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Abstract #25167 Published in IGR 12-1
Functional role of proteolytic processing of recombinant myocilin in self-aggregation
Aroca-Aguilar JD; Martínez-Redondo F; Sánchez-Sánchez F; Coca-Prados M; Escribano JInvestigative Ophthalmology and Visual Science 2010; 51: 72-78
PURPOSE: Recombinant myocilin expressed in cells in culture is endoproteolytically cleaved in the endoplasmic reticulum by calpain II, releasing an N-terminal and a C-terminal fragment. This proteolytic processing has been speculated to regulate the molecular interactions of myocilin. The main purpose of this study was to analyze the effect of the proteolytic cleavage on myocilin aggregation. METHODS: cDNAs encoding human myocilin and the N- and C-terminal fragments were transiently expressed in HEK-293T cells. Covalent interactions of recombinant myocilin were analyzed by SDS-PAGE and Western immunoblot analysis in different dissociating conditions. Noncovalent interactions were studied by solid-phase binding assays, performed with Ni-chelating HPLC-purified recombinant proteins, and by Far-Western blot analysis. RESULTS: Western blot analysis of recombinant myocilin aggregates under either increasing ionic strength or increasing concentration of reducing agent indicated that ionic interactions do not contribute to the stability of the molecular complexes linked by disulfide bridges. Disulfide myocilin homoaggregates decreased as the proteolytic processing increased. Solid-phase binding assays showed the existence of high-affinity (K(d) = 0.068 microM) noncovalent myocilin-myocilin interactions and that processed fragments bound to the full-length protein with significantly reduced affinity. Far-Western blot analysis confirmed noncovalent interactions between recombinant myocilin disulfide aggregates. CONCLUSIONS: The proteolytic processing of recombinant myocilin decreases myocilin homoaggregates. These data provide the first evidence of a functional role for this processing in myocilin aggregation and suggest that disulfide complexes of myocilin could organize into a dynamic extracellular network sustained by noncovalent N-terminal interactions.
Laboratorio de Genética Molecular Humana, Facultad de Medicina/Centro Regional de Investigaciones Biomédicas, Universidad de Castilla-La Mancha, Albacete, Spain.
Classification:
3.6 Cellular biology (Part of: 3 Laboratory methods)
3.5 Molecular biology incl. SiRNA (Part of: 3 Laboratory methods)
