Abstract
In holometabolous insects, the accumulation and utilization of storage proteins (SPs), including arylphorins and methionine-rich proteins, are critical for the insect metamorphosis. SPs function as amino acids reserves, which are synthesized in fat body, secreted into the larval hemolymph and taken up by fat body shortly before pupation. However, the detailed molecular mechanisms of digestion and utilization of SPs during development are largely unknown. Here, we report the crystal structure of Bombyx mori arylphorins at 2.8 Å, which displays a heterohexameric structural arrangement formed by trimerization of dimers comprising two structural similar arylphorins. Our limited proteolysis assay and microarray data strongly suggest that papain-like proteases are the major players for B. mori arylphorins digestion in vitro and in vivo. Consistent with the biochemical data, dozens of papain cleavage sites are mapped on the surface of the heterohexameric structure of B. mori arylphorins. Hence, our results provide the insightful information to understand the metamorphosis of holometabolous insects at molecular level.
Keywords: Bombyx mori, crystal structure, arylphorin, heterohexamer
Introduction
The accumulation and utilization of storage proteins (SPs) are critical for the insect metamorphosis. SPs function as an amino acids reserve for the production of adult proteins.1,2 Insect SPs are generally categorized into two groups: methionine-rich proteins and aromatic-rich proteins (arylphorins). In Bombyx mori, storage protein 1 (SP1) is named for methionine-rich protein, and storage protein 2 (SP2) is named for aromatic-rich protein (arylphorin).3,4
Insect SPs belong to hexamerin superfamily, which comprise many structural and sequence similar proteins, such as arthropod hemocyanins and arthropod phenoloxidases. Although these proteins have similar amino acid sequences and structures, they display different functions. For example, Panulirus hemocyanins contain the copper binding site, which are proposed to have oxygen delivery ability; whereas Maduca phenoloxidases are copper containing tyrosinases, which play a key role in humoral immune defense. B. mori SPs belong to hexamerin family, containing neither copper nor oxygen. These proteins consist of six identical or similar monomers with a total molecular weight around 500 kDa,4,5 which are synthesized in fat body of larvae and secreted into the hemolymph. When the insect begins wandering, SPs are gradually taken up into pupal fat body and proteolytically broken down during the development of the pharate adult.
Crystal structure of arylphorin from Chinese oak silkworm (Antheraea pernyi) reveals that arylphorin forms a stable homohexamer. Two glycosylation sites were identified from each monomer with one buried inside the hexamer and the other exposed to the surface. These two oligosaccharide chains are proposed to play important roles for folding and stabilization of arylphorin.6 Although B. mori arylphorin (SP2) shares 67% sequence similarity with A. pernyi arylphorin, B. mori SP2 contains only one glycosylation site (N211, corresponding to A. pernyi arylphorin residue N196) with the chemical formula of Glc1Man9GlcNAc2. The glycosylation site and the chemical formula of the oligosaccharide chain in B. mori SP2 are identical to the oligosaccharide chain buried inside the hexamer in A. pernyi arylphorin.7
Notably, our proteomics efforts on SPs involved in silkworm development have identified a new B. mori SP, which shares high sequence similarity (65%) with SP2 (named as SP3 thereafter).8 Remarkably, this newly discovered SP shares similar molecular weight, pI value and expression pattern with those of SP2. Similar to SP2, SP3 also contains one oligosaccharide chain attached to residue N208, corresponding to N196 in A. pernyi arylphorin. The highly correlated expression pattern of B. mori SP2 and SP3 suggests that these two proteins could form a stable complex in vivo, whose degradation provides the amino acids resource for adult development.1
Although arylphorins and other SPs are discovered in many insects, such as bees, ants, beetles, and cockroaches,9–12 the detailed molecular mechanisms of digestion and utilization of SPs during development are largely unknown. To investigate the structural features of SP2/SP3 complex and gain the structure insights into B. mori SP degradation in vivo, we determined the crystal structure of B. mori heterohexamer at 2.8 Å resolution. Our limited proteolytic assay demonstrate that B. mori SP2/SP3 are the substrates for the papain-like cysteine proteases in vitro, which is further validated by the expression pattern of papain-like proteases using microarray assay. In summary, our data demonstrate that B. mori arylphorin is a stable heterohexamer with multiple papain cleavage sites, whose cleavage in vivo is highly correlated with pupal to adult pharate development.
