Identification And Functional Characterization Of30K Protein In The Silkworm, Bombyx Mori

Bombyx mori, the domesticated silkworm, is a lepidopteran model insect. Similar to most lepidoptera, the silkworm has an open vessel system. Hemolymph is involved in many physiological function processes, such as nutrient and hormone transportation. Silkworm hemolymph comprises21%～25%of the body weight, whereas proteins account for6%of the hemolymph.30K proteins (30KP) are the most abundant protein components at the late fifth instar larval and pupal stages, and30K proteins are also plentiful in silkworm eggs.30K proteins are classified into lepidopteran low molecular weight lipoproteins family. To date,30K proteins homologs have only been identified in Bombyx mori, Manduca sexta and Pseudaletia separata.The9x coverage silkworm genome data has been assembled, as well as many publicly lepidopteran EST datasets are available. In our study, we identified lepidopteran low molecular weight lipoprotein genes based on these datasets, and then analyzed their structure and evolution. By using the published silkworm genome and microarray data, we analyzed the genome locations and the temporal-spatial expression patterns of lepidopteran low molecular weight lipoprotein genes.On this basis, the function of silkworm typical30K proteins of lepidopteran low molecular weight lipoproteins family was characterized. In our study, we performed systematic studies of30K proteins and revealed their synthesis, transportation and degradation by using the BmLP1and BmLP7antibody. The results provide valuable preferences for other30K proteins. We also purified several30K proteins, and then extracted lipids which bind to30K proteins, and identified them by LC-MS/MS.The main results are as follows:1. Identification of lepidopteran low molecular weight lipoprotein genes(1) Identification of lepidopteran low molecular weight lipoprotein genes:We performed an extensive survey of lepidopteran-derived genome and EST datasets. We identified7330KP homologous genes in12lepidopteran species (belong to Bombycoidea, Noctuoidea, and Pyraloidea), including B. mori (46), Samia cynthia ricini (5), Antheraea assama (6), Antheraea mylitta (4), M. sexta (3), Spodoptera frugiperda (1), Pseudaletia separate (1), Striacosta albicosta (1), Heliothis virescens (1), Mamestra brassicae (1), Trichoplusia ni (1), and Maruca vitrata (1). Among these identified genes,5630KP genes are novel members.(2) Classification of lepidopteran low molecular weight lipoproteins:The multiple sequence alignment results revealed that the identified30KP homologous genes could be classified into three groups:ENF-BP genes, typical30KP genes, and serine/threonine-rich30KP (S/T-rich30KP) genes. The C-terminal regions are common to all the three subfamilies (NTD and CTD), but the N-termini are highly variable. The N-terminal region of typical30KP is composed of the signal peptides and an unconserved region. S/T-rich30KP have an exclusive S/T-rich domain between signal peptides and NTD in the N terminus. ENF-BP was found to contain a special domain in the N terminus, which is homologous to Pp-0912of Pseudomonas putida.(3) Phylogenetic analysis of lepidopteran low molecular weight lipoprotein genes: In Bombycoidea insects, we found typical30KP genes, S/T-rich30KP and ENF-BP genes. However, in Noctuidea and Pyralidea, we only found ENF-BP genes. By searching against the whole genome data of H.virescens (unpublished data), we identify a total of four30KP genes, all of which belong to ENF-BP subfamily. Therefore, ENF-BP subfamily may be the origin of lepidopteran low molecular weight lipoprotein family. S/T-rich30KPs were only found in B. mori, and may be a Bombyx-specific low molecular weight lipoprotein subfamily.2. Characterization of silkworm low molecular weight lipoproteins(1) Genome locations of silkworm low molecular weight lipoprotein genes:46low molecular weight lipoprotein genes were identified in the silkworm, and43genes could be located on the chromosomes.24typical30KP genes (Bmlpl-Bmlp24) and8S/T-rich30KP genes (BmIp24-Bmlp32) exist in nscaf2795of Chr.20. The32genes are confined within970Kbp (1380K-2350K) and display a tandem pattern. Four sense and antisense transcnptional pairs (Bmlp37/38and Bmlp40/39and Bmlp41/42and Bmlp44/45) of ENF-BPs exist on chromosome7,22, and24in B. mori.