title: Cloning of a cDNA encoding the Gly m Bd 28K precursor and its vacuole transport in tobacco BY2 suspension-cultured cells.
abstract: Gly m Bd 28K (Gm28K), a soybean allergen, is formed as a preproprotein consisting of a predicted signal peptide, Gm28K, and the 23-kDa peptide (Gm23K). Gm28K and Gm23K are found in the protein-storage vacuoles (PSVs) of developing soybean seeds. However, the complete structure of Gm28K has not yet been identified and its processing and transport to the vacuoles has never been clarified. In the present study, we elucidated the 5'-nucleotide sequence of cDNA encoding the Gm28K precursor and identified a putative signal peptide (SP) with 24 N-terminal amino acid residues. We expressed peptides from the Gm28K precursor as fusion proteins with enhanced green fluorescent protein (EGFP) in tobacco BY2 suspension-cultured cells. BY2 cells transformed by an expression vector for SP-EGFP-Gm28-Gm23K (SP-EGFP-Gm28-Gm23K/BY2 cells) and SP-Gm28-Gm23K-EGFP/BY2 cells produced the EGFP fused-Gm28K precursor, and the EGFP-fluorescence in their vacuoles were recorded. In the experiments with SP-EGFP/BY2 and SP-EGFP-Gm28K/BY2 cells, large amounts of the EGFP segments were secreted into the medium. On the other hand, the fluorescence of EGFP in SP-EGFP-Gm23K/BY2 cells was shown to accumulate only in the endoplasmic reticulum without secretion into the medium. These findings show that the SP signals the precursor to enter the lumen of the endoplasmic reticulum and that both the Gm28K and Gm23K components may be involved in the transport from the endoplasmic reticulum (ER) lumen via the Golgi to the vacuoles in a proprotein form.
authors: International Center for Biotechnology, Osaka University.
authors: Hitomi
authors: H
authors: Yumioka-Ito
authors: Ryo
authors: R
authors: Misaki
authors: Miyuki
authors: M
authors: Yokoro
authors: Makiko
authors: M
authors: Suzuki
authors: Hiromi
authors: H
authors: Yamashita
authors: Miki
authors: M
authors: Hiemori-Kondo
authors: Masumi
authors: M
authors: Kimoto
authors: Ko
authors: K
authors: Kato
authors: Kazuhito
authors: K
authors: Fujiyama
authors: Hideaki
authors: H
authors: Tsuji
mesh_terms: D000485:Allergens / Q000235:genetics*; D000595:Amino Acid Sequence; D052179:Antigens, Plant / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005512:Food Hypersensitivity / Q000235:genetics*; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics*; D012639:Seeds / Q000378:metabolism*; D015398:Signal Transduction; D030262:Soybean Proteins / Q000235:genetics* / Q000378:metabolism; D013025:Glycine max / Q000235:genetics* / Q000378:metabolism; D014026:Nicotiana / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism*
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D018076:DNA, Complementary; C107967:Gly m Bd 28K allergen, Glycine max; D006023:Glycoproteins; D021382:Protein Sorting Signals; D030262:Soybean Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins