N-Terminomics identifies HtrA1 cleavage of thrombospondin-1 with generation of a proangiogenic fragment in the polarized retinal pigment epithelial cell model of age-related macular degeneration

Chia-Yi Chen, Esther Melo, Peter Jakob, Arno Friedlein, Brigitta Elsässer, Peter Goettig, Verena Kueppers, Frederic Delobel, Corinne Stucki, Tom Dunkley, Sascha Fauser, Oliver Schilling, Roberto Iacone

Research output: Contribution to journalArticle

Abstract

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population. Variants in the HTRA1-ARMS2 locus have been linked to increased AMD risk. In the present study we investigated the impact of elevated HtrA1 levels on the retina pigment epithelial (RPE) secretome using a polarized culture system. Upregulation of HtrA1 alters the abundance of key proteins involved in angiogenesis and extracellular matrix remodeling. Thrombospondin-1, an angiogenesis modulator, was identified as a substrate for HtrA1 using terminal amine isotope labeling of substrates in conjunction with HtrA1 specificity profiling. HtrA1 cleavage of thrombospondin-1 was further corroborated by in vitro cleavage assays and targeted proteomics together with small molecule inhibition of HtrA1. While thrombospondin-1 is anti-angiogenic, the proteolytically released N-terminal fragment promotes the formation of tube-like structure by endothelial cells. Taken together, our findings suggest a mechanism by which increased levels of HtrA1 may contribute to AMD pathogenesis. The proteomic data has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier. For quantitative secretome analysis, project accession: PXD007691, username: reviewer45093@ebi.ac.uk, password: 1FUpS6Yq. For TAILS analysis, project accession: PXD007139, username: reviewer76731@ebi.ac.uk, password: sNbMp7xK.

Original languageEnglish
Pages (from-to)84-101
Number of pages18
JournalMatrix Biology
Volume70
DOIs
Publication statusPublished - Sep 2018

Bibliographical note

Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Fields of Science and Technology Classification 2012

  • 106 Biology

Keywords

  • Aged
  • Amino Acid Sequence
  • Angiogenesis Inducing Agents/chemistry
  • Culture Media, Conditioned/chemistry
  • Diffusion Chambers, Culture
  • Electric Impedance
  • Epithelial Cells/metabolism
  • Extracellular Matrix/metabolism
  • Gene Expression Profiling
  • Gene Expression Regulation
  • High-Temperature Requirement A Serine Peptidase 1/genetics
  • Human Umbilical Vein Endothelial Cells/cytology
  • Humans
  • Macular Degeneration/genetics
  • Models, Molecular
  • Peptide Fragments/chemistry
  • Primary Cell Culture
  • Proteolysis
  • Proteome/genetics
  • Retinal Pigments/genetics
  • Thrombospondin 1/chemistry

Cite this