Results
Identification and purification of Bombyx mori SP2/SP3 complex
Our full-genome sequence data of B. mori revealed two arylphorin proteins (SP2 and SP3) with high sequence similarity (Fig. 1). Follow-up proteomics analysis on B. mori SPs demonstrated that these two SPs displayed the similar pI value and co-migrated at 2D gel.8 Hence, we speculated that these two SPs (SP2 and SP3) may form stable complex. To investigate this possibility, we separated the fat body from newly pupated female silkworm and purified the SPs by the combination of ammonium sulfate precipitation, heat treatment, and ion exchange chromatography purification approaches. Most of the methionine-rich SP1 were precipitated by 20–35% saturated ammonium sulfate, and arylphorin proteins (SP2 and SP3) were precipitated by 45–65% saturated ammonium sulfate. As we expected, B. mori arylphorin proteins were purified as a stable complex by ammonium sulfate precipitation and chromatographic purification. Follow-up matrix-assisted laser desorption/ ionization time of flight mass spectrometry (AB SCIEX,4700 MALDI TOF/TOF, USA) analysis on the purified arylphorin proteins showed that B. mori arylphorin proteins indeed are composed by SP2 and SP3 proteins unambiguously (Supporting Information Fig. S1). Interestingly, a band with apparent molecular weight of ∼150 kDa was observed at sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel even at the denatured conditions (data not shown). Such observation suggests that SP2 and SP3 form a stable complex in vitro.
Figure 1.
Sequence alignment of five lepidopteran insect arylphorins. The invariable residues are shaded in black, whereas the highly conserved residues are shaded in gray. Secondary structure elements are drawn on the basis of structures of Bm_SP2 and shown at the top of the aligned sequences. α-Helices are shown as bars in red, whereas β-sheets are shown as arrows in blue. Bm, An, Ma, Hy, and Sp represent Bombyx mori, Antheraea pernyi, Manduca sexta, and Spodoptera litura. The accession numbers in Genebank for the sequences are as following: Bombyx mori SP2 (NP_001037590.1), Bombyx mori SP3 (AFD02109.1), Antheraea pernyi (ABQ96634.1), Manduca sexta (AAA29305.1), Hyalophora cecropia (AAB86644.1), and Spodoptera litura (CAB55605.1).
Overall structure of Bombyx mori SP2/SP3 heterohexamer
To investigate the structural arrangement of SP2/SP3, we successfully determined the crystal structure of B. mori SP2/SP3 complex at 2.8 Å resolution at space group P21. We are able to trace residues 24–693 and 5 oligosaccharides for SP2 and residues 21–688 and up to 8 oligosaccharides for SP3 without ambiguity [Fig. 2(C,D) and Supporting Information Fig. S2].
Figure 2.
Overall structure of Bombyx mori arylphorins. A: Cartoon view of Bombyx mori SP2. Bombyx mori SP2 contains three domains, which are colored in yellow, magenta, and green, respectively. The appendix structural module containing the oligosaccharide chain is colored in red and the bound oligosaccharide chain is shown in stick mode and colored in cyan. B: Cartoon view of Bombyx mori SP3 with the same presentation view and color code as (A). C: The composite omit map electron densities of Bombyx mori SP2 at 1σ are shown in pink. The bound oligosaccharide chain and surrounding residues are shown in stick and colored based on element types. D: The composite omit map electron densities of Bombyx mori SP3 at 1σ are shown in pink.
Similar to A. pernyi arylphorin, B. mori arylphorin contains three domains [Fig. 2(A,B)]. Domain 1 (aa. 24–213 and aa. 21–210 for B. mori SP2 and SP3, respectively) contains an imperfect helical bundle (aa. 24–170 and aa. 21–167 for B. mori SP2 and SP3, respectively) and an appendix (aa. 171–213 and aa. 168–210 for B. mori SP2 and SP3, respectively), connected by a flexible random coil. The appendix structure contains an oligosaccharide attached site (N211 for SP2 and N208 for SP3), to which the characteristic Glc1Man9GlcNAc2 chain is covalently bound (Fig. 2). Domain 2 (aa. 214–446 and aa. 211–443 for B. mori SP2 and SP3, respectively) of B. mori SP2 is an α-helices dominant domain, comprising largely anti-parallel helical bundle [Fig. 2(A,B)]. Domain 2 locates to the center of the hexamer, making numerous contacts not only with the other two monomers within the same trimer but also with the two monomers at the other trimer. Domain 3 (aa. 447–693 and aa. 444–692 for B. mori SP2 and SP3, respectively) of B. mori SP2 contains a seven-stranded β-barrel packed against domain 2, two extended long loops embracing a large part of domain 1 and a unique long a-helix packed against the helical bundle of domain 1 [Fig. 2(A,B)].
