Isolation, purification and characterization of silk protein sericin from cocoon peduncles of tropical tasar silkworm, Antheraea mylitta

A high molecular weight water-soluble glue protein, sericin was identified in the cocoon peduncle (a strong thread connecting the cocoons to the branches of the tree with a ring) of the tropical tasar silkworm, Antheraea mylitta. The sericin was isolated by 8 M urea containing 1% sodium dodecyl sulfate and β-mercaptoethenol (2%) or by 1% sodium chloride. The protein was purified by gel filtration chromatography. In SDS-PAGE, a single band of approximately 200 kDa was detected both in non-reducing and reducing conditions. Amino acid analysis showed that the protein is enriched in glycine and serine. There is a slight difference observed in amino acid composition between the sericin from cocoon peduncle and cocoon of A. mylitta. Secondary structure estimation by circular dichroism spectrometry showed 36.7% β-sheets, 52.7% random coils, 10.6% turns and no helices.     

Purification and characterization of fibroin from the tropical Saturniid silkworm, Antheraea mylitta

The fibroin protein isolated from the posterior silkgland of the tropical Saturniid silkworm Antheraea mylitta, was solubilized in lithium dodecyl sulfate and purified by gel filtration. The major fraction from gel filtration was analyzed by SDS-PAGE under non-reducing and reducing conditions. One major protein band of ca 395 kDa was obtained under non-reducing conditions and a doublet band of approximately 197 kDa under reducing conditions. The appearance of a single spot in two-dimensional electrophoresis confirmed the purity of the protein indicating that it may be a homodimeric protein of two similar sized polypeptides. Amino acid composition analysis showed that, like other Saturniid fibroins, it is rich in glycine, alanine and serine amino acids. N-terminal amino acid sequence shows significant homology with other Antheraea species. The enzymatic deglycosylation analysis indicates that the fibroin protein is glycosylated and the oligosaccharides are O-linked to the protein backbone by N-acetylgalactoseamine moiety which conforms to a Core 1 mucin-type glycosylation pattern.     

Biochemical characterization of sericin isolated from cocoons of tropical tasar silkworm Antheraea mylitta raised on three different host plants for its prospective utilization

Sericin were isolated and characterized from tasar cocoons raised in three different food plants i.e. Terminalia arjuna, T. tomentosa and Shorea robusta for its applications. Their molecular composition, structure and physical nature were determined by elemental analysis, amino acid analysis, Fourier Transform Infrared Spectroscopy (FTIR) and Differential Scanning Calorimetry (DSC) analysis. These results show that tasar food plants influence the physical and chemical properties of sericin. Further, sericin isolated from cocoons of S. robusta food plant shows better antioxidant potential and inhibition of tyrosinase, elastase and glutathione-S-transferase activity than other food plants. This may be attributed to its amino acid variations and associated phenolic content. The present study appears to be useful in utilizing tasar sericin as a potential bio-molecule for its prospective utilization in pharmaceuticals and its associated fields.     

Development of random amplified polymorphic DNA markers for tropical tasar silkworm Antheraea mylitta

Antheraea mylitta (Drury) is a tropical tasar-silk producing insect. Its populations occupying different ecological and geographical regions show a certain degree of phenotypic variability, for which they are known as "eco-races." The eco-races are exploited for tasar silk production, and they are classified on the basis of their geographical distribution and morphology, which is often misleading when their systematic position is considered. To understand the genetic variability among the different eco-races, we used the random amplified polymorphic DNA (RAPD) method. Eighty random decamer primers were taken for RAPD amplifications. In total, 415 reproducible bands were used to generate a distance matrix, and for the subsequent clustering with unweighted pair-group method with arithmetic average. The number of polymorphic bands detected by each primer ranged from 5 to 24, with a mean value of 14.1 per primer. Percentage polymorphism was 81.9, and genetic distance values ranged from a minimum of 0.0108 between Modal and Nalia eco-races to a maximum of 0.0244 between Modal and Andhra local. The RAPD profiles obtained using A14, BC07, and C17 primers substantially differentiate all 10 commercially important eco-races, and the phylogenetic tree obtained from the data closely follows their geographical separations.     

