Non-lysosomal degradation of misfolded human lysozymes with and without an asparagine-linked glycosylation site.
Human lysozyme is a monomeric secretory protein composed of 130 amino acid residues, with four intramolecular disulfide bonds and no oligosaccharides. In this study, a mutant protein, [Ala128] lysozyme, which cannot fold because it lacks a disulfide bond, Cys6-Cys128, was expressed in mouse fibroblasts and was found to be mostly degraded in the cells, whereas the control wild-type lysozyme was quantitatively secreted into the media. The degradation of [Ala128]lysozyme was independent of the transport from the endoplasmic reticulum to the Golgi apparatus. The degradation was greatly inhibited by incubation of cells at 15 degrees C, but was minimally affected by treatment of cells with the lysosomotropic agent, chloroquine, implying a non-lysosomal process. Additional mutations (Gly48-->Ser or Met29-->Thr) were created to make asparagine-linked (N-linked) glycosylation site in the [Ala128]lysozyme, and the resultant double mutants, [Ser48, Ala128]lysozyme and [Thr29, Ala128]lysozyme, were analyzed with respect to their intracellular degradation. These mutant proteins were susceptible to N-linked glycosylation, and were degraded in a similar manner to that of [Ala128] lysozyme, except that the onset of degradation of [Ser48, Ala128]lysozyme and [Thr29, Ala128] lysozyme, but not of [Ala128]lysozyme, was preceded by a lag period of up to 60 min. Furthermore, the degradative double mutants, [Ser48, Ala128]lysozyme and [Thr29, Ala128]lysozyme, were glycosylated post-translationally as well as co-translationally. These observations suggest that there is some interaction between the mechanisms of glycosylation and degradation.
Omura
F
F
Protein Engineering Research Institute, Osaka, Japan.
Otsu
M
M
Yoshimori
T
T
Tashiro
Y
Y
Kikuchi
M
M
D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D020126:Brefeldin A; D002236:Carbohydrate Conformation; D003517:Cyclopentanes / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D014162:Transfection; D014709:Vero Cells
D003517:Cyclopentanes; D004220:Disulfides; D020126:Brefeldin A; D001216:Asparagine; D009113:Muramidase