Bicarbonate-dependent secretion and proteolytic processing of recombinant myocilin.
Myocilin is an extracellular glycoprotein of poorly understood function. Mutations of this protein are involved in glaucoma, an optic neuropathy characterized by a progressive and irreversible visual loss and frequently associated with elevated intraocular pressure. We previously showed that recombinant myocilin undergoes an intracellular proteolytic processing by calpain II which cleaves the central region of the protein, releasing one N- and one C-terminal fragment. Myocilin cleavage is reduced by glaucoma mutations and it has been proposed to participate in intraocular pressure modulation. To identify possible factors regulating the proteolytic processing of recombinant myocilin, we used a cellular model in which we analyzed how different culture medium parameters (i.e., culture time, cell density, pH, bicarbonate concentration, etc.) affect the presence of the extracellular C-terminal fragment. Extracellular bicarbonate depletion associated with culture medium acidification produced a reversible intracellular accumulation of full-length recombinant myocilin and incremented its intracellular proteolytic processing, raising the extracellular C-terminal fragment percentage. It was also determined that myocilin intracellular accumulation depends on its N-terminal region. These data suggest that aqueous humor bicarbonate variations could also modulate the secretion and cleavage of myocilin present in ocular tissues.
Laboratorio de Genética Molecular Humana, Facultad de Medicina/Instituto de Investigación en Discapacidades Neurológicas-IDINE, Universidad de Castilla-La Mancha, Albacete, Spain.
José-Daniel
JD
Aroca-Aguilar
Francisco
F
Martínez-Redondo
Alba
A
Martín-Gil
Jesús
J
Pintor
Miguel
M
Coca-Prados
Julio
J
Escribano
D001082:Aqueous Humor / Q000378:metabolism; D001639:Bicarbonates / Q000494:pharmacology*; D015153:Blotting, Western; D002460:Cell Line; D003470:Culture Media / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D005136:Eye Proteins / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D059748:Proteolysis / Q000187:drug effects; D000099179:Myocilin
D001639:Bicarbonates; D003470:Culture Media; D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D000099179:Myocilin