Heterologous expression of wildtype and mutant myocilin in High Five insect cells shows comparable effects to cultivated trabecular meshwork cells.
Myocilin (MYOC, TIGR) variations are associated with juvenile and primary open angle glaucoma (POAG). To investigate consequences of MYOC wildtype overexpression and selected mutations, we established a heterologous insect cell system (High Five). Wildtype, Pro370Leu, Gln368X and Lys423Glu were cloned into a modified pIB/V5-His (pEXIV) vector with and without downstream GFP in frame fusion. Mutations were introduced by in vitro mutagenesis. Heterologous expression was shown and analysed by RT-PCR, Western blotting, immunocytochemistry and fluorescence microscopy. Extended cultivation (>14 days) resulted in accumulation of MYOC protein for all variants in growing dilated cisterns of the rough endoplasmic reticulum. Finally cell death for overexpressed wildtype and mutants occurs. A direct attachment of ribosomes to these growing vesicles preceding the cell death was observed by electron microscopy. Our observations indicate that this system is suitable to trace the intracellular effects of MYOC mutants.
Oezbey
Sevinc
S
University Hospital Erlangen, Institute of Human Genetics, Schwabachanlage 10, 91054, Erlangen, Germany.
Stengel
Corinna
C
Schlötzer-Schrehardt
Ursula
U
Ekici
Arif
A
Rautenstrauss
Bernd
B
D000818:Animals; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000235:genetics* / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism*; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007915:Lepidoptera / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D014129:Trabecular Meshwork / Q000166:cytology* / Q000378:metabolism*; D000099179:Myocilin
D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D011994:Recombinant Proteins; D000099179:Myocilin