Protein purification, cDNA cloning and characterization of a protease inhibitor from the Indian tasar silkworm, Antheraea mylitta

An inhibitor of Aspergillus oryzae fungal protease was purified to homogeneity from the hemolymph of fifth instar larvae of Antheraea mylitta by ammonium sulfate precipitation, anion exchange and gel filtration (FPLC) chromatography, and termed as AmFPI-1. The extent of purification was checked by two-dimensional gel electrophoresis, and the molecular weight of purified inhibitor was determined by SDS-PAGE as 10.4 kDa. Fifteen N-terminal amino acid sequences of this protein were determined, and degenerate oligonucleotides were synthesized on the basis of these sequences. A cDNA library of A. mylitta integument was constructed, and protease inhibitor cDNA was partially amplified by PCR using degenerate oligonucleotides and CDS primers. A full-length inhibitor cDNA clone obtained by screening the library with PCR amplified DNA as probe was sequenced. The cDNA consists of 543 nucleotides with an ORF of 315 bp and encodes a protein of 105 amino acids. The sequence exhibits similarity to several Bombyx mori ESTs, and in particular to N-terminal amino acid sequence of an inducible serine protease inhibitor (ISPI-1) from Galleria mellonella indicating its relatedness to ISPI-1 of G. mellonella. The presence of this protease inhibitor in the hemolymph may play an important role as a natural defense system against invading microorganisms.     

Molecular identification of tropical tasar silkworm (Antheraea mylitta) ecoraces with RAPD and SCAR markers

The tropical tasar silkworm, Antheraea mylitta, has several ecoraces, 10 of which are commercially exploited for the production of tasar silk. These ecoraces are identified by morphological markers that are greatly influenced by photoperiod, humidity, altitude, and host plants. The DNA markers, random amplification of polymorphic DNA (RAPD), and sequence-characterized amplified region (SCAR) are identified to complement the existing morphological markers. Seven RAPD bands are selected that identify 8 of the 10 ecoraces. These identified RAPD fragments are sequenced and primers are designed for SCAR markers. Of the seven sets of primers, a single primer pair produced polymorphic SCAR bands that diagnose 5 of the 10 ecoraces. All 10 ecoraces are identified by the use of RAPD and SCAR markers together.     

Biological relevance of host plant-derived terpenoid in the cocoons of the tropical tasar silkworm Antheraea mylitta

We have characterized and studied the biological functions of a terpenoid derivative in the Indian tropical tasar silkworm, Antheraea mylitta reared on the primary host plant Arjun, Terminalia arjuna. The compound from insect cocoon turned out to be a terpenoid derivative which resembled oleanane type triterpene (Arjunolic acid) present in the host plant. The plant and cocoon compounds were anti-oxidative as determined by bleaching of beta carotene in vitro. UV-exposure is the major form of peroxidative insult encountered by this wild tropical silkworm. The life cycle comprising five larval stages and the cocoon stage lasts for about 30–45 days. Hence the sequestration of antioxidant and UV-protectant molecule from the host plant commands great biological significance.     

Effect of aminotriazole on antioxidant defense system of tasar silkworm Antheraea mylitta

This study was designed to find out the metabolic consequences of H₂O₂ following catalase inhibition by aminotriazole in the fat body of an Antheraea mylitta pupa. H₂O₂ content in the pupal fat body exhibited a decreasing trend over the experimental period (up to 48 h). However, a substantial decrease in its level was marked after 12, 24 and 48 h of treatment. The level of lipid peroxidation was elevated within 4 h of aminotriazole injection. Nevertheless, its level significantly decreased after 12, 24 and 48 h of treatment. Superoxide dismutase activity was elevated within 4 h, followed by a transient decrease in its activity at 12 h of treatment and again increased over the experimental period. Catalase activity was found to decline in the fat body within 4 h of aminotriazole treatment compared to the control. However, it was surprising to observe that there was a two‐fold increase in catalase activity compared to its previous experimental group after 12 h, followed by a rapid decline in its activity at 24 h of aminotriazole injection and non‐detectable catalase activity at 48 h. Ascorbic acid content was found to be elevated after 12 h of injection and maintained an increasing trend over the rest of the experimental period compared to the respective control. Despite the progressive inhibition of catalase activity beyond 12 h of treatment, H₂O₂ accumulation was not observed as a consequence of catalase inhibition. Hence, catalase depletion by aminotriazole involves compensatory changes in other components of the antioxidant system for the efficient removal of H₂O₂.     

