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Dual localization of wild-type myocilin in the endoplasmic reticulum and extracellular compartment likely occurs due to its incomplete secretion.

Sohn S, Joe MK, Kim TE, Im JE, Choi YR, Park H, Kee C - Mol. Vis. (2009)

Bottom Line: The expression pattern and cytopathic effect of myocilin were analyzed in individual cells by immunocytochemistry.The intracellular protein was found to be present in the lumen of the ER where it appeared to be retained without further export to the Golgi apparatus.Based on these findings and the fact that myocilin has no known ER retention signals, the ER localization of wild-type myocilin is likely a consequence of its incomplete secretion due to its misfolding.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea.

ABSTRACT

Purpose: Wild-type myocilin is known to be secreted extracellularly, but a significant amount of the protein is also present in the endoplasmic reticulum (ER). The present study was undertaken to address whether intracellular myocilin is a true ER resident protein.

Methods: Human wild-type myocilin was adenovirally expressed in human trabecular meshwork cells, and general characteristics of both intracellular and extracellular myocilins including molecular weight, pI, glycosylation state, and cleavage site of the signal peptide were examined by biochemical analyses. Topology and decay kinetics of myocilin were also examined by protease protection assay and pulse chase analysis, respectively. The expression pattern and cytopathic effect of myocilin were analyzed in individual cells by immunocytochemistry.

Results: Intracellular myocilin were very similar to secreted myocilin in characteristics such as molecular weight, pI, glycosylation state, and cleavage site of the signal peptide. The intracellular protein was found to be present in the lumen of the ER where it appeared to be retained without further export to the Golgi apparatus. The kinetics of myocilin turnover clearly showed that it was intrinsically a very stable but incompletely secreted protein. The expression of myocilin was confined to a subset of cells and accompanied by the upregulation of a 78 kDa glucose-regulated protein, suggesting that it was not properly folded or processed in the ER.

Conclusions: Based on these findings and the fact that myocilin has no known ER retention signals, the ER localization of wild-type myocilin is likely a consequence of its incomplete secretion due to its misfolding.

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Related in: MedlinePlus

Evaluation of the potential cytotoxicity of myocilin expression. A: HTM cells were transduced with Ad-myocilin-GFP (WT), Ad-myocilin-FLAG (FLAG), Ad-Y437H myocilin-GFP (437), or Ad-stromelysin-GFP (Str) and stained for GRP78. FITC-conjugated anti-FLAG antibody and DAPI was used to stain myocilin-FLAG and nuclei, respectively. Each column represents the same cells. The staining patterns of each myocilin (green) and GRP78 (red) merged with DAPI stains are shown in the upper and lower rows, respectively. Representative images are shown of three independent experiments. B: HTM cells were transduced with Ad-GFP (GFP), Ad-stromelysin-GFP (Str), Ad-myocilin-GFP (WT), or Ad-Y437H myocilin-GFP (437) and cultured for 48 h (upper row) or 96 h (lower row). After fixing and staining with DAPI, the green fluorescence of GFP or GFP fusion proteins were merged with DAPI stains. Representative images are shown of three independent experiments. Bar: 50 μm. The graph is plotted values showing the ratio of GFP-expressing cells to nontransduced cells. Data represents means±standard deviation from 200 cells.
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f7: Evaluation of the potential cytotoxicity of myocilin expression. A: HTM cells were transduced with Ad-myocilin-GFP (WT), Ad-myocilin-FLAG (FLAG), Ad-Y437H myocilin-GFP (437), or Ad-stromelysin-GFP (Str) and stained for GRP78. FITC-conjugated anti-FLAG antibody and DAPI was used to stain myocilin-FLAG and nuclei, respectively. Each column represents the same cells. The staining patterns of each myocilin (green) and GRP78 (red) merged with DAPI stains are shown in the upper and lower rows, respectively. Representative images are shown of three independent experiments. B: HTM cells were transduced with Ad-GFP (GFP), Ad-stromelysin-GFP (Str), Ad-myocilin-GFP (WT), or Ad-Y437H myocilin-GFP (437) and cultured for 48 h (upper row) or 96 h (lower row). After fixing and staining with DAPI, the green fluorescence of GFP or GFP fusion proteins were merged with DAPI stains. Representative images are shown of three independent experiments. Bar: 50 μm. The graph is plotted values showing the ratio of GFP-expressing cells to nontransduced cells. Data represents means±standard deviation from 200 cells.

Mentions: In the present study, therefore, we reexamined using the immunocytochemistry myocilin-induced ER stress response. The results showed that the UPR could be activated with wild-type myocilin expression as demonstrated by the upregulation of GRP78 in the cells expressing myocilin-GFP but not in cells without myocilin-GFP expression. The same pattern of GRP78 induction was observed in the cultures expressing myocilin-FLAG as well as the myocilin mutant such as myocilin with the Y437H mutation (Figure 7A). By contrast, the induction was not observed in cells expressing GFP-tagged stromelysin, demonstrating that the observations were not an artifact of the GFP tagging, protein overexpression, or the viral infection.


