Isolation, Characterization, and cDNA Sequence of a Carotenoid Binding Protein from the Silk Gland of Bombyx mori Larvae

Hiroko Tabunoki,Hiromu Sugiyama,Yoshiro Tanaka,Hiroshi Fujii,Yutaka Banno,Zeina E Jouni,Masahiko Kobayashi,Ryoichi Sato,Hideaki Maekawa,Kozo Tsuchida

doi:10.1074/jbc.m204507200

Abstract

A carotenoid binding protein (CBP) has been isolated from the silk glands of Bombyx mori larvae. The protein has an apparent molecular mass of 33 kDa and binds carotenoids in a 1:1 molar ratio. Lutein accounts for 90% of the bound carotenoids, whereas alpha-carotene and beta-carotene are minor components. Immunological analysis demonstrated the presence of CBP only in the yellow-colored tissues of the silk gland, midgut, testis, and ovary. Several phenotypes of B. mori mutants linked to carotenoid transport have been utilized to characterize CBP. The Y (yellow hemolymph) gene controls uptake of carotenoids from the midgut lumen into the midgut epithelium, and larvae with the +(Y) gene lack this property. Immunoblotting analysis confirmed the presence of CBP in mutants with the dominant Y gene only. Immunohistochemistry verified the localization of CBP in the villi of the midgut epithelium, indicating that CBP might be involved in absorption of carotenoids. A cDNA clone for CBP encoding a protein of 297 amino acids has been isolated from the B. mori silk gland cDNA library. The deduced amino acid sequence revealed that CBP is a novel member of the steroidogenic acute regulatory (StAR) protein family with its unique structural feature of a StAR-related lipid transfer domain, known to aid in lipid transfer and recognition. Lutein-binding capacity of the recombinant CBP (rCBP) determined by incubating rCBP with lutein followed by immunoprecipitation using anti-CBP IgG conjugated to protein A-Sepharose, demonstrated the formation of a lutein-rCBP complex. Sequence analyses coupled with binding specificity suggest that CBP is a new member of the StAR protein family that binds carotenoids rather than cholesterol.

Highlights

Immunohistochemistry—The midgut from day 4 of 5th instar larvae, corresponding to the time when the tissue becomes most yellow in color and the point of maximal tissue size, was used for immunohistochemistry (Fig. 10)
Summary—In this report we described the purification, characterization, and sequence analysis of a carotenoid binding protein from the wild type of silk gland of B. mori
Sitedirected mutagenesis and cDNA-CBP transient infection of the silk gland cells of white cocoon mutants will be investigated in our future studies

Summary

Introduction

Immunohistochemistry—The midgut from day 4 of 5th instar larvae (wild type, N4), corresponding to the time when the tissue becomes most yellow in color and the point of maximal tissue size, was used for immunohistochemistry (Fig. 10). The frozen sections of the midgut were slide-mounted, fixed in acetone, and incubated with anti-CBP antibody.

Results

Conclusion