(2) Temporal-spatial expression patterns of silkworm low molecular weight lipoprotein genes:The three groups have their respective temporal-spatial expression patterns, which revealed the functional divergence of three subfamilies. ENF-BP genes are mainly expressed in the hemocyte. Typical30KP genes are expressed mainly in the fat body and integument. The cluster analysis on30KP genes suggested that they show five different temporal expression profiles in the whole life cycle. S/T-rich30KP genes are expressed highly in testis, whereas lowly in ovary during the metamorphosis in which a pupa turns into an adult. S/T-rich30KPs may be involved in the process of spermatogenesis or fertilization because they are expressed highly in testis.3. The synthesis, transportation, degradation and utilization of30K proteins(1) The synthesis and secretion of30K proteins:RT-PCR was used to analyze the developmental expression profile of30K protein genes in the fat body. The protein level of30K protein in the hemolymph was detected by western blotting using the antibody. Comparative analysis revealed that30K proteins were synthesized in fat body and then secreted into hemolymph.(2) The transportation of30K proteins:In the pupal and adult stage, the protein level of30K proteins decreases gradually in the hemolymph, whereas increasing in the ovary. Our results revealed that the30K proteins in the ovary were transported mostly from the hemolymph and not the result of mRNA translation in the ovary. We made a30K proteins affinity chromatography column. The result of pull-down assays of membrane proteins of ovary showed that30K proteins could interact with vitellogenin receptor. We speculated that vitellogenin receptor is the receptor of30K proteins, but further investigation is needed before we address a definite conclusion. We used immunohistochemistry to confirm the location of30K proteins in the ovary and found that most of30K proteins were located in the oocyte of eggs.(3) The utilization of30K proteins:Western blotting analysis showed that30K proteins were present at only very low levels at days9and10during embryogenesis. The proteolytic activity of the crude extracts from eggs or newly hatched larvae was measured throughout embryogenesis of the silkworm, and suggested that protease activity increase from day5eggs and attained a maximum in the newly hatched larvae. The densitometry showed that30K proteins were degraded after being incubated with the crude extracts of the newly hatched larvae. Immunohistochemistry revealed that strong signals for30K proteins were detected in the extraembryonic yolk granules at day6eggs. At day10eggs,30K proteins filled the whole gut lumen. Integrated these data suggest that3OK proteins first loacted in the extraembryonic yolk granules at day6eggs, when the embryonic reverse (blastokinesis) is completed,30K proteins are then ingested into the gut lumen. Our data clearly shows that degradation of30K proteins takes place in the gut lumen. We considered that degraded30K proteins may provide essential nutrients for hatching newly larvae.4. Purification of30K proteins and identification of the lipids which bind to30K proteins(1) Purification of30K proteins:We purified several30K proteins from day7fifth instar larvae by using ammonium sulfate precipitation, hydrophobic chromatography and cation exchange chromatography. The30K proteins including fraction of2kinds of30K proteins, fraction of4kinds of30K proteins, BmLPl and BmLP7.(2)30K proteins can bind to lipids:The purified30K proteins were separated by SDS-PAGE. The result of oil red-O staining suggested that all purified30K proteins could bind to lipids.(3) Identification of the lipids of30K proteins:The lipids of30K proteins were extracted by using chloroform/methanol (2/1) mixture and then analyzed by LC-MS/MS. In total, we identified12lipids, and three lipids (molecular formulas are C22H35O6, C25H31O8, and C12H13O4) are abundant component. The lipid whose molecular formula is C25H31O8is the most abundant, and it has various isomerides. However, these lipids are too stable to obtain more information about their structure formula. So, we can not detecte their structure formula and we will detect the structure formula in future.(4) The utilization of lipids during embryogenesis:Thin-layer chromatography was performed to analyze the change pattern of lipids during embryogenesis. We found that the lipids decrease in the anaphase of embryogenesis. Therefore, we suggested that30K proteins together with the lipids were utilized in the anaphase of embryogenesis to provide essential nutrients for hatching newly larvae.Taken together with3and4, we found that30K protein genes(Bmlp1-Bmlp4, Bmlp7) were synthesized in fat body and then secreted into hemolymph from the fifth instar larval stage to wandering stage.30K proteins attained the maximum in the anaphase of the fifth instar, and then transported to the oocyte of eggs of ovary. Vitellogenin receptor is the possible receptor of30K proteins.30K proteins did not be utilized until the next generation.30K proteins were first loacted in the extraembryonic yolk granules, when the embryonic reverse is completed,30K proteins could be easily ingested into the gut lumen.30K proteins were degraded by de novo proteases in the gut lumen.30K proteins together with their binding lipids were utilized in the anaphase of embryogenesis to provide essential nutrients for embryogenesis.