Beta-fructofuranosidase genes of the silkworm, Bombyx mori: insights into enzymatic adaptation of B. mori to toxic alkaloids in mulberry latex

Abstract

Mulberry latex contains extremely high concentrations of alkaloidal sugar mimic glycosidase inhibitors, such as 1,4-dideoxy-1,4-imino-D-arabinitol (D-AB1) and 1-deoxynojirimycin (DNJ). Although these compounds do not harm the silkworm, Bombyx mori, a mulberry specialist, they are highly toxic to insects that do not normally feed on mulberry leaves. D-AB1 and DNJ are strong inhibitors of alpha-glucosidases (EC 3.2.1.20); however, they do not affect the activity of beta-fructofuranosidases (EC 3.2.1.26). Although alpha-glucosidase genes are found in a wide range of organisms, beta-fructofuranosidase genes have not been identified in any animals so far. In this study, we report the identification and characterization of beta-fructofuranosidase genes (BmSuc1 and BmSuc2) from B. mori. The BmSuc1 gene was highly expressed in the midgut and silk gland, whereas the expression of BmSuc2 gene was not detected. BmSuc1 encodes a functional beta-fructofuranosidase, whose enzymatic activity was not inhibited by DNJ or D-AB1. We also showed that BmSUC1 protein localized within the midgut goblet cell cavities. Collectively, our data clearly demonstrated that BmSuc1 serves as a sugar-digesting enzyme in the silkworm physiology. This anomalous presence of the beta-fructofuranosidase gene in the B. mori genome may partly explain why the silkworm can circumvent the mulberry's defense system.

FIGURE 1.

Amino acid sequences of BmSUC1 and BmSUC2. Amino acid sequences were compared with the sequence of β-fructofuranosidase from the bacterium B. licheniformis ATCC 14580 (GenBank™ accession number AAU25612). Sequences were aligned with the ClustalX program (19). Using the Boxshade program, identical and similar residues, in more than two sequences, have been highlighted in black and gray, respectively. Putative signal peptides are boxed, and the active sites of the yeast invertase (23) are underlined.

FIGURE 2.

Phylogenetic analysis of β-fructofuranosidases. Amino acid sequences of β-fructofuranosidases were aligned by the ClustalX program, and the phylogenetic tree was constructed by the neighbor-joining method using the MEGA3 program package (20). Bootstrap values after 1,000 replications are shown. GenBank™ or SwissProt accession numbers of sequences are shown in parentheses.

FIGURE 3.

Expression and purification of the His-tagged BmSUC1 protein. Recombinant BmSUC1 protein with the His tag at the C terminus was expressed using a baculovirus expression system. BmSUC1 was purified from the medium of virus-infected cells by nickel chromatography. Protein samples were analyzed by SDS-PAGE and stained with CBB (a), and the same samples were analyzed by immunoblot with the anti-His antibody (b). The molecular masses of the protein standards are shown on the left, and the estimated molecular mass of BmSUC1 is shown on the right. The following are the protein samples used. Mock, the medium from mock-infected cells; WT, the medium from parental AcMNPV-infected cells; BmSUC1-His, the medium from cells infected with the recombinant AcMNPV that expressed BmSUC1 protein; purified, the purified BmSUC1 protein.

FIGURE 4.

Enzymatic properties of recombinant BmSUC1 protein. BmSUC1 protein was incubated with selected substrates (sucrose, isomaltose, maltose, raffinose, or stachose), and the substrate specificity of BmSUC1 was measured. a, the liberated glucose was measured by the glucose oxidase method; b, the reducing sugars released were estimated by the Somogyi-Nelson method. Bars, mean ± S.D. (n = 3). c, pH-activity relationship of the recombinant BmSUC1, determined using sucrose as a substrate. The points indicate the mean ± S.D. (n = 3). d, inhibitory assay of DNJ on the recombinant BmSUC1 (filled circle) and the α-glucosidase (open triangle) from a bacterium (Sigma), using sucrose as a substrate. The same unit (nmol of glucose produced/min) of the BmSUC1 and bacterial α-glucosidase was used for the analysis. The points indicate the mean ± S.D. (n = 3).