Attacin gene sequence variations in different ecoraces of tasar silkworm Antheraea mylitta

Attacin gene exists as paralogous conversion and is being used for identification of strain variations in insects based on thesequence variation. Hence, a study was undertaken to analyze the sequence variation of the attacin gene isoforms in the tasarsilkworm Anthereae mylitta that exists in the form of different ecoraces depending upon the environment, food plant and location.Comparison of the previously reported attacin sequences with the DNA sequences of attacin A and B genes revealed six aminoacid substitutions among the sequences of the ecoraces which however did not affect the functional domain of Attacin. Thegenerated dendrogram clearly indicated unique branches for each ecorace with two separate gene clusters for attacin A and B. TheSarihan ecorace formed a separate sub-group under both the gene clusters. The present study also revealed the presence ofAttacin_N Superfamily domain exclusively in Exon I separated from the Attacin_C Superfamily domain that was present in Exon IIand part of Exon III, a prominent character of attacin gene. The phylogenetic reconstruction analysis of attacin gene in A.mylittasupported the common evolutionary origin of attacin genes belonging to the Lepidoteran and Dipteran families that formed twoseparate clusters.     

Identification of RAPD and SCAR markers associated with yield traits in the Indian tropical tasar silkworm Antheraea mylitta drury

The tropical tasar silkworm, Antheraea mylitta, is a semi-domesticated vanya silk-producing insect of high economic importance. To date, no molecular marker associated with cocoon and shell weights has been identified in this species. In this report, we identified a randomly amplified polymorphic DNA (RAPD) marker and examined its inheritance, and also developed a stable diagnostic sequence-characterized amplified region (SCAR) marker. Silkworms were divided into groups with high (HCSW) and low (LCSW) cocoon and shell weights, and the F₂ progeny of a cross between these two groups were obtained. DNA from these silkworms was screened by PCR using 34 random primers and the resulting RAPD fragments were used for cluster analysis and discriminant function analysis (DFA). The clustering pattern in a UPGMA-based dendogram and DFA clearly distinguished the HCSW and LCSW groups. Multiple regression analysis identified five markers associated with cocoon and shell weights. The marker OPW16_{905 bp} showed the most significant association with cocoon and shell weights, and its inheritance was confirmed in F₂ progeny. Cloning and sequencing of this 905 bp fragment showed 88% identity between its 134 nucleotides and the Bmc-1/Yamato-like retroposon of A. mylitta. This marker was further converted into a diagnostic SCAR marker (SCOPW 16_{826 bp}). The SCAR marker developed here may be useful in identifying the right parental stock of tasar silk-worms for high cocoon and shell weights in breeding programs designed to enhance the productivity of tasar silk.     

Structure of the induced antibacterial protein from tasar silkworm, Antheraea mylitta. Implications to molecular evolution

The crystal structure of an antibacterial protein of immune origin (TSWAB), purified from tasar silkworm (Antheraea mylitta) larvae after induction by Escherichia coli infection, has been determined. This is the first insect lysozyme structure and represents induced lysozymes of innate immunity. The core structure of TSWAB is similar to c-type lysozymes and alpha-lactalbumins. However, TSWAB shows significant differences with respect to the other two proteins in the exposed loop regions. The catalytic residues in TSWAB are conserved with respect to the chicken lysozyme, indicating a common mechanism of action. However, differences in the noncatalytic residues in the substrate binding groove imply subtle differences in the specificity and the level of activity. Thus, conformational differences between TSWAB and chicken lysozyme exist, whereas functional mechanisms appear to be similar. On the other hand, alpha-lactalbumins and c-type lysozymes exhibit drastically different functions with conserved molecular conformation. It is evident that a common molecular scaffold is exploited in the three enzymes for apparently different physiological roles. It can be inferred on the basis of the structure-function comparison of these three proteins having common phylogenetic origin that the conformational changes in a protein are minimal during rapid evolution as compared with those in the normal course of evolution.     

Occurrence of immunoreactive estradiol-positive material in the haemolymph and ovary of the tasar silkworm,Antheraea mylitta

Summary

Estradiol from the haemolymph and ovaries of tasar silkworm, Antheraea mylitta, was assayed using radio immunoassay. The estimated concentration of estradiol was 143 pg/mg in the ovaries and 102 pg/ml in the haemolymph.     

