Site-Specific N-Glycosylation Analysis of Bone Morphogenic Protein Receptor-2

Introduction and Objective. Pulmonary hypertension (PH) is an irreversible condition defined by elevated blood pressure within the lung vasculature. If left untreated, PH may lead to right heart failure and eventually death. Mutations in the gene encoding bone morphogenetic protein receptor type-2 (BMPR-2) have been linked to inherited forms of pulmonary artery hypertension. BMPR-2 is expressed in multiple cell types including endothelial cells and mediates cell signaling following ligand binding of the transforming growth factor beta (TGF-β) family ligands. BMPR-2 activation can contribute to cell proliferation, apoptosis, and angiogenesis. Glycosylation of BMPR-2 reportedly influences BMPR-2 ligand binding and receptor activation. Glycosylation is the co- and/or post-translational addition of glycans (sugars) to proteins. Here, we sought to define the glycan structures at three putative N-glycosylation sites on the extracellular domain of BMPR-2 using tandem mass spectrometry. Methods. To optimize conditions for the observation of BMPR-2 glycosylation sites via bottom up nano liquid chromatography tandem mass spectrometry (nLC-MS/MS), we tested three conditions for proteolysis (trypsin, GluC, and a combination of trypsin and GluC). Following proteolysis, nLC-MS/MS were performed to reveal N-glycosylation sites and glycan compositions on human BMPR-2. Data were analyzed using ProteomeDiscoverer and Byonic software. Results. We observed N-glycopeptides spanning all three putative BMPR-2 N-glycosylation sites (N56, N110, and N126). High BMPR-2 amino acid sequence coverage was observed for all three proteolytic conditions. We report high coverage of the BMPR-2 extracellular domain, with 100%, 90.4%, and 90.4% coverage achieved utilizing trypsin, GluC, or Trypsin plus GluC, respectively. Glycopeptide analyses revealed complex glycoforms with abundant fucosylation and sialylation. Conclusions-Implications. The best workflow for the analysis of BMPR-2 N-glycosylation involved the use of trypsin for proteolysis, which resulted in the highest amino acid coverage and enabled the observation of all three glycosylation sites of BMPR-2 (N55, N110, and N126). Further, tryptic glycopeptides were assigned with higher confidence compared to glycopeptides from the other digests. It is crucial to understand the composition of BMPR-2 N-glycans, in order to define and elucidate their role in BMPR-2 signaling and in pulmonary hypertension. The nLC-MS/MS method established here will facilitate the study of BMPR-2 N-glycosylation in pulmonary hypertension.