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A proteomic view on the developmental transfer of homologous 30 kDa lipoproteins from peripheral fat body to perivisceral fat body via hemolymph in silkworm, Bombyx mori.

Pakkianathan BC, Singh NK, Krishnan M, König S - BMC Biochem. (2012)

Bottom Line: In B. mori larvae and pupae, 30 kDa lipoproteins LP1 to LP5 and L301/302 were detected in PPFB and PVFB tissue as well as in hemolymph.The amino acid sequences of all known 30 kDa proteins showed very high homology.A first attempt to that end allowed isolation of a B. mori LP3-like protein, the complete structure, properties and function of which will now be elucidated in detail.

View Article: PubMed Central - HTML - PubMed

Affiliation: Integrated Functional Genomics, Interdisciplinary Center for Clinical Research, University of Münster, Röntgenstr, 21, 48149 Münster, Germany.

ABSTRACT

Background: A group of abundant proteins of ~30 kDa is synthesized in silkworm larval peripheral fat body (PPFB) tissues and transported into the open circulatory system (hemolymph) in a time-depended fashion to be eventually stored as granules in the pupal perivisceral fat body (PVFB) tissues for adult development during the non-feeding stage. These proteins have been shown to act anti-apoptotic besides being assigned roles in embryogenesis and defense. However, detailed protein structural information for individual PPFB and PVFB tissues during larval and pupal developmental stages is still missing. Gel electrophoresis and chromatography were used to separate the 30 kDa proteins from both PPFB and PVFB as well as hemolymph total proteomes. Mass spectrometry (MS) was employed to elucidate individual protein sequences. Furthermore, 30 kDa proteins were purified and biochemically characterized.

Results: One- and two-dimensional gel electrophoresis (1/2D-PAGE) was used to visualize the relative changes of abundance of the 30 kDa proteins in PPFB and PVFB as well as hemolymph from day 1 of V instar larval stage to day 6 of pupal stage. Their concentrations were markedly increased in hemolymph and PVFB up to the first two days of pupal development and these proteins were consumed during development of the adult insect. Typically, three protein bands were observed (~29, 30, 31 kDa) in 1D-PAGE, which were subjected to MS-based protein identification along with spots excised from 2D-gels run for those proteomes. Gas phase fragmentation was used to generate peptide sequence information, which was matched to the available nucleotide data pool of more than ten highly homologous insect 30 kDa lipoproteins. Phylogenetic and similarity analyses of those sequences were performed to assist in the assignment of experimentally identified peptides to known sequences. Lipoproteins LP1 to LP5 and L301/302 could be matched to peptides extracted from all bands suggesting the presence of full length and truncated or modified protein forms in all of them. The individual variants could not be easily separated by classical means of purification such as 2D-PAGE because of their high similarity. They even seemed to aggregate as was indicated by native gel electrophoresis. Multistep chromatographic procedures eventually allowed purification of an LP3-like protein. The protein responded to lipoprotein-specific staining.

Conclusions: In B. mori larvae and pupae, 30 kDa lipoproteins LP1 to LP5 and L301/302 were detected in PPFB and PVFB tissue as well as in hemolymph. The concentration of these proteins changed progressively during development from their synthesis in PPFB, transport in hemolymph to storage in PVFB. While the 30 kDa proteins could be reproducibly separated in three bands electrophoretically, the exact nature of the individual protein forms present in those bands remained partially ambiguous. The amino acid sequences of all known 30 kDa proteins showed very high homology. High-resolution separation techniques will be necessary before MS and other structural analysis can shed more light on the complexity of the 30 kDa subproteome in B. mori. A first attempt to that end allowed isolation of a B. mori LP3-like protein, the complete structure, properties and function of which will now be elucidated in detail.

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MS/MS spectra of peptides unique for specific 30 kDa lipoproteins. Fragment ion series from the N- and C-terminus were observed as well as immonium ions. A) L302. B/C) from purified lipoprotein (fraction 60, Fig. 4). B) LP3/L301, C) LP3/L301, modified (phosphorylated or O-sulfonated).
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Figure 3: MS/MS spectra of peptides unique for specific 30 kDa lipoproteins. Fragment ion series from the N- and C-terminus were observed as well as immonium ions. A) L302. B/C) from purified lipoprotein (fraction 60, Fig. 4). B) LP3/L301, C) LP3/L301, modified (phosphorylated or O-sulfonated).