Silk sericin protein of tropical tasar silkworm inhibits UVB-induced apoptosis in human skin keratinocytes

The silk protein sericin has been identified as a potent antioxidant and photoprotective agent against ultraviolet B (UVB) irradiation in mouse skin model. In this study, we have investigated the anti-apoptotic effect of sericin in UVB (30 mJ/cm²)-irradiated human keratinocytes. Flow cytometry analysis has shown that pre-treatment with sericin inhibits UVB-induced apoptosis. The pre-treatment with sericin suppresses bax expression, up-regulates the expression of bcl-2, prevents both the activation of caspase-3 and cleavage of Poly (ADP-ribose) polymerase. Generation of intracellular hydrogen peroxide in UVB-treated keratinocytes is inhibited through pre-treatment with sericin suggesting that sericin probably prevents mitochondrial damage. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ultrastructure of posterior silk gland cells and liquid silk in Indian tasar silkworm,Antheraea mylitta drury (Lepidoptera : Saturniidae)

The ultrastructural characteristics of the posterior silk glands of the mature Antheraea mylitta (Lepidoptera : Saturniidae) larvae were clarified. Fibroin globules containing a small dense mass of fibroin fibers are produced in Golgi vacuoles, and released from the apical surface into the lumen by exocytosis. Bundles of microfilaments, which serve as a dynamic skeleton, are well developed. Numerous autophagosomes originating; from mitochondria accumulate in both basal and apical ends of the gland cell; the degenerated materials are released in the basement membrane and into the gland lumen, respectively. These materials invade the central fibroin column, leaving digested vacuoles in the fibroin cocoon filament. Ours may be the first finding of this rare phenomenon in which the degenerated lysosomes originated from mitochondria in both liquid silk in the lumen and cocoon filament.     

Parasitization of fifth instar tasar silkworm, Antheraea mylitta, by the uzi fly, Blepharipa zebina; a host-parasitoid interaction and its effect on host's nutritional parameters and parasitoid development

The uzi fly, Blepharipa zebina, is a well-known larval endoparasitoid of the tropical tasar silkworm, Antheraea mylitta. The present study dealt with the effect of the number of maggots developing per host on host nutritional parameters, parasitoid development and reproduction. Nutritional indices for ingestion, digestion, approximate digestibility, relative consumption rate, relative growth rate, and gain in body weight declined significantly with the increase in parasitoid burden, but the efficiency of conversion of digested food recorded a significant increase. The efficiency of conversion of ingested food remained little affected. The developmental period was significantly extended in larvae parasitized with 5 and 10 maggots per larva (mpl). Cocoon shell weight decreased by 27-63.5% in parasitized groups (1, 2, and 5 mpl) while larvae parasitized with 10 mpl could not spin cocoons. The maggot development period, recovery percentage, and fecundity of the uzi fly declined significantly with the increase in number of maggots developing per host.     

Purification, characterization, and localization of 70 kDa calcium-sensitive protein (calelectrin) from mammary glands

Mammary glands contain a group of calcium-sensitive proteins that bind to membranes in a calcium-dependent manner. Using the calcium-dependent binding to hydrophobic surfaces in combination with conventional techniques, we have purified the 70 kDa mammary calcium-binding protein (70 kDa M-CBP) to homogeneity. Antisera prepared to the 70 kDa M-CBP or to bovine liver 67 kDa calelectrin reacted in immunoblot analysis with the 70 kDa M-CBP antigen and with several additional mammary CBP species in crude tissue homogenates. Limited proteolysis of the 70 kDa M-CBP produced smaller immunoreactive species; extensive proteolysis resulted in more complete degradation of the protein. Identical data were obtained with digestion of 67 kDa calelectrin. The pl for the 70 kDa M-CBP was determined to be approximately 5.8; the same value reported for 67 kDa calelectrin. Phosphorylation of 70 kDa M-CBP was not detected in epithelial cell culture metabolic labeling. Immunohistochemical localization showed the protein to be located in ductal epithelia of virgin mouse mammary glands with a pattern of increased staining of the basal portions of the cells. Some stromal cells were also reactive. Apparently, the 70 kDa M-CBP and 67 kDa calelectrin are the same protein. Furthermore, like the 32.5 calelectrin (endonexin) and calpactin I/p36/lipocortin II, the 70 kDa protein appears to be a ductal epithelial cell associated protein in the mammary gland.     

