title: Assembly of the silk fibroin elementary unit in endoplasmic reticulum and a role of L-chain for protection of alpha1,2-mannose residues in N-linked oligosaccharide chains of fibrohexamerin/P25.
abstract: Silk fibroin of Bombyx mori is secreted from the posterior silk gland (PSG) as a 2.3-MDa elementary unit, consisting of six sets of a disulfide-linked heavy chain (H-chain)-light chain (L-chain) heterodimer and one molecule of fibrohexamerin (fhx)/P25. Fhx/P25, a glycoprotein, associates noncovalently with the H-L heterodimers. The elementary unit was found and purified from the endoplasmic reticulum (ER) extract of PSG cells. A substantial amount of fhx/P25 unassembled into the elementary unit was also present in ER. In normal-level fibroin-producing breeds (J-139 and C108), the elementary unit contained fhx/P25 of either 30 kDa (major) or 27 kDa (minor). The 27-kDa fhx/P25 was produced from the 30-kDa form by digestion with the bacterial alpha1,2-mannosidase in vitro. The elementary unit in the ER extract contained only the 30-kDa fhx/P25, whereas both 30- and 27-kDa forms of fhx/P25 were present in the ER plus Golgi mixed extracts. In naked-pupa mutants [Nd(2), Nd-s and Nd-sD], extremely small amounts of fibroin were produced and they consisted of one molecule of 27-kDa fhx/P25 and six molecules of H-chain but no L-chain. When the Nd-sD mutant was subjected to transgenesis with the normal L-chain gene, the (H-L)6fhx1-type elementary unit containing the 30-kDa fhx/P25, was produced. These results suggest that fhx/P25 in the elementary unit is largely protected from digestion with Golgi alpha1,2-mannosidases when L-chains are present in the unit. Models suggesting a role of L-chain for the protection of alpha1,2-mannose residues of fhx/P25 are presented.
authors.affiliation: Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Inoue
authors.forename: Kazunori
authors.initials: K
authors.lastname: Tanaka
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Tanaka
authors.forename: Kohei
authors.initials: K
authors.lastname: Ohtomo
authors.forename: Toshio
authors.initials: T
authors.lastname: Kanda
authors.forename: Morikazu
authors.initials: M
authors.lastname: Imamura
authors.forename: Guo-Xing
authors.initials: GX
authors.lastname: Quan
authors.forename: Katsura
authors.initials: K
authors.lastname: Kojima
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Yamashita
authors.forename: Tasuku
authors.initials: T
authors.lastname: Nakajima
authors.forename: Hideharu
authors.initials: H
authors.lastname: Taira
authors.forename: Toshiki
authors.initials: T
authors.lastname: Tamura
authors.forename: Shigeki
authors.initials: S
authors.lastname: Mizuno
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D015153:Blotting, Western; D012831:Bombyx / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005349:Fibroins / Q000378:metabolism* / Q000502:physiology*; D006023:Glycoproteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology; D019476:Insect Proteins / Q000378:metabolism* / Q000502:physiology*; D007814:Larva / Q000235:genetics / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D008361:Mannosidases / Q000378:metabolism*; D011679:Pupa / Q000235:genetics / Q000378:metabolism; D047011:Silk; D014170:Transformation, Genetic; D019076:Transgenes
chemical_list: D006023:Glycoproteins; D019476:Insect Proteins; C432480:L-chain, fibroin protein, insect; D047011:Silk; C418613:fhx protein, Bombyx mori; D005349:Fibroins; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase; D008358:Mannose