Comprehensive Mapping and Dynamics of Site-Specific Prolyl-Hydroxylation, Lysyl-Hydroxylation and Lysyl O-Glycosylation of Collagens Deposited in ECM During Zebrafish Heart Regeneration

Abstract

Cardiac fibrosis-mediated heart failure (HF) is one of the major forms of end-stage cardiovascular diseases (CVDs). Cardiac fibrosis is an adaptive response of the myocardium upon any insult/injury. Excessive deposition of collagen molecules in the extracellular matrix (ECM) is the hallmark of fibrosis. This fibrotic response initially protects the myocardium from ventricular rupture. Although in mammals this fibrotic response progresses towards scar-tissue formation leading to HF, some fishes and urodeles have mastered the art of cardiac regeneration following injury-mediated fibrotic response. Zebrafish have a unique capability to regenerate the myocardium after post-amputation injury. Following post-amputation, the ECM of the zebrafish heart undergoes extensive remodeling and deposition of collagen. Being the most abundant protein of ECM, collagen plays important role in the assembly and cell-matrix interactions. However, the mechanism of ECM remodeling is not well understood. Collagen molecules undergo heavy post-translational modifications (PTMs) mainly hydroxylation of proline, lysine, and glycosylation of lysine during biosynthesis. The critical roles of these PTMs are emerging in several diseases, embryonic development, cell behavior regulation, and cell-matrix interactions. The site-specific identification of these collagen PTMs in zebrafish heart ECM is not known. As these highly modified peptides are not amenable to mass spectrometry (MS), the site-specific identification of these collagen PTMs is challenging. Here, we have implemented our in-house proteomics analytical pipeline to analyze two ECM proteomics datasets (PXD011627, PXD010092) of the zebrafish heart during regeneration (post-amputation). We report the first comprehensive site-specific collagen PTM map of zebrafish heart ECM. We have identified a total of 36 collagen chains (19 are reported for the first time here) harboring a total of 95 prolyl-3-hydroxylation, 108 hydroxylysine, 29 galactosyl-hydroxylysine, and 128 glucosylgalactosyl-hydroxylysine sites. Furthermore, we comprehensively map the three chains (COL1A1a, COL1A1b, and COL1A2) of collagen I, the most abundant protein in zebrafish heart ECM. We achieved more than 95% sequence coverage for all the three chains of collagen I. Our analysis also revealed the dynamics of prolyl-3-hydroxylation occupancy oscillations during heart regeneration at these sites. Moreover, quantitative site-specific analysis of lysine-O-glycosylation microheterogeneity during heart regeneration revealed a significant (p < 0.05) elevation of site-specific (K1017) glucosylgalactosyl-hydroxylysine on the col1a1a chain. Taken together, these site-specific PTM maps and the dynamic changes of site-specific collagen PTMs in ECM during heart regeneration will open up new avenues to decode ECM remodeling and may lay the foundation to tinker the cardiac regeneration process with new approaches.

Integrative proteomics and phosphoproteomics profiling on osteogenic differentiation of periodontal ligament stem cell

“…Analogously, our data identified that the protein level and phosphorylation on Ser1124 and Ser1247 of COL1A2 significantly decreased. Numerous PTMs occur in the biosynthesis of collagen molecules, including hydroxylation of proline and glycosylation of lysine [22]. However, there are few reports on the phosphorylation modification of collagen molecules, which suggests that it may be an overlooked but important research area.…”

Exploring the cardiac ECM during fibrosis: A new era with next-gen proteomics

“…Collagen biosynthesis involves heavy post-translational modifications (PTMs). Proteomics studies have shown that these PTMs have a role in the stability, function, and communication of collagen I with other ECM components and cell-surface receptors to regulate cell adhesion, migration, and cardiac regeneration ( Pokidysheva et al, 2014 ; Basak et al, 2016 ; Merl-Pham et al, 2019 ; Sarohi et al, 2022b ). Injury and scars are non-repairable in the mammalian heart due to a lack of regeneration capability ( Cutie and Huang, 2021 ).…”

“…In an interesting proteomic study done by Sarohi et al, they detected 36 collagen chains in zebrafish heart. The levels of collagen PTMs were found to be altered during the regeneration process in the zebrafish heart ( Sarohi et al, 2022b ). This hints toward the role of collagen PTMs in the zebrafish heart-regeneration process.…”

“…The role of the lysyl and prolyl-hydroxylation and O-glycosylation (G-HyK and GG-HyK) sites in collagens is poorly understood during fibrosis progression. Proteomics provides broader opportunities to identify these PTMs in a site-specific manner in order to study the roles of these PTMs during cardiac fibrosis ( Merl-Pham et al, 2019 ; Sarohi et al, 2022b ). Apart from hydroxylation and O-glycosylation, lysine can also be modified to allysines by oxidation.…”

“…On the contrary, the expressional abundances, post-translational modifications (hydroxylations, glycosylations, etc. ), proteolytic processing, protein assembly network (crosslink analysis) of the core, and associated ECM proteins can only be depicted with the mass-spectrometry (MS)-based approaches ( Barallobre-Barreiro et al, 2012 ; Terajima et al, 2014 ; Basak et al, 2016 ; Sarohi et al, 2022b ). Mass-spectrometry (MS)-based proteomics has been useful in identifying primary sequences, protein–protein interactions, and PTMs ( Aebersold and Mann, 2003 ).…”