Structure And Function Of 30K Proteins And The Proteomic Analysis Of The Hemolymph Of Bombyx Mori At The Fifth Instar Larvae Stage

The silkworm Bombyx mori has been domesticated as not only a major economic insect, also its ability to metamorphosis from a worm to a moth makes it a good model for lepidopteran genetic and molecular biology research. Silkworm has an open circulatory hemolymph system, especially at the fifth instar, silkworm becomes more activity for metamorphosis, a lot of hemolymph proteins are synthesized in fat bodies and secreted into the hemolymph, and they are required to participate diverse biochemical and physiological processes.The molecular mechanisms of these proteins are still not well-understood. The silkworm"30K"proteins are a group of plasma proteins with a molecular weight of approximately 30 kDa, termed B. mori low molecular weight lipoproteins (Bmlps). These so-called"30K proteins"share a highly conserved primary sequence, they are synthesized in fat bodies and secreted into the hemolymph in the later stage of the fourth through to the fifth instar larvae, and eventually vanished after the larval hatching. The functions of these 30K proteins remain unclear, and the lipid content of these proteins and their functional relationships are unknown. In addition, 30K proteins had been found to exhibit anti-apoptotic activity. 30K proteins had also been shown to bind to glucose, maltose and glucans, suggesting their involvement in insect self-defense systems.To elucidate their molecular functions, we have successfully purified the natural Bmlp7 from silkworm hemolymph for crystallization and overexpressed five of them (Bmlp1–3, Bmlp7 and Bmlp8), the Bmlp7 diffraction was phased against the multi-wavelength anomalous diffraction (MAD) data of the Se-Met-substituted protein and determined the crystal structure of Bmlp7, at 1.91 ?. Bmlp7 has two distinct domains: an all-αN-terminal domain (NTD) and an all-βC-terminal domain (CTD) of theβ-trefoil fold. It reveals a novelβ-trefoil family, in addition to the current 14 families of theβ-trefoil superfamily (http://pfam.sanger.ac.uk/), which are proposed to arise from a common ancestor, despite sharing no characteristic binding sites or subcellular localization motif. Structural analysis in combination with simulation indicated a putative lipid-binding cavity at the NTD and one potential sugar-binding site at the CTD; however, the in vitro binding for neither lipid nor sugar was detected. Therefore, further investigations are needed to characterize the molecular function of this protein.The completion of the B. mori genome sequences made it possible to understand the information between gene expression and function and the proteomic analysis can be actualized through this database. To improve the understanding of this important bioprocess and gene expression situation, we utilized the proteomic approach to investigate the proteome of the fifth instar hemolymph during growth and development. The One-Dimensional gradient acrylamide SDS-PAGE Electrophoresis method (1D-SDS-PAGE) in gel digestion and the different pH values of hemolymph in solution digestion, combination with the Two Dimensional Liquid Chromatography Electrospray Ionization Tandem-Mass Spectrometry (2DLC-MS) technique were used here to analyze the proteome of the hemolymph of the Bombyx mori in 5th instar. In total, 289 proteins are identified; the isoelectric point values of these proteins range from 4–12. Most of the proteins are soluble in water, have a molecular weight of 10–60 kDa. The functions of these identified proteins were categorized by NCBI BLAST and Gene Ontology. These proteins belong to 12 functional categories and most of them involved in storage, defense, transport and metabolism. Furthermore, the levels of expression of 30K proteins were investigated. The results showed that its highest expression was reached at the early of the pupa stage, then disappeared in hemolymph of both sex moths, and the expression level correlates well with their functions. The results of the hemolymph proteomics will help us for the future study of these'hot spot'proteins.