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Functional role of proteolytic processing of recombinant myocilin in self-aggregation

José-Daniel Aroca-Aguilar1, Francisco Martínez-Redondo, Francisco Sánchez-Sánchez

  • 1Laboratorio de Genética Molecular Humana, Facultad de Medicina/Centro Regional de Investigaciones Biomédicas, Universidad de Castilla-La Mancha, Albacete, Spain.

Investigative Ophthalmology & Visual Science
|August 22, 2009
PubMed
Abstract

Insights

Proteolytic cleavage of myocilin reduces its aggregation by affecting disulfide bonds. This processing influences myocilin

Area of Science:

  • Molecular biology
  • Protein biochemistry

Background:

  • Myocilin is endoproteolytically cleaved in the endoplasmic reticulum by calpain II.
  • This processing generates N-terminal and C-terminal fragments.
  • Proteolytic processing is speculated to regulate myocilin's molecular interactions.

Purpose of the Study:

  • To analyze the effect of proteolytic cleavage on myocilin aggregation.
  • Investigate how processing impacts myocilin's molecular interactions.

Main Methods:

  • Transient expression of human myocilin and fragments in HEK-293T cells.
  • Analysis of covalent interactions using SDS-PAGE and Western immunoblot.
  • Study of noncovalent interactions via solid-phase binding assays and Far-Western blot.

Main Results:

  • Disulfide myocilin homoaggregates decreased with increased proteolytic processing.
  • High-affinity noncovalent myocilin-myocilin interactions were identified (K(d) = 0.068 microM).
  • Processed fragments exhibited reduced affinity for full-length myocilin.

Conclusions:

  • Proteolytic processing of myocilin reduces its homoaggregates.
  • Provides evidence for a functional role of processing in myocilin aggregation.
  • Suggests disulfide complexes form an extracellular network sustained by noncovalent N-terminal interactions.

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