FIGURE 5.

Expression profiles of BmSuc1 mRNA. a, RT-PCR analysis of BmSuc1 expression. Total RNA from the 3rd day of the fifth instar larvae were used in the RT-PCR analysis. The B. mori actin 3 (BmA3) gene was used as the control. Template cDNAs used for RT-PCR experiments were synthesized with (+) or without (-) reverse transcriptase. Tissues used for analysis were as follows. Lane 1, foregut; lane 2, anterior part of the midgut; lane 3, middle part of the midgut; lane 4, posterior part of the midgut; lane 5, anterior part of the intestine; lane 6, posterior part of the intestine; lane 7, rectum; lane 8, testis; lane 9, anterior silk gland; lane 10, anterior part of the MSG; lane 11, middle part of the MSG; lane 12, posterior part of the MSG; lane 13, posterior silk gland; lane 14, malphigian tubules; lane 15, fat bodies; lane 16, trachea; lane 17, epidermis. b, RNA blot analysis of BmSuc1. Total RNA (5 μg) of the midgut, silk gland, fat bodies, epidermis, and trachea from day 0–5 of fifth instar larvae (V0–V5, respectively) and from larvae at wandering stage (W; day 6) were analyzed. rRNA stained with ethidium bromide was used as a loading control.

FIGURE 6.

Immunoblot analysis of BmSUC1 protein. Immunoblot analysis using anti-BmSUC1 antiserum was performed. Protein samples were isolated from the epidermis (E), midgut (MG), soluble fraction (supernatant of the homogenate) of peritrophic membrane (PM), fat bodies (FB), silk gland (SG), trachea (T), and hemolymph (H) from the 3-day-old fifth instar larvae. 2 μg of protein was loaded in each lane. As a positive control, the medium from cells infected with AcMNPV that expressed BmSUC1 protein was also loaded (P). The size and position of the protein standards are indicated on the left. BmSUC1 protein signals are shown by an arrow. An arrowhead shows the signal from putative BmSUC1 protein from the peritrophic membrane, which did not enter the running gel.

FIGURE 7.

Immunohistochemical analysis of BmSUC1 protein in the midgut. A, midgut cells stained with DAPI (blue) and AlexaFluor594-conjugated phalloidin (red). B, schematic representation of midgut cells of B. mori. C, columnar cell; G, goblet cell; GC, goblet cell cavity; N, nucleus; B, basal lamina; M, microvilli. C, control experiments using preimmune serum. D, immunofluorescence visualization of BmSUC1. Sections were incubated with anti-BmSUC1 antibody followed by the secondary antibody labeled with AlexaFluor488 (green) and counterstained with DAPI. Strong signals were detected in the goblet cell cavities. E and F, control experiments using antisera against sALP (green) and mALP (green), respectively. Note that localization of BmSUC1 is closely similar to that of sALP, which was shown to localize within goblet cell cavities. Arrows, localization of the protein; bar, 200 μm.

FIGURE 8.

Immunohistochemical analysis of BmSUC1 protein in the silk gland. A, schematic representation of the silk gland of the silkworm. Sections were obtained from the middle part of the anterior silk gland (ASG), MSG, and posterior silk gland (PSG), as indicated by red lines. B, immunofluorescence visualization of BmSUC1 and CBP. Slides were incubated with anti-BmSUC1 or anti-CBP antiserum followed by the secondary antibody labeled with AlexaFluor546 (red) and counterstained with DAPI (blue). Control experiments were also preformed using preimmune serum. Arrows, localization of the protein; bar, 200 μm. Schematic representations of sections are also shown (top). N, nucleus; C, cuticle layer; S, sericin layer; F, fibroin layer.