There are three SP2 molecules and three SP3 molecules per asymmetric unit assembled slightly different from 32 point group symmetry, which is best described as a trimer of “tight SP2/SP3 dimers” (Fig. 3). There are two trimers stacked each other with one trimer consisting of one SP2 molecule and two SP3 molecules, whereas the other timer consisting of two SP2 molecules and one SP3 molecule [Figs. 3(A,B) and 4(A)]. The contacts between the SP2/SP3 dimer are numerous, which suggests that SP2/SP3 dimer could serve as the nuclei to form the hexamer [Fig. 3(C,D)]. Interestingly, the contact area nearest to the center of the hexamer is most extensive and consists mainly of residues that are quite conserved among arylphorin proteins. The six molecules have essentially maintained the same structure with r.m.s.d. ranging from ∼0.3 Å (within SP2 and SP3) to ∼1.1 Å (between SP2 and SP3) [Fig. 3(A,B)]. The buried-surface area between SP2/SP3 dimer is ∼7,538 Å2 as calculated using PDBe PISA (http://www.ebi.ac.uk/msd-srv/prot_int/), which strongly suggests that SP2/SP3 dimer could be the physiological building unit for B. mori arylphorin formation [Fig. 4(F)]. We speculate that buried-surface areas between SP2/SP2 homodimer and SP3/SP3 homodimer should be smaller than that of SP2/SP3 heterodimer, although we have no direct evidence to prove it. Interestingly, analytic gel filtration analysis showed that B. mori arylphorin forms primarily a mixture of hexamer and dimer in solution, which suggests that B. mori arylphorin could be formed gradually from dimer to hexamer (Supporting Information Fig. S3).
Figure 3.
Structural arrangement of Bombyx mori SP2/SP3 heterohexamer. A: Cartoon view of Bombyx mori SP2/SP3 heterohexamer. The SP2 molecules are colored in magenta, hotpink, and violet, whereas SP3 molecules are colored in yellow, green, and lime, respectively. The bound oligosaccharide chains are shown in stick mode and colored in cyan. There are two SP/SP heterotrimers stacked each other, with one comprising 2 SP2 and 1 SP3 molecules and the other comprising 1 SP2 and 2 SP3 molecules. B: Surface view of Bombyx mori SP2/SP3 heterohexamer with the same presentation view and color code as (A). C: Cartoon view of Bombyx mori SP2/SP3 heterohexamer with a 90° rotation of (A) along X axis. D: Surface view of Bombyx mori SP2/SP3 heterohexamer with a 90° rotation of (B) along X axis.
Figure 4.
Structural arrangement of Bombyx mori SP2/SP3 complex. A: Cartoon view of Bombyx mori SP2/SP3 heterotrimer. The SP2 molecule is colored in magenta, whereas SP3 molecules are colored in yellow and green, respectively. The bound oligosaccharide chains are shown in stick mode and colored in cyan. Less intra-molecular contacts are observed. B: Cartoon view of Bombyx mori SP2/SP3 heterohexamer with a 90° rotation of (A) along X axis. The same color code as (A) for one Bombyx mori SP2/SP3 heterotrimer. The second Bombyx mori SP2/SP3 heterotrimer within the hexamer is colored in silver. C: Surface view of Bombyx mori SP2/SP3 heterohexamer with the same presentation view and color code as (B). D: Cartoon view of Bombyx mori SP2/SP3 dimer. The SP2 molecule is colored in magenta, whereas SP3 molecule is colored in green. The bound oligosaccharide chains are shown in stick mode and colored in cyan. Numerous intra-molecular contacts are observed. E: Cartoon view of Bombyx mori SP2/SP3 heterohexamer with the same presentation view of (D). The second Bombyx mori SP2/SP3 heterotrimer within the hexamer is colored in silver. F: Surface view of Bombyx mori SP2/SP3 heterohexamer with the same presentation view and color code as (B).
Similar to other insect arylphorins, B. mori arylphorins are aromatic residues abundant proteins. B. mori SP2 protein contains 7 Trp (1%), 54 Tyr (7.7%), and 65 Phe (9.2%), whereas B. mori SP3 protein contains 6 Trp (0.9%), 66 Tyr (9.5%), and 69 Phe (9.9%). Significantly, all these aromatic residues are evenly distributed within the B. mori SP2/SP3 complex structure spatially, which may provide an opportunity for B. mori pupa to generate small peptide fragments with similar content of aromatic residues as nutrition by protease treatment (Supporting Information Fig. S4).
Structural details of inter-subunit contacts
Structural determination of B. mori arylphorin revealed a heterohexamer structure formed by trimerization of “tight SP2/SP3 heterodimer.” There are two groups of interacting subunit contacts involving tight dimer and loose trimer formation, respectively (Fig. 4). The number of inter-subunit contacts within the tight dimer is much more constant [Fig. 4(A)]. There are 17 hydrogen bonding and 197 nonbonded contacts observed. By contrast, the contacts between the molecules of the trimer are relatively weak. There are only 13 hydrogen bonding, 7 salt bridges, and 101 nonbonded contacts observed. Notably, domain 2 contributes many inter-subunit contacts in stabilizing the hexamer.
