Combined proteomic and biochemical analyses redefine the consensus sequence requirement for epidermal growth factor-like domain hydroxylation

Journal article

Epidermal growth factor-like domains (EGFDs) have important functions in cell-cell signaling. Both secreted and cell surface human EGFDs are subject to extensive modifications, including aspartate and asparagine residue C3-hydroxylations catalyzed by the 2-oxoglutarate oxygenase aspartate/asparagine-β-hydroxylase (AspH). Although genetic studies show AspH is important in human biology, studies on its physiological roles have been limited by incomplete knowledge of its substrates. Here, we redefine the consensus sequence requirements for AspH-catalyzed EGFD hydroxylation based on combined analysis of proteomic mass spectrometric data and mass spectrometry-based assays with isolated AspH and peptide substrates. We provide cellular and biochemical evidence that the preferred site of EGFD hydroxylation is embedded within a disulfide-bridged macrocycle formed of 10 amino acid residues. This definition enabled the identification of previously unassigned hydroxylation sites in three EGFDs of human fibulins as AspH substrates. A non-EGFD containing protein, lymphocyte antigen-6/plasminogen activator urokinase receptor domain containing protein 6B (LYPD6B) was shown to be a substrate for isolated AspH, but we did not observe evidence for LYPD6B hydroxylation in cells. AspH-catalyzed hydroxylation of fibulins is of particular interest given their important roles in extracellular matrix dynamics. In conclusion, these results lead to a revision of the consensus substrate requirements for AspH and expand the range of observed and potential AspH-catalyzed hydroxylation in cells, which will enable future study of the biological roles of AspH.

Authors: Brewitz, L; Onisko, BC; Schofield, CJ

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Elsevier
• Journal: Journal of Biological Chemistry
• Volume: 298
• Issue: 8
• Article number: 102129
• Publication date: 2022-08-01
• Acceptance date: 2022-06-10
• DOI: 10.1016/j.jbc.2022.102129
• EISSN: 1083-351X
• ISSN: 0021-9258
• Pmid: 35700824
• Language: English
• Keywords: 2-oxoglutarate; fibulin; α-ketoglutarate oxygenase; fibrillin; protein hydroxylation; aspartate/asparagine-β-hydroxylase; FFR; latent-transforming growth factor β-binding protein; epidermal growth factor-like domain; proteomics