Mentions: The assignment of the MS data for pupal lipoproteins is summarized in Table 1. It is based on peptides, which are unique for certain protein sequences (for individual peptide matches see Additional file 5 and Additional file 14). An exemplary spectrum for a unique peptide of L302 is shown in Figure 3A demonstrating confident sequence determination. The Q-TOF mass spectrometer allows measurements with a resolution of ~10.000 (full width at half maximum) and mass accuracies < 10 ppm, but the assignment of amino acid residues very similar in mass such as lysine (128.095 mass units) and glutamine (128.056 mass units) may remain ambiguous. Isobaric amino acids residues like leucine and isoleucine (both 113.084 mass units) were also not distinguishable in the chosen measurement approach. None of the three bands at ~29, 30 and 31 kDa could be assigned to only one protein sequence even though they were well separated in 1D-PAGE. In fact, characteristic peptides for every single lipoprotein L1-L5 could be detected in hemolymph day 1 (bands at 30, 31 kDa) and day 5 (bands at 29 and 30 kDa). The observation that L2 and G3 were absent on day 1 in the 29 kDa band and L1 was not found in the 31 kDa band at day 5 may in principal be attributed to a lower concentration of those proteins during certain periods of development; more likely, however, are difficulties in finding unique peptides in the complex mixtures present in each band. This is especially true, since hemolymph is the link between synthesis and storage site and all lipoproteins L1-L5 have arrived in PVFB at day 5. Essentially, all lipoproteins L1 to L5 could be found in all three bands, possibly at different individual lengths or in modified forms. Obviously, 1D-PAGE technology is not sensitive enough to visualize subtle concentration changes of one of several proteins in a single band. Therefore, we hoped to achieve better separation in 2D-PAGE experiments of FB and hemolymph methanol/chloroform precipitated protein samples from final instar stadium (day 3, V instar; non-linear pI range 3-10; Figure 4). This technology separates proteins not only by molecular weight but also by their isoelectric point and it has been used to analyze silkworm hemolymph proteins, skeletal muscle, silk glands and FB (for review see [2]). Approximately 250 proteins spots were visible in the gel image for PPFB, 210 for hemolymph and 190 for PVFB. A relatively uniform staining of the spots in the PPFB image infered that many proteins were produced at the synthesis site at about equal rate. In hemolymph, however, about a dozen spots across the whole gel were more intensively stained including spots in the 30 kDa region. This pattern is almost mirrored for PVFB demonstrating again the protein flow synthesis > transport > storage. Thirty-two protein spots were excised from the gel of the hemolymph proteome in the 30 kDa region and subjected to proteomic analysis. Unfortunately, the 30 kDa proteins were distributed over an area of pI ~5-9 with low resolution (for spot assignment see Additional file 15). Lipoproteins L1 to L5 were detected in parallel in single spots, but also in different spots at varying pI. That effect was repeatedly observed although other proteins such as 27 kDa glycoprotein, juvenile hormone binding protein and 14-3-3 protein could be well separated. Possibly, functional moieties of varying size such as lipids, attached to the protein backbone, were responsible for this property of the 30 kDa proteins. It became clear in these experiments that traditional gel electrophoretic separation techniques were not suitable to properly elucidate the primary structure of these proteins.


A proteomic view on the developmental transfer of homologous 30 kDa lipoproteins from peripheral fat body to perivisceral fat body via hemolymph in silkworm, Bombyx mori.

Pakkianathan BC, Singh NK, Krishnan M, König S - BMC Biochem. (2012)

MS/MS spectra of peptides unique for specific 30 kDa lipoproteins. Fragment ion series from the N- and C-terminus were observed as well as immonium ions. A) L302. B/C) from purified lipoprotein (fraction 60, Fig. 4). B) LP3/L301, C) LP3/L301, modified (phosphorylated or O-sulfonated).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3306753&req=5

Figure 3: MS/MS spectra of peptides unique for specific 30 kDa lipoproteins. Fragment ion series from the N- and C-terminus were observed as well as immonium ions. A) L302. B/C) from purified lipoprotein (fraction 60, Fig. 4). B) LP3/L301, C) LP3/L301, modified (phosphorylated or O-sulfonated).
Mentions: The assignment of the MS data for pupal lipoproteins is summarized in Table 1. It is based on peptides, which are unique for certain protein sequences (for individual peptide matches see Additional file 5 and Additional file 14). An exemplary spectrum for a unique peptide of L302 is shown in Figure 3A demonstrating confident sequence determination. The Q-TOF mass spectrometer allows measurements with a resolution of ~10.000 (full width at half maximum) and mass accuracies < 10 ppm, but the assignment of amino acid residues very similar in mass such as lysine (128.095 mass units) and glutamine (128.056 mass units) may remain ambiguous. Isobaric amino acids residues like leucine and isoleucine (both 113.084 mass units) were also not distinguishable in the chosen measurement approach. None of the three bands at ~29, 30 and 31 kDa could be assigned to only one protein sequence even though they were well separated in 1D-PAGE. In fact, characteristic peptides for every single lipoprotein L1-L5 could be detected in hemolymph day 1 (bands at 30, 31 kDa) and day 5 (bands at 29 and 30 kDa). The observation that L2 and G3 were absent on day 1 in the 29 kDa band and L1 was not found in the 31 kDa band at day 5 may in principal be attributed to a lower concentration of those proteins during certain periods of development; more likely, however, are difficulties in finding unique peptides in the complex mixtures present in each band. This is especially true, since hemolymph is the link between synthesis and storage site and all lipoproteins L1-L5 have arrived in PVFB at day 5. Essentially, all lipoproteins L1 to L5 could be found in all three bands, possibly at different individual lengths or in modified forms. Obviously, 1D-PAGE technology is not sensitive enough to visualize subtle concentration changes of one of several proteins in a single band. Therefore, we hoped to achieve better separation in 2D-PAGE experiments of FB and hemolymph methanol/chloroform precipitated protein samples from final instar stadium (day 3, V instar; non-linear pI range 3-10; Figure 4). This technology separates proteins not only by molecular weight but also by their isoelectric point and it has been used to analyze silkworm hemolymph proteins, skeletal muscle, silk glands and FB (for review see [2]). Approximately 250 proteins spots were visible in the gel image for PPFB, 210 for hemolymph and 190 for PVFB. A relatively uniform staining of the spots in the PPFB image infered that many proteins were produced at the synthesis site at about equal rate. In hemolymph, however, about a dozen spots across the whole gel were more intensively stained including spots in the 30 kDa region. This pattern is almost mirrored for PVFB demonstrating again the protein flow synthesis > transport > storage. Thirty-two protein spots were excised from the gel of the hemolymph proteome in the 30 kDa region and subjected to proteomic analysis. Unfortunately, the 30 kDa proteins were distributed over an area of pI ~5-9 with low resolution (for spot assignment see Additional file 15). Lipoproteins L1 to L5 were detected in parallel in single spots, but also in different spots at varying pI. That effect was repeatedly observed although other proteins such as 27 kDa glycoprotein, juvenile hormone binding protein and 14-3-3 protein could be well separated. Possibly, functional moieties of varying size such as lipids, attached to the protein backbone, were responsible for this property of the 30 kDa proteins. It became clear in these experiments that traditional gel electrophoretic separation techniques were not suitable to properly elucidate the primary structure of these proteins.