Structure and function of oligosaccharide attached to Asn residue
Notably, most sugar moieties of this oligosaccharide chain are buried inside the cleft formed between SP2-SP3 interface and could be responsible for inter- and intra-molecular hydrogen bonds and hydrophobic interactions. Therefore, we speculate that the Glc1Man9GlcNAc2 oligosaccharide chain attached to the invariable Asn residue (N211 for SP2 and N208 for SP3) should play significant role in hexamer formation by stabilizing the tight SP2/SP3 dimer.
However, in our B. mori arylphorin structure determined at 2.8 Å, although the Asn-glycan is located in the deep cleft of SP2/SP3 interface, there are no apparent strong interactions between the sugar moieties of the attached oligosaccharide chain of SP2 with its dimeric SP3 partner, vice versa, as revealed from the well-defined electron-density map. This observation suggested that the oligosaccharide chain might not be involved in protein stabilization. On the other hand, interface analysis between the molecules of B. mori arylphorin hexamer revealed that the interface area between SP2/SP3 dimer interface (∼7500 Å2) is much larger than that between nondimer (∼4600 Å2). Therefore, the dimeric pair of SP2/SP3 should be the key interaction responsible for the formation of the hexameric arylphorin.
Identification of papain cleavage sites in Bombyx mori SP2/SP3 complex
Like other insect SPs, B. mori arylphorin is up-taken by fat body during a brief time period shortly before and after pupation and digested gradually as an amino acids pool for pharate development.2 Notably, there is no particular protease reported to play the critical role for B. mori arylphorin degradation, although the activities of B. mori cysteine protease inhibitors are dramatically increased at the time of spinning and maintain this level of activity during pupation.13
To investigate whether B. mori arylphorin indeed is the substrate of cysteine protease, we performed limited proteolytic assay by incubation of purified B. mori SP2/SP3 proteins with different proteases at different time scale, respectively. Surprisingly, B. mori SP2/SP3 proteins showed strong resistance to most of the proteases, including trypsin, subtilisim, elastase, and α-chymotrypsin after incubation at 37°C for 12 h [Fig. 5(A)]. In contrast, B. mori SP2/SP3 proteins are extremely sensitive to papain, which is able to completely digest B. mori SP2/SP3 proteins within 1 min [Fig. 5(A)]. Consistent to this notion, sequence analysis of B. mori SP2 and SP3 proteins reveals numerous papain cleavage sites. Remarkably, most of these papain cleavage sites are readily mapped to the solvent accessible surface of B. mori SP2/SP3 heterohexamer [Fig. 5(B,C)].
Figure 5.
Bombyx mori SP2/SP3 complex is the substrate for papain-like protease. A: Limited proteolytic assay on purified Bombyx mori SP2/SP3 complex (left) and SP1 (right) by incubation with Papain, Trypsin, Subtlisin, Elastase, and α-chymotrypsin at different time scale. B: Surface view of Bombyx mori SP2/SP3 heterohexamer with Papain cleavage sites mapping at the surface. The SP2 molecules are colored in cyan, whereas the SP3 molecules are colored in yellow. The bound oligosaccharide chain is shown in stick mode and colored in green. C: Surface view of Bombyx mori SP2/SP3 heterohexamer with a 90° rotation of (B) along Y axis. The same color code as that in (B). D: Expression profile of papain-like proteases in Bombyx mori during different developmental stages. The columns represents 20 different sample time points: day 3, 4, 5, 6, 7 of the fifth instar, start of wandering, 14 different times after wandering: 12 h, 24 h, 36 h, 48 h, 60 h, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, and adult. “M” represents the sample collected from male silkworm, whereas “F” represents the sample collected from female silkworm. E: Expression profile of Bombyx mori arylphorins at different pupa developmental stages. Arylphorin proteins extracted from pupa fat body (20 μg) at each stage were separated by SDS-PAGE and transferred to membrane, followed by hybridized using SP2/SP3 polyclonal antibodies. 1–9 represent the samples extracted from 1st to 9th day of pupa, respectively. F: Protease activity of Bombyx mori fat at different pupa developmental stages. Protein samples extracted from the fat body of pupa were used to measure cathespin B and Cathespin L proteolytic activity against Z-Phe-Arg-MCA. One unit of enzyme activity represents the fluorescence released from 5 nmol substrate over per 10 min.
