library using probes for 34 sequence-tagged sites (STSs). Between 3 and 11 (6.1 hits on average) clones were isolated with each STS, in good agreement with the library size, 5.8 genome equivalents. The previously reported close linkage between the Bombyx homologs of the invected (Bm in) and engrailed (Bm en) genes was confirmed by construction of a BAC contig that contained both. Moreover, screening revealed novel information about the chromosomal organization of the sericin-1 and DH-PBAN genes, which were localized within a 22-kb interval and are divergently oriented. These results show that it is possible to construct contigs and analyze chromosome organization using these libraries. Received: 12 October 1998 / Accepted: 10 February 1999     

Peptide amidation in an invertebrate: Purification,characterization, and inhibition of peptidylglycine α-hydroxylating monooxygenase from the heads of honeybees (apis mellifera)

Peptidylglycine α-hydroxylating monooxygenase (PHM), an enzyme involved in formation of neuropeptides with a C-terminal amide functionality in mammals and amphibians, was isolated from the head of an invertebrate, the honeybee, Apis mellifera, and purified 220-fold in 1% overall yield. The bee PHM has a molecular weight of 71,000, is membrane associated but can be solubilized with a detergent (n-octyl-β-D-glucopyranoside), and cross-reacts with rabbit antibodies generated toward bacterially expressed rat PHM. In the presence of copper, oxygen, and ascorbic acid, the enzyme hydroxylates model tripeptides such as dansyl-L-Phe-L-Phe-Gly on the methylene carbon of the glycine residue with retention of configuration. Using this tripeptide as substrate, the K_m is 1.7 μM and the V_max is 2.3 nmol ? μg^?1 ? h^?1. Treatment of the insect PHM with D-Phe-L-Phe-D-vinylglycine, a substrate analogue and mechanism-based inactivator of PHM from pig pituitary, results in irreversible loss of activity. The diastereomeric analogue, D-Phe-L-Phe-L-vinylglycine, is only a competitive inhibitor (lC₅₀ = 320 μM). © 1994 Wiley-Liss, Inc.     

Evidences for a stage-specific juvenile hormone binding protein in the hemolymph of the silkworm,Bombyx mori L.: Identification and characterization by photoaffinity labeling and immunological analyses

Two molecular forms of juvenile hormone binding proteins were identified in the larval hemolymph of Bombyx mori by photoaffinity labeling. One form having an Mr of 33 kDa was present constantly in the hemolymph of the third to the fifth instar larvae while the other form having an Mr of 35 kDa was detected in the hemolymph until in the early fifth instar larvae but not in the prewandering larvae and prepupae. A 33 kDa binding protein was purified by hydrophobic interaction chromatography, gel filtration, and native PAGE. Antiserum against 33 kDa binding protein cross-reacted with 35 kDa binding protein on Western blots, suggesting that these binding proteins shared the same epitopes. From the results of saturation binding assays, it was inferred that 33 and 35 kDa binding proteins had a similar binding affinity for JH 1. It was revealed that one of these binding proteins, 35 kDa binding protein, was produced in the fat body in a stage-specific manner: fat body of the early fifth instar larvae synthesized both 33 and 35 kDa binding proteins while that of prewandering larvae synthesized only 33 kDa binding protein. © 1996 Wiley-Liss, Inc.     

Demarcation of diapause development by cold and its relation to time-interval activation of TIME-ATPase in eggs of the silkworm, Bombyx mori

We investigated the mode of action of winter cold in the termination of diapause by investigating Time-Interval-Measuring Enzyme (TIME). First, we determined the period of cold required for the completion of diapause development. Synchronously developing egg batches of a pure strain (C108 Bombyx mori silkworm) were used to minimize variations in hatching time. Hatching occurred with only 18 days chilling at 5 degrees C when the incubation at 25 degrees C after the chilling was elongated. The 18-day period was much shorter than we expected; diapause in B. mori is known to terminate completely with about 100 days of chilling. Even in such a short period of chilling, no sporadic hatching occurred. Moreover, we determined that a temperature-insensitive stage, which we called "Neboke", followed the short cold-requiring stage. Thus, the stage of diapause development was demarcated from other stages of diapause. While the length of diapause development was elongated when chilling was delayed after oviposition, the Neboke stage length was invariant. Cold evidently exerts its effect only on diapause development. When TIME was purified from eggs and chilled in test tubes, a transitory burst of its ATPase activity occurred at a time equivalent to shortly before the completion of diapause development; this was an interval-timer activation. The mechanism by which cold activates TIME to measure the time interval may help explain in biochemical terms the insect's adaptation to its seasonal environments.     