Purification and characterization of a cysteine proteinase from silkworm eggs

Eggs of the silkworm, Bombyx mori, contain a high level of a proteinase which is most active in acidic pH region. The proteinase was purified from an extract of eggs by a six-step procedure which included conventional chromatographic fractionations. The molecular mass of the proteinase was estimated to be 350 kDa by gel filtration and 47 kDa by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels, suggesting an octameric structure. The amino acid composition was found to resemble that of mammalian lysosomal cysteine proteinases, in particular cathepsin L. The NH2-terminal 10-residue sequence is Val-Gln-Phe-Phe-Asp-Leu-Val-Lys-Glu-Glu-. The enzyme appears to be a member of the class of cysteine proteinases since it was strongly inhibited by sulfhydryl-reactive compounds and N-[N-(1,3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). The enzyme hydrolyzed various protein substrates, such as hemoglobin, vitellogenin, vitellin, and lipophorin, with maximal activity around pH 3-3.5. The specificity of the cleavage sites in the oxidized B chain of insulin was rather well defined and there was high affinity for hydrophobic residues at the P2 and P3 positions. The cysteine proteinase is thought to be involved in protein degradation during embryonic development of silkworm eggs.     

Genetic variation in ecoraces of tropical tasar silkworm, <Emphasis Type="Italic">Antheraea mylitta</Emphasis> using SSR markers

The tropical tasar silkworm, Antheraea mylitta, polyphagous sericigenous insect mostly found in the tropical areas of India. It is found in these regions as ecotypes or ecoraces. It feeds primarily on plants, a variety of secondary plants like Terminalia arjuna and T. tomentosa. Tasar culture is a traditional livelihood for lakhs of tribal populace in the areas of Jharkhand, Chhatisgarh, Orissa, Maharashtra, Andhra Pradesh, West Bengal and Uttar Pradesh. In the present study, the genetic diversity of these ecoraces is identified by DNA markers, namely simple sequence repeats (SSRs), most of which produced polymorphic bands.     

Purification and characterization of a 54 kDa chitinase from Bombyx mori

The 54 kDa protein that was suggested to be processed from the 65 kDa and 88 kDa chitinases of Bombyx mori [Koga et al., Insect Biochem. Mol. Biol. 27, 757–767 (1997)] was purified and proved to be a third chitinase (EC 3.2.1.14). This chitinase was purified from the fifth larval instar of B. mori by chromatography on DEAE-Cellulofine A–500, hydroxylapatite, Butyl-Toyopearl 650M, and Fractogel EMD DEAE 650(M) columns. The apparent molecular mass was confirmed to be 54 kDa by SDS–PAGE. Its optimum pH was 6.0 toward a short substrate, N-acetylchitopentaose (GlcNAc₅), while in its reaction with a longer substrate, glycolchitin, the enzyme showed a wide pH-range between 4.0 and 10. Kinetic parameters for the chitinase could be obtained in the hydrolysis of glycolchitin but not in that of N-acetylchitooligosaccharides (GlcNAc_n, n=2–6) because of substrate inhibition. The chitinase hydrolyzed N-acetylchitooligosaccharides except for dimer as follows: trimer to monomer plus dimer, tetramer to two molecules of dimer, pentamer to dimer plus trimer, and hexamer to dimer plus tetramer as well as two molecules of trimer. These results suggest that the 54 kDa chitinase is an endo-type hydrolase and preferred the longer-chain N-acetylchitooligosaccharides. Moreover, the anomeric forms of N-acetylchitooligosaccharides were analyzed in the reaction with the 54-kDa chitinase. It was revealed that this enzyme cleaves the substrate to produce the β anomeric product. With respect to inhibition of the 54 kDa chitinase, it was specifically inhibited by allosamidin in a competitive way with K_i values depending on the pH of the reaction mixture (K_i=0.013−0.746 μM). Comparing the properties and kinetic behavior of this chitinase with those of the 88 and 65 kDa chitinases from B. mori, regarding the specific activity of the three enzymes, the 65-kDa chitinase was 2.15 and 2.8 times more active than the 88 and 54-kDa chitinases, respectively. However, in the overall reaction of glycolchitin (k_cat/K_m), the 88-kDa enzyme was 4 and 40 times more active than the 65-kDa and the 54-kDa enzymes, respectively. Concerning the affinity (1/K_m) to glycolchitin, the 88 kDa chitinase affinity (at pH 6.5) was 5.8 times higher than that of the 65 kDa chitinase (at pH 5.5) and 4.0 times higher than that of the 54 kDa chitinase (at pH 6.0). These kinetic results suggest that B. mori chitinases are processed during ecdysis from the larger chitinase to smaller ones that leads to changes in their kinetic properties such as K_m, k_cat and k_cat/K_m successively.