Temporal expression profiles of Bombyx mori papain genes
Previous biochemical analysis demonstrated that one of the cysteine proteases encoded by B. mori genome shares functional similarity to cathepsin L and papain and is different from those of cathepsin D, pepsin, trypsin, and chymotrypsin.14–16 Sequence blast of this cysteine protease and other insect papain genes against silkworm genome revealed 9 papain-like genes encoded by B. mori.
To further investigate whether papain-like cysteine proteases play an important role for digestion of B. mori arylphorin in vivo, we systematically examined the gene expression level of papain-like proteases in B. mori from day 3 of 5th instar to moth by microarray approach. Our data showed that most silkworm papain-like cysteine proteases genes are highly expressed in metamorphosis not only in wandering stage involving in molting and metamorphosis17,18 but also in later pupa stage and adult cuticle formation stage, which are highly correlated to the digestion time scale of B. mori arylphorin in vivo [Fig. 5(D)]. In addition, we examined the concentration of B. mori arylphorin from day 1 of pupa to moth by western blotting. Similar to the results revealed in the literature, the concentration of B. mori arylphorin decreases gradually [Fig. 5(E)].
To investigate whether B. mori fat body contains papain-like proteases for fat digestion, we measured the protease activity in pupa fat body at the same time points shown for the microarray results of mRNA levels. As expected, the measured protease activity is elevated significantly during pupa development [Fig. 5(F)]. Notably, the protease activities in female pupa fat are 2- and 3-fold higher than those in male pupa fat at day 8 and day 9, respectively.
The highly reverse correlation of concentration of B. mori arylphorin and expression of papain-like cysteine protease in vivo prompted us to speculate that B. mori arylphorin could be one of the physiological substrates digested by papain-like cysteine as amino acid resource for pharate development in vivo.
Discussion
Structural comparison of other insect storage proteins
Insect SPs are mainly divided into two categories in lepidoptera: arylphorin proteins containing more than 15% tyrosine and phenylalanine content and “methionine-rich SPs.” Notably, some insects, including Plutella xylostella and Manduca sexta, are reported to contain two arylphorins, although the structures of these arylphorins are not reported. Dissimilar to structural arrangement of A. pernyi arylphorin homohexamers, B. mori arylphorins comprise two similar arylphorins, which are confirmed by genomics, proteomics, and structural investigations. Hence, our heterohexameric arylphorin structure could serve as a starting template to study the arylphorin function in other insect systems.
Although the overall hexameric arrangement of B. mori arylphorins is similar to that of A. pernyi arylphorin, our crystal structure of B. mori arylphorins demonstrated that B. mori arylphorins form a heterohexamer, which is composed of a trimer of the tight SP2/SP3 heterodimer [Fig. 3(A,B)]. Such arrangement is closer to those of hexameric hemocyanins, some of which are composed by several different subunits5 [Fig. 6(A–D)].
Figure 6.
Structural comparison of Bombyx mori Arylphorins with other similar proteins. A–D: Overall structural comparison. The models of Bombyx mori SP3, A. pernyi arylphorins, and Panulirus hemocyanins were aligned to Bombyx mori SP2 and shown at the same direction. The protein structures are shown in cartoon view and colored in green. The bound oligosaccharide chains are shown in stick mode and colored in yellow. E–H: Structural details in cooper coordination site. Structural comparisons of the di-copper binding center in Panulirus hemocyanins (PDB code 1HC1), the corresponding site of Bombyx mori SP2, Bombyx mori SP3, and Antheraea pernyi arylphorins (PDB code 3GWJ).
However, hemocyanins contain di-copper centers, where two copper atoms are coordinated by six histidine residues involving in oxygen transport throughout arthropod bodies.19 By contrast, the corresponding residues of B. mori SP2 and SP3 are primarily consisted of Tyr residues, which are not capable of copper coordination [Fig. 6(E–H)].
Dissimilar to A. pernyi arylphorin, B. mori arylphorin contains only one oligosaccharide chain, which could be involved in overall protein stability [Fig. 6(A–D)], and lacks of the second oligosaccharide chain, which is proposed to be critical for the correct folding of A. pernyi arylphorin6 [Fig. 6(A–D)].
Strikingly, the trimeric arrangement of the tight SP2/SP3 dimer is novel and unexpected. In contrast to follow 3-fold axis by forming SP2 homotrimer as one layer and SP3 homotrimer as another layer, B. mori arylphorins form a SP2:SP3 (2:1) heterotrimer as one layer and SP2:SP3 (1:2) heterotrimer as another layer. Therefore, one SP2/SP3 dimer is surrounding by two SP2/SP3 dimers at both sides in a head-to-tail manner [Fig. 4(E,F)]. We speculate that in order to form the hexameric arrangement observed in structure, one SP2/SP3 dimer should serve the nucleus to recruit other two SP2/SP3 dimers, mediated by SP2-SP3 interactions.