Syntheses of glycine and L-serine by their interconversion in the posterior silkgland of the silkworm, Bombyx mori

Summary. It was observed by solution-state ¹³C NMR spectroscopy that a great portion of the ¹³C of [1-¹³C]L-serine fed to the 5th instar larvae of the silkworm, Bombyx mori was incorporated into C1 of glycine in silk fibroin. [1-¹³C]Glycine was detected along with [1-¹³C]serine in fibroin of the posterior silkgland cultured in a medium containing [1-¹³C]serine. This formation of [1-¹³C]glycine was inhibited by addition of aminopterin to the culture medium. These findings suggest that an active conversion from serine to glycine, which needs tetrahydrofolate, occurs in the posterior silkgland for fibroin synthesis. Moreover, the solid-state ¹³C CP/MAS spectrum of the fibroin prepared from cocoons spun by larvae fed with [¹³C]formate revealed that serine C3 was labelled specifically with ¹³C, suggesting that the reverse conversion from glycine to serine took place in the silkworm. The posterior silkgland has the ability to synthesize not only fibroin but also its major materials, glycine and serine. Received May 4, 1999 Accepted December 10, 1999     

The consumption and utilization of the spiral blue-green algae Spirulina platensis in artificial diets by the silkworm, Bombyx mori

The consumption and utilization of various concentrations of the spiral blue-green algae, Spirulina platensis (Nordst.) Geitl., by the fourth-instar larvae of the silkworm, Bombyx mori L., was studied by incorporation into an artificial diet. The silkworms ingested more food on the low algal-powder diets, whereas they digested more efficiently on the high algal levels. The larvae fed on diets containing 30–40% algae had the shortest larval duration compared to those on low algal-powder diets. The freeze-dried powder of S. platensis is well utilized by B. mori larvae when added to artificial diets at levels between 15% and 30%, although the high algal content at 40% was slightly better for the efficiency of conversion of ingested diet into body substance (E.C.I.) and the cocoon production. However, analysis of the protein content in feces indicated an excess of the algal powder at 40%. Measurements of data on the food consumption and utilization in this study proposed that this spiral alga is dietetically utilizable as a protein source for rearing the silkworm in smaller quantities than on a soybean meal.

---

Resúmé La consommation et l'utilisation de différentes concentrations de Spirulina platensis par les chenilles de 4ème stade de Bombyx mori, ont été étudiées par incorporation dans un aliment artificiel. Les vers à soie ont ingéré plus d'aliment avec les régimes à faible concentration en algue, tandis qu'ils ont digéré plus efficacement les régimes à haute concentration d'algue. Les chenilles alimentées sur des régimes contenant 30 à 40% d'algue ont eu un développement larvaire plus bref que celles alimentées sur des régimes à faible concentration. La poudre congelée et séchée de S. platensis incorporée aux taux de 15 à 30% dans le régime artificiel est bien utilisée par les chenilles de B. mori, quoiqu'une teneur de 40% soit légèrement meilleure pour l'efficacité de conversion de l'aliment en matière vivante (E.C.I.) et la production de cocons. Cependant, l'analyse du contenu protéique dans les excréments révélait un excès de poudre d'algue à 40%. L'analyse des données au cours de cette étude, sur la consommation et l'utilisation des aliments, montre que S. platensis est utilisable diét étiquement en plus petite quantité que la farine de soja comme source de protéines pour l'élevage du ver à soie.

     

Controlled ecdysteroid accumulation in eggs of the silkworm,Bombyx mori,by an imidazole compound (KK-42), and embryogenesis in these eggs

