title: Recognition of signal peptide by protein translocation machinery in middle silk gland of silkworm Bombyx mori.
abstract: To investigate the functions of signal peptide in protein secretion in the middle silk gland of silkworm Bombyx mori, a series of recombinant Autographa californica multiple nucleopolyhedroviruses containing enhanced green fluorescent protein (egfp) gene, led by sericin-1 promoter and mutated signal peptide coding sequences, were constructed by region-deletions or single amino acid residue deletions. The recombinant Autographa californica multiple nucleopolyhedroviruses were injected into the hemocoele of newly ecdysed fifth-instar silkworm larvae. The expression and secretion of EGFP in the middle silk gland were examined by fluorescence microscopy and Western blot analysis. Results showed that even with a large part (up to 14 amino acid residues) of the ser-1 signal peptide deleted, the expressed EGFP could still be secreted into the cavity of the silk gland. Western blot analysis showed that shortening of the signal peptide from the C-terminal suppressed the maturation of pro-EGFP to EGFP. When 8 amino acid residues were deleted from the C-terminal of the signal peptide (mutant 13 aa), the secretion of EGFP was incomplete, implicating the importance of proper coupling of the h-region and c-region. The deletion of amino acid residue(s) in the h-region did not affect the secretion of EGFP, indicating that the recognition of signal peptide by translocation machinery was mainly by a structural domain, but not by special amino acid residue(s). Furthermore, the deletion of Arg2 or replacement with Asp in the n-region of the signal peptide did not influence secretion of EGFP, suggesting that a positive charge is not crucial.
authors.affiliation: State Key Laboratory of Molecular Biochemistry, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Xiuyang
authors.initials: X
authors.lastname: Guo
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Xue
authors.initials: X
authors.lastname: Zhang
authors.forename: Shengpeng
authors.initials: S
authors.lastname: Wang
authors.forename: Changde
authors.initials: C
authors.lastname: Lu
mesh_terms: D000818:Animals; D012831:Bombyx / Q000378:metabolism*; D005088:Exocrine Glands / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology*; D047030:Sericins / Q000378:metabolism*
chemical_list: D047030:Sericins