title: Lysozyme Mutants Accumulate in Cells while Associated at their N-terminal Alpha-domain with the Endoplasmic Reticulum Chaperone GRP78/BiP.
abstract: Amyloidogenic human lysozyme variants deposit in cells and cause systemic amyloidosis. We recently observed that such lysozymes accumulate in the endoplasmic reticulum (ER) with the ER chaperone GRP78/BiP, accompanying the ER stress response. Here we investigated the region of lysozyme that is critical to its association with GRP78/BiP. In addition to the above-mentioned variants of lysozyme, we constructed lysozyme truncation or substitution mutants. These were co-expressed with GRP78/BiP (tagged with FLAG) in cultured human embryonic kidney cells, which were analyzed by western blotting and immunocytochemistry using anti-lysozyme and anti-FLAG antibodies. The amyloidogenic variants were confirmed to be strongly associated with GRP78/BiP as revealed by the co-immunoprecipitation assay, whereas N-terminal mutants pruned of 1-41 or 1-51 residues were found not to be associated with the chaperone. Single amino acid substitutions for the leucine array along the α-helices in the N-terminal region resulted in wild-type lysozyme remaining attached to GRP78/BiP. These mutations also tended to show lowered secretion ability. We conclude that the N-terminal α-helices region of the lysozyme is pivotal for its strong adhesion to GRP78/BiP. We suspect that wild-type lysozyme interacts with the GRP at this region as a step in the proper folding monitored by the ER chaperone.
authors.affiliation: Laboratory of Biochemistry and Bioscience, The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065, Japan.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Kamada
authors.affiliation: Laboratory of Biochemistry and Bioscience, The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Nawata
authors.affiliation: Laboratory of Biochemistry and Bioscience, The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Sugimoto
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D057809:HEK293 Cells; D006360:Heat-Shock Proteins / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D009113:Muramidase / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D000072417:Protein Domains; D017510:Protein Folding; D017384:Sequence Deletion; D013312:Stress, Physiological
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D009113:Muramidase
keywords: GRP78/BiP; aggregate; amyloidosis; endoplasmic reticulum stress; lysozyme; variants