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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

Electrophoretic mobility and glycosylated status of intracellular and extracellular wild-type myocilins. HTM cells were plated into a 24 well plate, grown to 70% confluence, and transduced for 2 h with Ad-myocilin-FLAG at an MOI of 10 pfu. After 48 h, the cells and culture media were harvested and the myocilin in the samples were analyzed by western blot using polyclonal anti-myocilin antibody. A: Cells and culture media were harvested together (lane 1), or cells and culture media were separately harvested (lane 2 and 3), or cells were replenished after culture with fresh culture media and then cells and culture media were harvested together (lane 4), or cells were cultured with serum-free culture media and then the conditioned media was harvested (lane 5). Cells were harvested with 0.4 ml of 1X Laemmli sample buffer whereas culture media (0.3 ml) was harvested with 0.1 ml of 4X Laemmli sample buffer. B: Myocilin in cells (lane 2 and 3) or media (lane 5 and 6) was treated with Endo H (lane 2 and 5) or PNGase F glycosidase (lane 3 and 6) as described in Methods. Undigested myocilins in cells (lane 1) and culture media (lane 4) served as controls. C: Myocilin in cells was either treated (lane 1) or not treated (lane 2) with Endo D glycosidase.
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f1: Electrophoretic mobility and glycosylated status of intracellular and extracellular wild-type myocilins. HTM cells were plated into a 24 well plate, grown to 70% confluence, and transduced for 2 h with Ad-myocilin-FLAG at an MOI of 10 pfu. After 48 h, the cells and culture media were harvested and the myocilin in the samples were analyzed by western blot using polyclonal anti-myocilin antibody. A: Cells and culture media were harvested together (lane 1), or cells and culture media were separately harvested (lane 2 and 3), or cells were replenished after culture with fresh culture media and then cells and culture media were harvested together (lane 4), or cells were cultured with serum-free culture media and then the conditioned media was harvested (lane 5). Cells were harvested with 0.4 ml of 1X Laemmli sample buffer whereas culture media (0.3 ml) was harvested with 0.1 ml of 4X Laemmli sample buffer. B: Myocilin in cells (lane 2 and 3) or media (lane 5 and 6) was treated with Endo H (lane 2 and 5) or PNGase F glycosidase (lane 3 and 6) as described in Methods. Undigested myocilins in cells (lane 1) and culture media (lane 4) served as controls. C: Myocilin in cells was either treated (lane 1) or not treated (lane 2) with Endo D glycosidase.

Mentions: It has been reported that in the standard conditions of SDS–PAGE, the mobility of secreted myocilin is slightly increased compared to intracellular myocilin, although both myocilins are resolved as a doublet at a molecular weight of approximately 55−57 kDa (compare lane 2 with lane 3 in Figure 1A) [15,24]. The different mobility of the intracellular myocilin compared to the extracellular myocilin implies different molecular weights, which can be accepted as evidence for the presence of myocilin isoforms. However, the mobility of the intracellular myocilin also increased when cell lysates were added with culture media containing FBS (lane 4 in Figure 1A). By contrast, the mobility of the secreted myocilin decreased when cells were cultured without FBS (lane 5 in Figure 1A) or only increased mobility was observed when the cell lysate and medium were combined (lane 1 in Figure 1A). Therefore, the fast mobility of the secreted myocilin must have been an artifact.


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)

Electrophoretic mobility and glycosylated status of intracellular and extracellular wild-type myocilins. HTM cells were plated into a 24 well plate, grown to 70% confluence, and transduced for 2 h with Ad-myocilin-FLAG at an MOI of 10 pfu. After 48 h, the cells and culture media were harvested and the myocilin in the samples were analyzed by western blot using polyclonal anti-myocilin antibody. A: Cells and culture media were harvested together (lane 1), or cells and culture media were separately harvested (lane 2 and 3), or cells were replenished after culture with fresh culture media and then cells and culture media were harvested together (lane 4), or cells were cultured with serum-free culture media and then the conditioned media was harvested (lane 5). Cells were harvested with 0.4 ml of 1X Laemmli sample buffer whereas culture media (0.3 ml) was harvested with 0.1 ml of 4X Laemmli sample buffer. B: Myocilin in cells (lane 2 and 3) or media (lane 5 and 6) was treated with Endo H (lane 2 and 5) or PNGase F glycosidase (lane 3 and 6) as described in Methods. Undigested myocilins in cells (lane 1) and culture media (lane 4) served as controls. C: Myocilin in cells was either treated (lane 1) or not treated (lane 2) with Endo D glycosidase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Electrophoretic mobility and glycosylated status of intracellular and extracellular wild-type myocilins. HTM cells were plated into a 24 well plate, grown to 70% confluence, and transduced for 2 h with Ad-myocilin-FLAG at an MOI of 10 pfu. After 48 h, the cells and culture media were harvested and the myocilin in the samples were analyzed by western blot using polyclonal anti-myocilin antibody. A: Cells and culture media were harvested together (lane 1), or cells and culture media were separately harvested (lane 2 and 3), or cells were replenished after culture with fresh culture media and then cells and culture media were harvested together (lane 4), or cells were cultured with serum-free culture media and then the conditioned media was harvested (lane 5). Cells were harvested with 0.4 ml of 1X Laemmli sample buffer whereas culture media (0.3 ml) was harvested with 0.1 ml of 4X Laemmli sample buffer. B: Myocilin in cells (lane 2 and 3) or media (lane 5 and 6) was treated with Endo H (lane 2 and 5) or PNGase F glycosidase (lane 3 and 6) as described in Methods. Undigested myocilins in cells (lane 1) and culture media (lane 4) served as controls. C: Myocilin in cells was either treated (lane 1) or not treated (lane 2) with Endo D glycosidase.
Mentions: It has been reported that in the standard conditions of SDS–PAGE, the mobility of secreted myocilin is slightly increased compared to intracellular myocilin, although both myocilins are resolved as a doublet at a molecular weight of approximately 55−57 kDa (compare lane 2 with lane 3 in Figure 1A) [15,24]. The different mobility of the intracellular myocilin compared to the extracellular myocilin implies different molecular weights, which can be accepted as evidence for the presence of myocilin isoforms. However, the mobility of the intracellular myocilin also increased when cell lysates were added with culture media containing FBS (lane 4 in Figure 1A). By contrast, the mobility of the secreted myocilin decreased when cells were cultured without FBS (lane 5 in Figure 1A) or only increased mobility was observed when the cell lysate and medium were combined (lane 1 in Figure 1A). Therefore, the fast mobility of the secreted myocilin must have been an artifact.

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