Bottom Line: In B. mori larvae and pupae, 30 kDa lipoproteins LP1 to LP5 and L301/302 were detected in PPFB and PVFB tissue as well as in hemolymph.The amino acid sequences of all known 30 kDa proteins showed very high homology.A first attempt to that end allowed isolation of a B. mori LP3-like protein, the complete structure, properties and function of which will now be elucidated in detail.

View Article: PubMed Central - HTML - PubMed

Affiliation: Integrated Functional Genomics, Interdisciplinary Center for Clinical Research, University of Münster, Röntgenstr, 21, 48149 Münster, Germany.

ABSTRACT

Background: A group of abundant proteins of ~30 kDa is synthesized in silkworm larval peripheral fat body (PPFB) tissues and transported into the open circulatory system (hemolymph) in a time-depended fashion to be eventually stored as granules in the pupal perivisceral fat body (PVFB) tissues for adult development during the non-feeding stage. These proteins have been shown to act anti-apoptotic besides being assigned roles in embryogenesis and defense. However, detailed protein structural information for individual PPFB and PVFB tissues during larval and pupal developmental stages is still missing. Gel electrophoresis and chromatography were used to separate the 30 kDa proteins from both PPFB and PVFB as well as hemolymph total proteomes. Mass spectrometry (MS) was employed to elucidate individual protein sequences. Furthermore, 30 kDa proteins were purified and biochemically characterized.

Results: One- and two-dimensional gel electrophoresis (1/2D-PAGE) was used to visualize the relative changes of abundance of the 30 kDa proteins in PPFB and PVFB as well as hemolymph from day 1 of V instar larval stage to day 6 of pupal stage. Their concentrations were markedly increased in hemolymph and PVFB up to the first two days of pupal development and these proteins were consumed during development of the adult insect. Typically, three protein bands were observed (~29, 30, 31 kDa) in 1D-PAGE, which were subjected to MS-based protein identification along with spots excised from 2D-gels run for those proteomes. Gas phase fragmentation was used to generate peptide sequence information, which was matched to the available nucleotide data pool of more than ten highly homologous insect 30 kDa lipoproteins. Phylogenetic and similarity analyses of those sequences were performed to assist in the assignment of experimentally identified peptides to known sequences. Lipoproteins LP1 to LP5 and L301/302 could be matched to peptides extracted from all bands suggesting the presence of full length and truncated or modified protein forms in all of them. The individual variants could not be easily separated by classical means of purification such as 2D-PAGE because of their high similarity. They even seemed to aggregate as was indicated by native gel electrophoresis. Multistep chromatographic procedures eventually allowed purification of an LP3-like protein. The protein responded to lipoprotein-specific staining.

Conclusions: In B. mori larvae and pupae, 30 kDa lipoproteins LP1 to LP5 and L301/302 were detected in PPFB and PVFB tissue as well as in hemolymph. The concentration of these proteins changed progressively during development from their synthesis in PPFB, transport in hemolymph to storage in PVFB. While the 30 kDa proteins could be reproducibly separated in three bands electrophoretically, the exact nature of the individual protein forms present in those bands remained partially ambiguous. The amino acid sequences of all known 30 kDa proteins showed very high homology. High-resolution separation techniques will be necessary before MS and other structural analysis can shed more light on the complexity of the 30 kDa subproteome in B. mori. A first attempt to that end allowed isolation of a B. mori LP3-like protein, the complete structure, properties and function of which will now be elucidated in detail.

Show MeSH
Related in: MedlinePlus