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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

Two dimensional analysis of myocilin expression. Myocilin isoforms in cell lysate (upper panel) and culture medium (lower panel) from HTM cells transduced with Ad-myocilin-FLAG were first separated by their isoelectric points and then resolved according to their molecular weights. Myocilin detection was performed by western blot analysis using anti-FLAG antibody. This experiment was performed two times, and representative gels are shown.
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f3: Two dimensional analysis of myocilin expression. Myocilin isoforms in cell lysate (upper panel) and culture medium (lower panel) from HTM cells transduced with Ad-myocilin-FLAG were first separated by their isoelectric points and then resolved according to their molecular weights. Myocilin detection was performed by western blot analysis using anti-FLAG antibody. This experiment was performed two times, and representative gels are shown.

Mentions: Previously, two dimensional (2D) PAGE analysis of myocilin expression showed that multiple isoforms could be detected in cell lysates of human optic nerve head cells or in the media of human lamina cells as well as in cell lysates and media of dexamethasone-treated HTM cells [22,23,33]. Because 2D-PAGE is the only method used to describe myocilin isoforms in the literature, we also used it to identify myocilin isoforms that are specific to the intracellular or extracellular compartments. To precisely compare the myocilin isoforms in the cell lysate with those in the culture media, we simultaneously performed all experimental procedures including the sample preparations and the electrophoresis. In agreement with previous results, western blot analysis following 2D-PAGE of cell lysates showed five to six different myocilin isoforms ranging in pI from 5.0 to 5.4 and molecular weight from 55 to 57 kDa (Figure 3, upper panel) whereas the myocilin in concentrated media was separated as multiple isoforms with pI ranging from 4.9 to 5.3 and molecular weight from 55 to 57 kDa (Figure 3, lower panel). It is important to point out that a significant amount of the myocilin isoforms with same mass and pI were found in both the cell lysate and medium, although it is evident that about half of the myocilin isoforms in the culture medium were more acidic than those in the cell lysate. In addition to the preceding data, these results consistently showed that there were no myocilin isoforms found only in the ER or extracellular compartment.


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)

Two dimensional analysis of myocilin expression. Myocilin isoforms in cell lysate (upper panel) and culture medium (lower panel) from HTM cells transduced with Ad-myocilin-FLAG were first separated by their isoelectric points and then resolved according to their molecular weights. Myocilin detection was performed by western blot analysis using anti-FLAG antibody. This experiment was performed two times, and representative gels are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Two dimensional analysis of myocilin expression. Myocilin isoforms in cell lysate (upper panel) and culture medium (lower panel) from HTM cells transduced with Ad-myocilin-FLAG were first separated by their isoelectric points and then resolved according to their molecular weights. Myocilin detection was performed by western blot analysis using anti-FLAG antibody. This experiment was performed two times, and representative gels are shown.
Mentions: Previously, two dimensional (2D) PAGE analysis of myocilin expression showed that multiple isoforms could be detected in cell lysates of human optic nerve head cells or in the media of human lamina cells as well as in cell lysates and media of dexamethasone-treated HTM cells [22,23,33]. Because 2D-PAGE is the only method used to describe myocilin isoforms in the literature, we also used it to identify myocilin isoforms that are specific to the intracellular or extracellular compartments. To precisely compare the myocilin isoforms in the cell lysate with those in the culture media, we simultaneously performed all experimental procedures including the sample preparations and the electrophoresis. In agreement with previous results, western blot analysis following 2D-PAGE of cell lysates showed five to six different myocilin isoforms ranging in pI from 5.0 to 5.4 and molecular weight from 55 to 57 kDa (Figure 3, upper panel) whereas the myocilin in concentrated media was separated as multiple isoforms with pI ranging from 4.9 to 5.3 and molecular weight from 55 to 57 kDa (Figure 3, lower panel). It is important to point out that a significant amount of the myocilin isoforms with same mass and pI were found in both the cell lysate and medium, although it is evident that about half of the myocilin isoforms in the culture medium were more acidic than those in the cell lysate. In addition to the preceding data, these results consistently showed that there were no myocilin isoforms found only in the ER or extracellular compartment.

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