LBD1 of Vitellogenin Receptor Specifically Binds to the Female-Specific Storage Protein SP1 via LBR1 and LBR3

Abstract

Storage proteins are the major protein synthesized in the fat body, released into hemolymph and re-sequestered into the fat body before pupation in most insect species. Storage proteins are important amino acid and nutrition resources during the non-feeding pupal period and play essential roles for the metamorphosis and oogenesis of insects. The sequestration of storage protein is a selective, specific receptor-mediated process. However, to date, the potential receptor mediating the sequestration of storage protein has not been determined in Bombyx mori. In this study, we expressed and purified the first ligand binding domain of Bombyx mori vitellogenin receptor (BmVgR), LBD1, and found LBD1 could bind with an unknown protein from the hemolymph of the ultimate silkworm larval instar via pull-down assay. This unknown protein was subsequently identified to be the female-specific storage protein SP1 by mass spectrometry. Furthermore, far western blotting assay, immunoprecipitation and isothermal titration calorimetry analysis demonstrated LBD1 specifically bound with the female-specific SP1, rather than another unisex storage protein SP2. The specific binding of LBD1 with SP1 was dependent on the presence of Ca2+ as it was essential for the proper conformation of LBD1. Deletion mutagenesis and ITC analysis revealed the first and third ligand binding repeats LBR1 and LBR3 were indispensable for the binding of LBD1 with SP1, and LBR2 and LBR4 also had a certain contribution to the specific binding. Our results implied BmVgR may mediate the sequestration of SP1 from hemolymph into the fat body during the larval-pupal transformation of Bombyx mori.

Patterns of Proteins at Different Stages of Red Flour Beetle, Tribolium castaneum

“…The presence of the 25K and 21K proteins is sex-linked and they are present only in the adult females. Initial appearance of 25K mRNA is closely coordinated in time with that of Vgs, 21K and 19K, all of which are induced by JH implying possible involvement of JH in the regulation of synthesis of the 25K protein [17]. Interesting finding of the present study is that the similar type of protein is also found in the female adult of T. castaneum which reached a maximum level over days 10-15 after the insect molted into adult.…”

“…It is a dynamic tissue involved in multiple metabolic functions. The uptake of these proteins by the fat body tissue is stage specific process occurs before pupation by a receptor mediated endocytotic process [17]. One of these functions is to store and release energy in response to the energy demands of the insect.…”

Vitellogenin receptor selectively endocytoses female-specific and highly-expressed hemolymph proteins in the silkworm,Bombyx mori

“…However, few studies have attempted to identify or characterize the BmSP1 receptor. A recent study showed that LBD1 of BmVgR can specifically bind to the BmSP1 (Liu et al 2016).…”

“…BmSP1 was purified as described previously (Liu et al 2016). BmVg was purified using a method similar to that used for BmSP1.…”

Expressional analysis of the silkworm storage protein 1 and identification of its interacting proteins

“…Usually synthesized in the fat body of larvae, storage proteins are released into the haemolymph. During pupation, they are selectively reabsorbed by the fat body, where they form dense protein particles in the cell (Liu et al, 2016). Storage proteins play important physiological and biochemical roles in insects.…”