Keratoconjunctivitis Sicca: The importance of protein glycosylation for tear film stability
Final Report Abstract
Dry eye disease (DES), also known as Keratoconjunctivitis sicca, is defined as a multifactorial disease of the tear film and ocular surface. Its symptoms include visual disturbance, tear film instability accompanied by morphological changes and inflammation of the ocular surface. The glycosylation of proteins is one of the most common post-translational modifications (PTMs) and plays important regulatory functions in diverse biological processes such as protein stability or cell signalling. Therefore, in this study we developed a lectin-based affinity method for the enrichment of tear glycoproteins to characterize the specific N-glycosylation sites by high-resolution mass spectrometry (MS). Firstly, we have identified a total of 26 N glycosylation sites of 11 N-glycoproteins in the tear sample pools of healthy individuals utilizing the multilectin column system. Most of these N-glycoproteins are well established DES markers, namely, LTF (N497 & N642), IGHA1 (N144 & N340), PIP (N105), and LACRT (N105). Next, we have investigated the quantitative differences in healthy and DES subgroups utilizing individual tear samples. To accomplish this task, we have encountered a significant challenge due to the scarcity of available tear samples and limitations of the enrichment of the multi-lectin column system in identifying glycopeptides/glycoproteins. To address this gap, we have optimized and established a robust and cost-effective new method to identify the N- glycosylation sites and non-glycosylated peptides simultaneously without lectin-enrichment for the individual samples utilizing a very low amount of tear proteins. In this study, tear samples from a total of 182 subjects were utilized to investigate the quantitative differences of N- glycopeptides in individual samples of healthy and DES subgroups. To date, this is the largest number of individual tear samples used for a tandem MS-based discovery proteomics in unravelling the tear N-glycopeptides markers for DES. For the first time, we were able to demonstrate the significant differences in the N-glycopeptides in tears of DES subgroups, especially severe aqueous-deficient and evaporative DES (sDRYaqlip) compared to healthy individual. Importantly, we have successfully identified 19 N-glycosylated peptides to be differently abundant in the sDRYaqlip, namely, SERPINA3 (site, N271), AHSG (site, N157), CFH (site, N911), HP (site, N241), LACRT (site, N119), LTF (site, N445), SCGB21 (site, N68) and PIP (site, N105). To the best of our knowledge, the present study identified the highest number of N-glycopeptides annotated DES markers. The large majority of these N- glycopeptides were significantly implicated with various biological functions, namely, inflammatory response, apoptosis, and antibacterial response. In summary, this study unravelled numerous N-glycoprotein markers annotated to DES that could be potentially used for further diagnosis, prognosis, and drug development.
Publications
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Lectin-Based Affinity Enrichment and Characterization of N-Glycoproteins from Human Tear Film by Mass Spectrometry. Molecules, 28(2), 648.
Schmelter, Carsten; Brueck, Alina; Perumal, Natarajan; Qu, Sichang; Pfeiffer, Norbert & Grus, Franz H.