Notably, the crystal structure of silkworm SP2/SP3 complex determined in 2.9 Å was reported recently.20 In this structure, there is only one SP2/SP3 dimer per asymmetric unit. The heterohexamer generated by symmetric operation was formed by one SP2 trimer and one SP3 trimer. Significantly, our SP2/SP3 complex was crystallized in space group P21 and determined at 2.8 Å resolution, whereas the published SP2/SP3 complex was crystallized in space group P6322 at 2.9 Å resolution. Therefore, the structural arrangement deviations observed from these two structures could be derived from crystal packing. Nevertheless, both structures demonstrated that the heterohexamer was generated by the “tight SP2/SP3 heterodimer.”
Molecular insights into Bombyx mori arylphorins as amino acid sources
Like other arylphorin, B. mori arylphorins are reabsorbed into the fat body shortly before pupation, and digested as energy and amino acid sources during insect maturation, when no feeding is available. Ironically, arylphorins are highly stable with long half-life during the insect lifespan, which are high resistance to enzymes and temperature variation. B. mori arylphorins are started being digested for utilization as energy and amino acid sources only after being redirected to the larval fat body shortly before pupation. Therefore, specific proteases are required for the digestion and utilization of arylphorins.
Our microarray data revealed that gene expression of papain-like proteins in B. mori increased dramatically shortly after pupation and returned to base line before ecdysis, which is highly reverse correlated with B. mori arylphorins concentration level [Fig. 5(D,E)]. In addition, our biochemical screening of proteases on B. mori arylphorins digestion in vitro further demonstrated for the first time that papain is the protease responsible for SP digestion [Fig. 5(A)]. Moreover, the measured protease activity in pupa fat is elevated significantly during pupa development [Fig. 5(F)]. Taken together, these data suggested that papain-like proteases are the major players for insect arylphorins digestion to supply amino acids and energy necessary for adult development. Consistent to this hypothesis, multiple solvent accessible papain cleavage sites are identified at the surface of B. mori arylphorins heterohexamer [Fig. 5(B,C)]. Notably, limited proteolytic analysis also showed that B. mori methionine-rich SP1 had the similar behavior as B. mori arylphorins: sensitive to papain and resistant to other proteases [Fig. 5(A)]. Taken together, our data provide the strong evidence demonstrating that B. mori arylphorins form a stable heterohexamer, which are digested only by papain-like protease in vivo, providing energy and amino acids source for adult development. Because accumulation and utilization of SPs are critical for the metamorphosis of holometabolous insects, our results should provide the insightful information to understand this important event at molecular level.
Materials and Methods
Separation and purification of Bombyx mori arylphorins
B. mori (strain p50, DaZao) were reared on artificial silkworm food at 25°C. B. mori SPs were separated and purified following the published method.1 The fat body from newly pupated female was collected and grinded in liquid nitrogen, suspended in buffer A (20 mM NaCl, 25 mM Tris-HCl, pH 7.5), followed by ultrasonic homogenization on ice. The mixture was centrifuged at 16,000g at 4°C for 20 min, and the supernatant collected was submitted for heat treatment at 76°C for 10 min, followed by centrifugation at 10,000g at 4°C for 20 min. Ammonium sulfate powder was added to supernatant collected to precipitate SPs. B. mori arylphorins were precipitated as a stable complex by ammonium sulfate at the concentration of 45–65% saturation and were further purified by Q column, followed by HiLoad Superdex S-200 26/60 column purification (GE health care). The purified protein was concentrated to 20–25 mg/mL in a Centriprep-30 (Amicon).
Crystallization, data collection, and structure determination
More than 500 unique conditions were screened for crystallization of B. mori arylphorins (SP2/SP3 complex). X-ray quality crystals were grown at 20°C by mixture of 1.0 μL of protein in buffer A (20 mM NaCl, 25 mM Tris-HCl, pH 7.5) with 1.0 μL of reservoir containing 9% PEG3350, 100 mM MgCl2, and 100 mM Acetate buffer (pH 5.0). These crystals grew to a maximum size of 0.3 mm × 0.1 mm × 0.1 mm over the course of 3 days.