An imidazole compound (KK-42), a potent inhibitor of ecdysone synthesis, was applied to the female pharate adult of the silkworm, Bombyx mori, to control ecdysteroid accumulation in developing ovaries and mature eggs. KK-42 applied on day 2 or later completely suppressed an increase in ecdysteroid content in developing ovaries. The inhibitory action of KK-42 was restricted to vitellogenic follicles, i.e., those in which active ecdysteroid synthesis is occurring. Ecdysteroid content in the mature eggs of moths remained at the level accumulated in ovaries before KK-42 application. Thus, KK-42 was shown to be a novel agent to suppress the ecdysteroid accumulation in eggs. Eggs containing different amounts of ecdysteroids showed different levels of embryonic development. About 80% of the eggs which contained less than 10 ng free ecdysteroids/g eggs were not fertilized. More than 80% of the eggs containing less than 40 ng/g eggs of free ecdysteroids initiated embryogenesis but failed to hatch. Larvae hatched from almost all eggs which accumulated free ecdysteroids of more than 150 ng/g. Thus, maternal ecdysteroids appear to be required at different titers for fertilization, embryogenesis, and hatching of the silkworm larvae. © 1994 Wiley-Liss, Inc.     

Synthesis and structure-activity relationship of beta-defensins, multi-functional peptides of the immune system.

beta-defensins are a large family of multiple disulfide-bonded peptides occurring in mammals and birds. They play an important role in the innate immune system, directly killing microbial organisms. Recent research has demonstrated that beta-defensins are important for other biological functions beyond antimicrobial effects, including inhibition of viral infection, interaction with Toll-like receptors, chemotactic effects, and sperm function. The corresponding broad spectrum of activities makes this peptide class an important subject and tool in immunologic research. In this review, we summarize the current status of the routes to obtain synthetic beta-defensins, their major structural properties and structure-activity relationship.     

Effect of the plant peptide regulator, phytosulfokine-alpha, on the growth and Taxol production from Taxus sp. suspension cultures

Phytosulfokine-alpha (PSK-alpha) is a small plant peptide (5 amino acids) that displays characteristics typically associated with animal peptide hormones. PSK-alpha was originally isolated based on its mitogenic activity with plant cultures; it has been reported to increase production of tropane alkaloids from Atropa belladonna, although its general influence on secondary metabolite production is unknown. The studies reported in this article were initiated to evaluate the effects of PSK-alpha supplementation on production of Taxol (paclitaxel) from plant cell cultures of Taxus sp. particularly when methyl jasmonate (MeJA) is added as an elicitor of secondary metabolism. The response to PSK-alpha supplementation was cell line dependent. Taxus cuspidata P93AF showed no statistically significant response to PSK-alpha supplementation while Taxus canadensis C93AD and T. cuspidata PO93X displayed a concentration-dependent response (up to 100 nM PSK-alpha added in first 24 h of culture) with a decrease in initial growth rate, an increase in cell density (dry weight/fresh weight), and increased Taxol production. More remarkably with T. canadensis (C93AD), a very strong synergistic response of PSK-alpha (100 nM) and methyl jasmonate (MeJA, 100 microM) elicitation was observed, resulting in Taxol level of 35.3 +/- 2.1 mg/L or 1.83 +/- 0.02 mg Taxol/g dry cell weight achieved at day 21, a level of approximately 10-fold higher than for either treatment by itself. Although the level of Taxol production achieved is not remarkable, this synergistic treatment was able to partially revive taxane production in cultures that have lost productivity due to extended time (over 10 years) in continuous subculture.     

Kinetics of Ampicillin Synthesis Catalyzed by Penicillin Acylase from E. coli in Homogeneous and Heterogeneous Systems. Quantitative Characterization of Nucleophile Reactivity and Mathematical Modeling of the Process

Kinetic regularities of the enzymatic acyl group transfer reactions have been studied using ampicillin synthesis catalyzed by E. coli penicillin acylase as an example. It was shown that ampicillin synthesis proceeds through the formation of an acylenzyme–nucleophile complex capable of undergoing hydrolysis. The relative nucleophile reactivity of 6-aminopenicillanic acid (6-APA) is a complex parameter dependent on the nucleophile concentration. The kinetic analysis showed that the maximum yield of antibiotic being synthesized depended only on the nucleophile reactivity of 6-APA, the ratio between the enzyme reactivities with respect to the target product and acyl donor, and the initial concentrations of reagents. The parameters characterizing the nucleophile reactivity of 6-APA have been determined. The algorithm of modeling the enzymatic synthesis has been elaborated. The proposed algorithm allows the kinetics of the process not only in homogeneous, but also in heterogeneous (aqueous solution–precipitate) systems to be quantitatively predicted and described based on experimental values of parameters of the reaction. It was shown that in heterogeneous aqueous solution–precipitate systems PA-catalyzed ampicillin synthesis proceeds much more efficiently compared to the homogeneous solution.