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

Abstract

BackgroundA 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.ResultsOne- 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.ConclusionsIn 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 a...

Vitellogenin from the Silkworm, Bombyx mori: An Effective Anti-Bacterial Agent

“…The changes in the total proteome upon infection were quite striking. Several of the normally highly abundant proteins such as the 30 kDa lipoproteins [29] seemed drastically lowered in concentration, if not removed, from the proteome, possibly due to proteolytical degradation.…”

“…Unless otherwise noted, sample collection and experiments (gel electrophoresis, Western blotting, lipoprotein staining of native gel) were performed as described earlier [29]. The monoclonal antibody against Vg of Pteromalus puparum was a generous gift from Prof. Gong-Yin Ye; State Key Laboratory of Rice Biology, Institute of Insect Sciences, College of Agricultural and Biotechnology, Zhejiang University, China.…”

Two Crystal Structures of Bombyx mori Lipoprotein 3 - Structural Characterization of a New 30-kDa Lipoprotein Family Member

“…Most likely, all ten 30-kDa lipoprotein genes have a common ancestor that underwent differentiation and divergent evolution [12]. Moreover, differences in amino acid sequences are also observed between 30-kDa proteins from different silkworm strains [13]. Also, analysis of expressed sequence tags (ESTs) derived from several silkworm tissues indicated that the level of expression of the 30-kDa proteins varies.…”

Protein Patterns of Silkworm Bombyx mori L. Fed on Leaves of Wild and Cultivated Mulberry Varieties

“…These proteins are often called larval serum protein, larval specific serum proteins or larval haemolymph proteins. Comparative proteomics suggested that abundantly synthesized 30 K proteins in haemolymph of the fifth larval instar (the vigorous feeding stage) were taken up by and stored in fat body (storage tissue), to meet nutrient requirement of non-feeding stages, pupae, adults and eggs (Pakkianathan et al, 2012;Zhang et al, 2014).…”