Crystals were flash frozen (100 K) in the above reservoir solution supplemented with 30% ethylene glycol. A total of 180 frames with 0.5° oscillation were collected against a native B. mori SP2/SP3 crystal at wavelength 1.075 Å, and processed by HKL2000 (http://www.hkl-xray.com).21 The structure of B. mori SP2/SP3 complex was determined by molecular replacement software Molrep within CCP4 package (http://www.ccp4.ac.uk) using the crystal structure of A. pernyi arylphorin (PDB ID: 3GWJ) as the search model.6 The model was rebuilt by using the program O (http://xray.bmc.uu.se/alwyn)22 and refined by using REFMAC/CCP4 (http://www.ccp4.ac.uk).23 The crystallographic statistic details of these structures are listed in Table I. The structure was analyzed using the software PDBsum (https://www.ebi.ac.uk/pdbsum/) and PDBePISA (http://www.ebi.ac.uk/msd-srv/prot_int/pi_visual.html).24,25
Table I.
Data Collection and Refinement Statistics
| Data collection | |
|---|---|
| Space group | P2(1) |
| PDB ID | 3WJM |
| Cell dimensions | |
| a, b, c (Å) | 92.06, 205.02, 119.71 |
| Β (°) | 103.0 |
| Wavelength (Å) | 1.075 |
| Resolution (Å)a | 50–2.8 (2.85–2.8) |
| Rsym (%) | 7.7 (49.7) |
| I/σ(I) | 25.3 (4.4) |
| Completeness (%)a | 96.6 (89.8) |
| Redundancy | 3.5 (3.2) |
| Search model | 3GWJ |
| Refinement | |
| Resolution range (Å) | 50–2.8 (2.88–2.80) |
| No. reflections | 97,193 |
| Rwork (Rfree) (%) | 17.4/23.9 (22.2/32.1) |
| No. atoms | |
| Protein | 34,060 |
| Glycoside | 421 |
| Water | 120 |
| B-factors (Å2) | |
| Protein | 53.175 |
| Glycoside | 76.301 |
| Water | 37.677 |
| R.m.s. deviations | |
| Bond lengths (Å) | 0.012 |
| Bond angles (°) | 1.627 |
| % Favored (disallowed) in Ramachandran plot | 90.1 (0) |
Values for the highest resolution shell are in parentheses.
Whole-genome microarray on developmental expression pattern of Bombyx mori papain genes
Gene sequence of Carica papaya papain (sigma, Genebank accession number 1STF_E) was used as query to BLAST against the B. mori database (http://silkworm.genomics.org.cn).26 Identified genes were validated by blasting against the nonredundant gene dataset and EST database with threshold of E-value < 10−30, identities > 90%, and match lengths > 100 bp.27
Based on the nucleotide sequences, a genome-wide oligonucleotide microarray with more than 22,000 probes, including 9 papain-specific oligonucleotide probes, were constructed.28 To determine the developmental expression patterns, individuals were collected for both genders at 20 different time points, including day 3, 4, 5, 6, 7 of the fifth instar; start of wandering; 12th, 24th, 36th, 48th, 60th, 72nd, 96th, 120th hour after wandering; day 6, 7, 8, 9, 10 after wandering, and adult. Three individuals were collected and combined as one sample. Total RNAs were extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions.
The microarray hybridization and data normalization analysis were performed by CapitalBio Corp (http://www.capitalbio.com). Developmental expression pattern analyses were performed twice per sample by the dye reversal procedure. In the first experiment, the control sample (mixed male and female larvae from the third day of the fifth instar) was labeled with Cy3 and a male or female sample from each time point was labeled with Cy5. In the second experiment, the control sample was labeled with Cy5 and the male or female sample from each time point was labeled with Cy3. Gene expression levels were visualized using GeneCluster 2.0.28,29 The detailed experimental procedures for microarray and data analyses were described in previous report.
Limited proteolysis analysis
Purified SP2/SP3 complex at a concentration of 2.5 mg/mL was incubated with 10 μg/mL of Papain, Trypsin, Subtlisin, Elastase, and α-chymotrypsin at 37°C, respectively. Twenty microliters of each sample was aspirated out at times intervals of 1 min, 5 min, 30 min, 2 h, and 12 h and incubated with SDS-loading buffer at 100°C for 10 min. Subsequently, the treated samples were loaded onto a 12% SDS page gel and the gel was stained using coomasive blue.
