Transport of Carotenoids by a Carotenoid-Binding Protein in the Silkworm - Carotenoids: Physical, Chemical, and Biological Functions and Properties - page 512

Hemolymph

1 2

3

Gut

Mouth

Anus

Silk gland

FIGURE 24.1 The pathway of carotenoid transport in the silkworm. Carotenoids are absorbed from dietary
mulberry leaves into the intestinal mucosa, transferred to the hemolymph (1), transported in the hemolymph
by plasma lipoproteins (2), and accumulated in the silk gland (3).

poor. With rare exceptions, we do not know what genes control the concentrations and forms of
carotenoid in each tissue.
How can the molecular mechanism of the transport system of carotenoids be studied? A hint
comes from the recognition that carotenoids are generally hydrophobic. In order to move carote-
noids through the aqueous biological environment, carotenoid-binding proteins (CBPs) that cover
the hydrophobic surface of carotenoids are thought to be needed. In particular, selective uptake of
certain types of carotenoids likely demands highly specii c CBPs. Thus, identii cation and analysis
of CBPs are expected to provide new insights into the transport system of carotenoids (Bhosale and
Bernstein 2007).
The silkworm, Bombyx mori , is a good model organism for studying CBPs for the following
reasons. Wild-type silkworm larvae feed on carotenoid-rich mulberry leaves, where carotenoids are
absorbed into the intestinal mucosa and transferred to the hemolymphal lipoprotein called lipo-
phorin (Chino 1985). Here, the carotenoids are accumulated in the silk gland, the largest tissue in the
late stage of the last instar and the site of silk protein synthesis (Figure 24.1), resulting in the forma-
tion of a yellow cocoon. In addition, the silkworm is relatively large and easily reared in large num-
bers, allowing us to obtain substantial amounts of carotenoid-rich materials for purii cation of CBPs.
Furthermore, during the long history of sericulture, several mutants that produce white cocoons due
to defects in the carotenoid transport system have been found and maintained as genetic resources
(Tazima 1964, Banno et al. 2005, Fujii and Banno 2005). We can investigate the relationship between
CBPs and the mutants, which will enable us to dissect the transport system genetically.
The hemolymphal transport of carotenoids by lipophorin has been elucidated and documented
(Law and Wells 1989, Tsuchida et al. 1998, Arrese et al. 2001, Canavoso et al. 2001), as has plasma
transport by mammalian lipoproteins (Paker 1996, Yeum and Russell 2002). Lipophorin serves as
a shuttle that moves carotenoids from one tissue to another without itself entering the cells, in stark
contrast to the vertebrate low-density lipoprotein (LDL) (Brown and Goldstein 1986), which is
endocytosed and metabolized in the cell. Here, we focus on the recent biochemical and genetic stud-
ies of the intracellular CBP of the silkworm, which mainly transports lutein. We hope this review
provides insights into the studies of CBPs in other organisms.

24.2 IDENTIFICATION OF THE CAROTENOID-BINDING PROTEIN
IN THE SILK GLAND

24.2.1 P URIFICATION AND C LONING OF CBP
The purii cation strategy for CBPs is conceptually simple. Proteins from carotenoid-rich tissues are
separated under nondenaturing and relatively aqueous conditions where carotenoids are expected to
remain bound to the CBPs. CBPs are then detected by the color of the carotenoids.
Several researchers have tried to isolate cellular CBPs from the silkworm. In Nakajima's study
(1963), the whole midgut mucosa was homogenized and the proteins separated with a gel-i ltration
chromatography column. Carotenoids were found in certain fractions containing proteins, suggest-
ing the existence of CBPs in the midgut. Jouni and Wells purii ed a 35 kDa protein containing lutein