Molecular cloning and characterization of the ¦Á-glucosidase II from Bombyx mori and Spodoptera frugiperda

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• 作者：Satoko Watanabe^a ; ^b ; ^{satochan@affrc.go.jp} ; Akemi Kakudo^c ; Masato Ohta^b ; Kazuei Mita^d ; ¹ ; Kazuhito Fujiyama^c ; Shigeki Inumaru^b
• 关键词：Glucosidase II ; Bombyx mori ; Spodoptera frugiperda ; Sugar-mimic alkaloids ; ¦Á-glucosidase inhibitor
• 刊名：Insect Biochemistry and Molecular Biology
• 出版年：2013
• 出版时间：April, 2013
• 年：2013
• 卷：43
• 期：4
• 页码：319-327
• 全文大小：516 K

文摘

The ¦Á-glucosidase II (GII) is a heterodimer of ¦Á- and ¦Â-subunits and important for N-glycosylation processing and quality control of nascent glycoproteins. Although high concentration of ¦Á-glucosidase inhibitors from mulberry leaves accumulate in silkworms (Bombyx mori) by feeding, silkworm does not show any toxic symptom against these inhibitors and N-glycosylation of recombinant proteins is not affected. We, therefore, hypothesized that silkworm GII is not sensitive to the ¦Á-glucosidase inhibitors from mulberry leaves. However, the genes for B.?mori GII subunits have not yet been identified, and the protein has not been characterized. Therefore, we isolated the B.?mori GII ¦Á- and ¦Â-subunit genes and the GII ¦Á-subunit gene of Spodoptera frugiperda, which does not feed on mulberry leaves. We used a baculovirus expression system to produce the recombinant GII subunits and identified their enzyme characteristics. The recombinant GII ¦Á-subunits of B.?mori and S.?frugiperda hydrolyzed p-nitrophenyl ¦Á-d-glucopyranoside (pNP-¦ÁGlc) but were inactive toward N-glycan. Although the B.?mori GII ¦Â-subunit was not required for the hydrolysis of pNP-¦ÁGlc, a B.?mori GII complex of the ¦Á- and ¦Â-subunits was required for N-glycan cleavage. As hypothesized, the B.?mori GII ¦Á-subunit protein was less sensitive to ¦Á-glucosidase inhibitors than was the S.?frugiperda GII ¦Á-subunit protein. Our observations suggest that the low sensitivity of GII contributes to the ability of B.?mori to evade the toxic effect of ¦Á-glucosidase inhibitors from mulberry leaves.