Measurement of arylphorins concentration in Bombyx mori pupa fat at different pupa developmental stages
A quantitative fluorometric assay was modified for measuring cathepsin activities in pupa.30 The dissected fat body was homogenized in ice cold phosphate buffered saline (PBS) buffer. After centrifugation, the protein concentration of supernatants (tissue extract) was determined by the BCA assay. Proteolytic activity was assayed using a specific substrate of cathepsin B and L, Z-Phe-Arg-MCA (Z-F-R-MCA, Sigma). A total of 200 μL protein extracts were added to 600 μL buffer (contain 113 mmol/L Na2HPO4-citrate, 2.6 mmol/L dithiothreitol (DTT), 1.3 mmol/L ethylene diamine tetraacetic acid disodium salt (EDTA-Na2), and 1.7 mM l-cysteine, 0.3% Brij35) and 200 μL 25 µmol/L Z-F-R-MCA was added to the mixture and incubated for 10 min at 40°C. After incubation, 1.5 mL buffer (containing 0.1 mol/L Sodium acetate, 0.1 mol/L Chloroacetic acid sodium, pH 4.3) were added to terminate the reaction. After centrifugation, the fluorescent product was quantified in a fluorometer (Promega, E7031) at the excitation/emission of 360/460. One unit of enzyme activity represents the fluorescence released from 5 nmol substrate over per 10 min.
Determination of protease activity of Bombyx mori pupa fat at different pupa developmental stages
Protein extracts (20 μg) were separated in 12% SDS-PAGE gel and transferred on to a polyvinylidene fluoride (PVDF) membrane. After being blocked with blocking buffer (5% skimmed in tris-buffered saline (TBS)) for 1 h at room temperature, the membrane was incubated in anti-SP2/Sp3 serum (1:5000 dilution in blocking buffer) overnight at 4°C. After five-times tris buffered saline with tween 20 (TBST) (0.02% Tween in TBS) wash for 10 min each, the blot was probed by using peroxidase-conjugated goat-anti-rabbit IgG. The signals were detected by ECL advance Western Blotting Detection Reagents (GE Healthcare).
Acknowledgments
The authors thank J. He at Shanghai Synchrotron Radiation Facility (Bl17U, SSRF), H. Robinson at Brookhaven National Laboratory (NSLS, X29A) for assistance during data collection, and S. Panjikar at Australian Synchrotron for structure calculation. The coordinates have been deposited in the PDB under the accession code 3WJM.
Supporting Information
Additional Supporting Information may be found in the online version of this article.
Supplementary Information Figure 1. Bombyx mori Arylphorins contain two similar proteins revealed by Mass MALDI TOF/TOF spectroscopy (A) Mass spectrum results of Arylphorins treated by trypsin. A total of 37 out of 48 deisotoped peptides match the sequences of Bombyx mori arylphorins. Arrows indicate peptides from SP2, whereas arrowheads indicate peptides SP3.
Supplementary Information Figure 2. Structural differences between Bombyx mori SP2 and SP3 The omit map electron densities of α8 fragment of Bombyx mori SP2 at 2.5σ are shown in pink. The protein fragment is shown in stick and colored based on element types. The omit map electron densities of α8 fragment of Bombyx mori SP3 at 2.5σ are shown in pink. The protein fragment is shown in stick and colored based on element types.
Supplementary Information Figure 3. Analytical Gel filtration profile of Bombyx mori Arylphorins The elution profile of purified Bombyx mori Arylphorins from the analytic gel filtration column is shown.
Supplementary Information Figure 4. Cartoon view of aromatic residues distribution within Bombyx mori Arylphorins (A) The structures of Bombyx mori Arylphorins are shown in carton view and colored in yellow, the bound oligosaccharide chains are shown in stick mode and colored in cyan, whereas the aromatic residues are colored in red. (A) 90°rotation of (A) along X axis. The same color code as (A).
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supplementary Information Figure 1. Bombyx mori Arylphorins contain two similar proteins revealed by Mass MALDI TOF/TOF spectroscopy (A) Mass spectrum results of Arylphorins treated by trypsin. A total of 37 out of 48 deisotoped peptides match the sequences of Bombyx mori arylphorins. Arrows indicate peptides from SP2, whereas arrowheads indicate peptides SP3.
Supplementary Information Figure 2. Structural differences between Bombyx mori SP2 and SP3 The omit map electron densities of α8 fragment of Bombyx mori SP2 at 2.5σ are shown in pink. The protein fragment is shown in stick and colored based on element types. The omit map electron densities of α8 fragment of Bombyx mori SP3 at 2.5σ are shown in pink. The protein fragment is shown in stick and colored based on element types.
Supplementary Information Figure 3. Analytical Gel filtration profile of Bombyx mori Arylphorins The elution profile of purified Bombyx mori Arylphorins from the analytic gel filtration column is shown.
Supplementary Information Figure 4. Cartoon view of aromatic residues distribution within Bombyx mori Arylphorins (A) The structures of Bombyx mori Arylphorins are shown in carton view and colored in yellow, the bound oligosaccharide chains are shown in stick mode and colored in cyan, whereas the aromatic residues are colored in red. (A) 90°rotation of (A) along X axis. The same color code as (A).