#4547 Identification of Glycosylated Igf2 in Human Urinary Peptidome and Its Association With CKD

Abstract

Background and Aims Chronic kidney disease (CKD) is prevalent in 10% of world's adult population, with estimated glomerular filtration rate (eGFR) and albuminuria employed in its diagnosis. Role of protein glycosylation in causal mechanisms of CKD progression is largely unknown. Aim of this study was to identify urinary O-linked glycopeptides in association to CKD for better characterization of CKD molecular manifestations. Method Urine samples from eight CKD and two healthy subjects were analyzed by Capillary Electrophoresis-Tandem Mass Spectroscopy (CE-MS/MS) and glycopeptide analysis using Proteome Discoverer 1.4 was performed. Distribution of identified glycopeptides and correlation with Age, eGFR and Albuminuria were evaluated in 3810 datasets from the Human Urinary Proteome database using Spearman's rank correlation test and multiple linear regression analysis by statistical software R. The quantification of Insulin-like growth factor-II (IGF2) protein (7.5 kDa) was performed by enzyme-linked immunosorbent assay (ELISA) in two matched (for age, sex, no diabetes and no cardiovascular history) groups of 12 plasma samples each (eGFR < 30 ml/min/1.73m2 and eGFR > 90 ml/min/1.73m2). An open-source tool “Proteasix” was used to predict proteases involved in cleavage of the identified glycopeptides. The transcriptomics database and analysis tool “Nephroseq v4” were utilized to investigate the expression of predicted proteases in existing human transcriptomics CKD datasets. Results In total, 17 O-linked glycopeptides from 7 different proteins were identified, derived primarily from Insulin-like growth factor-II (IGF2) (n = 6). Glycosylation occurred at the surface exposed IGF2 Threonine 96 position. Further Investigation of the human proteome database containing 3810 datasets, showed that only three unglycosylated urinary peptides of IGF2 were identified, at significantly lower abundance and frequency (0-2.5% in the database) in comparison to the glycosylated forms (frequency of 23-90%). Interestingly, all identified IGF2 peptides belonged to E-domain of IGF2, i.e., 92 – 180 aa; which cleaves after undergoing O-linked glycosylation during the post-translational process yielding the “mature IGF2” (25-91 aa) protein. Results of multiple linear regression analysisa indicated that three IGF2 glycopeptides, DVStPPTVLPDNFPRYPVGKF [β(1024) = 0.08, p = 1.42*10−4], DVStPPTVLPDNFPRYPVG [β(560) = 0.01, p = 5.22*10−5] and DVStPPTVLPDNFPRYP [β(290) = 0.02, p = 1.07*10−4], exhibited positive correlation with Age and the glycopeptide tPPTVLPDNFPRYP showed strong negative association with eGFR [β(177) = -0.01, p = 4.21*10−9]. Along the same lines, the latter urinary IGF2 glycopeptide tPPTVLPDNFPRYP was found at increased [p < 2.2*10−16] abundance in CKD (n = 686) in comparison to healthy control (n = 229) datasets. In addition, IGF2 increased [p = 0.042] plasma levels were observed in CKD patients (eGFR < 30 ml/min/1.73m2 group) as supported by ELISA measurements. Protease predictions, considering also available transcriptomics data, suggest activation of cathepsin S with CKD potentially involved in the cleavage of the CKD-associated glycopeptide of IGF2. Conclusion Collectively, this study indicates that with aging and deteriorating kidney function, alterations in IGF2 proteoforms take place; which may be reflective of respective changes in the mature IGF2 protein abundance and function; as well as associated protease activity. Further, correlation analyses of the levels of the CKD-associated urinary IGF2 glycopeptide with the plasma IGF2 levels with CKD progression is planned. a Results of multiple linear regression where regression Co-efficient is β; degree of freedom is stated in (brackets); and p-value <0.05 was considered statistically significant