Mutation of Fibulin-3 (efemp-1) Alters the Protein Content of ARPE-19 Exosomes

Abstract

Purpose : Although drusen have been characterized extensively on a morphological basis, the molecular pathways involved in drusen formation remain largely unknown. A single point mutation, R345W, in the extracellular matrix protein fibulin-3 (efemp-1) results in an inherited macular dystrophy (Malattia Leventinese). The hypothesis of the current study is that Fib3^R345W promotes drusen formation by altering the content of vesicles secreted by RPE cells. To identify drusen-promoting proteins and pathways, ARPE-19 cells overexpressing Fib3-wt or Fib3-R345W will be purified and subjected to proteomic analysis.

Methods : Exosomes were isolated from human ARPE-19 cell culture media by sucrose cushion and OptiPrep gradient density ultracentrifugation. Exosome enrichment was verified by Western blotting, particle size and concentration were measured by NanoSight Tracking Analysis and vesicle morphology was visualized by transmission electron microscopy. Samples were separated on SDS-PAGE gels, cut into forty fragments and digested with trypsin. The resulting peptides were subjected to LC-MS/MS using a ThermoFisher QExactive mass spectrometer.

Results : Western blot analysis of purified exosomal fractions were positive for classic exomes markers (CD63, CD81, HSP70) as well as Fib3. TEM analysis revealed concave appearing vesicles measuring ~100nm in diameter. NTA analysis revealed that the vast majority of vesicles were 137nm in diameter with a concentration of 140 million particles per milliliter. A total of 2,906 proteins were identified in Fib3-WT exosomes and 2,899 were identified in Fib3-R345W exosomes. As expected prototypical exosome proteins, such as CD63, CD81, rabs and annexins, were abundant in both samples but Fib3 was only identified in exosomes purified from cells overexpressing Fib3-R345W. The top 20 cellular signaling pathways (Ingenuity Pathway Analysis) in both samples include EIF2, Axon Guidance, Ephrin signaling and Clathrin-Mediated Endocytosis pathways.

Conclusions : ARPE-19 cells secrete vesicles with a diameter, morphology and protein content typical of exosomes. Fib3-R345W, but not Fib3-WT, was identified in the exosomal fraction. Additional biological replicates and bioinformatic analysis will required to identify putative drusen-promoting proteins through differential expression analysis.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.