Dual localization of wild-type myocilin in the endoplasmic reticulum and extracellular compartment likely occurs due to its incomplete secretion.

Sohn S, Joe MK, Kim TE, Im JE, Choi YR, Park H, Kee C - Mol. Vis. (2009)

Evaluation of the potential cytotoxicity of myocilin expression. A: HTM cells were transduced with Ad-myocilin-GFP (WT), Ad-myocilin-FLAG (FLAG), Ad-Y437H myocilin-GFP (437), or Ad-stromelysin-GFP (Str) and stained for GRP78. FITC-conjugated anti-FLAG antibody and DAPI was used to stain myocilin-FLAG and nuclei, respectively. Each column represents the same cells. The staining patterns of each myocilin (green) and GRP78 (red) merged with DAPI stains are shown in the upper and lower rows, respectively. Representative images are shown of three independent experiments. B: HTM cells were transduced with Ad-GFP (GFP), Ad-stromelysin-GFP (Str), Ad-myocilin-GFP (WT), or Ad-Y437H myocilin-GFP (437) and cultured for 48 h (upper row) or 96 h (lower row). After fixing and staining with DAPI, the green fluorescence of GFP or GFP fusion proteins were merged with DAPI stains. Representative images are shown of three independent experiments. Bar: 50 μm. The graph is plotted values showing the ratio of GFP-expressing cells to nontransduced cells. Data represents means±standard deviation from 200 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654785&req=5

f7: Evaluation of the potential cytotoxicity of myocilin expression. A: HTM cells were transduced with Ad-myocilin-GFP (WT), Ad-myocilin-FLAG (FLAG), Ad-Y437H myocilin-GFP (437), or Ad-stromelysin-GFP (Str) and stained for GRP78. FITC-conjugated anti-FLAG antibody and DAPI was used to stain myocilin-FLAG and nuclei, respectively. Each column represents the same cells. The staining patterns of each myocilin (green) and GRP78 (red) merged with DAPI stains are shown in the upper and lower rows, respectively. Representative images are shown of three independent experiments. B: HTM cells were transduced with Ad-GFP (GFP), Ad-stromelysin-GFP (Str), Ad-myocilin-GFP (WT), or Ad-Y437H myocilin-GFP (437) and cultured for 48 h (upper row) or 96 h (lower row). After fixing and staining with DAPI, the green fluorescence of GFP or GFP fusion proteins were merged with DAPI stains. Representative images are shown of three independent experiments. Bar: 50 μm. The graph is plotted values showing the ratio of GFP-expressing cells to nontransduced cells. Data represents means±standard deviation from 200 cells.
Mentions: In the present study, therefore, we reexamined using the immunocytochemistry myocilin-induced ER stress response. The results showed that the UPR could be activated with wild-type myocilin expression as demonstrated by the upregulation of GRP78 in the cells expressing myocilin-GFP but not in cells without myocilin-GFP expression. The same pattern of GRP78 induction was observed in the cultures expressing myocilin-FLAG as well as the myocilin mutant such as myocilin with the Y437H mutation (Figure 7A). By contrast, the induction was not observed in cells expressing GFP-tagged stromelysin, demonstrating that the observations were not an artifact of the GFP tagging, protein overexpression, or the viral infection.

Bottom Line: The expression pattern and cytopathic effect of myocilin were analyzed in individual cells by immunocytochemistry.The intracellular protein was found to be present in the lumen of the ER where it appeared to be retained without further export to the Golgi apparatus.Based on these findings and the fact that myocilin has no known ER retention signals, the ER localization of wild-type myocilin is likely a consequence of its incomplete secretion due to its misfolding.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea.

ABSTRACT

Purpose: Wild-type myocilin is known to be secreted extracellularly, but a significant amount of the protein is also present in the endoplasmic reticulum (ER). The present study was undertaken to address whether intracellular myocilin is a true ER resident protein.

Methods: Human wild-type myocilin was adenovirally expressed in human trabecular meshwork cells, and general characteristics of both intracellular and extracellular myocilins including molecular weight, pI, glycosylation state, and cleavage site of the signal peptide were examined by biochemical analyses. Topology and decay kinetics of myocilin were also examined by protease protection assay and pulse chase analysis, respectively. The expression pattern and cytopathic effect of myocilin were analyzed in individual cells by immunocytochemistry.

Results: Intracellular myocilin were very similar to secreted myocilin in characteristics such as molecular weight, pI, glycosylation state, and cleavage site of the signal peptide. The intracellular protein was found to be present in the lumen of the ER where it appeared to be retained without further export to the Golgi apparatus. The kinetics of myocilin turnover clearly showed that it was intrinsically a very stable but incompletely secreted protein. The expression of myocilin was confined to a subset of cells and accompanied by the upregulation of a 78 kDa glucose-regulated protein, suggesting that it was not properly folded or processed in the ER.

Conclusions: Based on these findings and the fact that myocilin has no known ER retention signals, the ER localization of wild-type myocilin is likely a consequence of its incomplete secretion due to its misfolding.

Show MeSH
Related in: MedlinePlus