Combined proteomic and biochemical analyses redefine the consensus sequence requirement for epidermal growth factor-like domain hydroxylation
Bibliographic record
Abstract
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.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".