Browsing by Author "Mechref, Yehia"

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    Altered O-glycomes of Renal Brush-Border Membrane in Model Rats with Chronic Kidney Diseases
    (MDPI, 2021-10-21) Yu, Aiying; Zhao, Jingfu; Zhong, Jieqiang; Wang, Junyao; Yadav, Shiv Pratap S.; Molitoris, Bruce A.; Wagner, Mark C.; Mechref, Yehia; Medicine, School of Medicine
    Chronic kidney disease (CKD) is defined as a decrease in renal function or glomerular filtration rate (GFR), and proteinuria is often present. Proteinuria increases with age and can be caused by glomerular and/or proximal tubule (PT) alterations. PT cells have an apical brush border membrane (BBM), which is a highly dynamic, organized, and specialized membrane region containing multiple glycoproteins required for its functions including regulating uptake, secretion, and signaling dependent upon the physiologic state. PT disorders contribute to the dysfunction observed in CKD. Many glycoprotein functions have been attributed to their N- and O-glycans, which are highly regulated and complex. In this study, the O-glycans present in rat BBMs from animals with different levels of kidney disease and proteinuria were characterized and analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). A principal component analysis (PCA) documented that each group has distinct O-glycan distributions. Higher fucosylation levels were observed in the CKD and diabetic groups, which may contribute to PT dysfunction by altering physiologic glycoprotein interactions. Fucosylated O-glycans such as 1-1-1-0 exhibited higher abundance in the severe proteinuric groups. These glycomic results revealed that differential O-glycan expressions in CKD progressions has the potential to define the mechanism of proteinuria in kidney disease and to identify potential therapeutic interventions.
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    Changes in the Expression of Renal Brush Border Membrane N-Glycome in Model Rats with Chronic Kidney Diseases
    (MDPI, 2021-11-11) Yu, Aiying; Zhao, Jingfu; Yadav, Shiv Pratap S.; Molitoris, Bruce A.; Wagner, Mark C.; Mechref, Yehia; Medicine, School of Medicine
    Chronic kidney disease (CKD) is defined by a reduced renal function i.e., glomerular filtration rate (GFR), and the presence of kidney damage is determined by measurement of proteinuria or albuminuria. Albuminuria increases with age and can result from glomerular and/or proximal tubule (PT) alterations. Brush-border membranes (BBMs) on PT cells play an important role in maintaining the stability of PT functions. The PT BBM, a highly dynamic, organized, specialized membrane, contains a variety of glycoproteins required for the functions of PT. Since protein glycosylation regulates many protein functions, the alteration of glycosylation due to the glycan changes has attracted more interests for a variety of disease studies recently. In this work, liquid chromatography-tandem mass spectrometry was utilized to analyze the abundances of permethylated glycans from rats under control to mild CKD, severe CKD, and diabetic conditions. The most significant differences were observed in sialylation level with the highest present in the severe CKD and diabetic groups. Moreover, high mannose N-glycans was enriched in the CKD BBMs. Characterization of all the BBM N-glycan changes supports that these changes are likely to impact the functional properties of the dynamic PT BBM. Further, these changes may lead to the potential discovery of glycan biomarkers for improved CKD diagnosis and new avenues for therapeutic treatments.
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    Glycosylation of a key cubilin Asn residue results in reduced binding to albumin
    (Elsevier, 2022) Yadav, Shiv Pratap Singh; Yu, Aiying; Zhao, Jingfu; Singh, Jasdeep; Kakkar, Saloni; Chakraborty, Srinivas; Mechref, Yehia; Molitoris, Bruce; Wagner, Mark C.; Medicine, School of Medicine
    Kidney disease often manifests with an increase in proteinuria, which can result from both glomerular and/or proximal tubule injury. The proximal tubules are the major site of protein and peptide endocytosis of the glomerular filtrate, and cubilin is the proximal tubule brush border membrane glycoprotein receptor that binds filtered albumin and initiates its processing in proximal tubules. Albumin also undergoes multiple modifications depending upon the physiologic state. We previously documented that carbamylated albumin had reduced cubilin binding, but the effects of cubilin modifications on binding albumin remain unclear. Here, we investigate the cubilin-albumin binding interaction to define the impact of cubilin glycosylation and map the key glycosylation sites while also targeting specific changes in a rat model of proteinuria. We identified a key Asn residue, N1285, that when glycosylated reduced albumin binding. In addition, we found a pH-induced conformation change may contribute to ligand release. To further define the albumin-cubilin binding site, we determined the solution structure of cubilin's albumin-binding domain, CUB7,8, using small-angle X-ray scattering and molecular modeling. We combined this information with mass spectrometry crosslinking experiments of CUB7,8 and albumin that provides a model of the key amino acids required for cubilin-albumin binding. Together, our data supports an important role for glycosylation in regulating the cubilin interaction with albumin, which is altered in proteinuria and provides new insight into the binding interface necessary for the cubilin-albumin interaction.
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    Identification of Glycopeptides with Multiple Hydroxylysine O-Glycosylation Sites by Tandem Mass Spectrometry
    (ACS, 2015-11) Zhang, Yanlin; Yu, Chuan-Yih; Song, Ehwang; Li, Shuai Cheng; Mechref, Yehia; Tang, Haixu; Liu, Xiaowen; Department of Biohealth Informatics, IU School of Informatics and Computing
    Glycosylation is one of the most common post-translational modifications in proteins, existing in ∼50% of mammalian proteins. Several research groups have demonstrated that mass spectrometry is an efficient technique for glycopeptide identification; however, this problem is still challenging because of the enormous diversity of glycan structures and the microheterogeneity of glycans. In addition, a glycopeptide may contain multiple glycosylation sites, making the problem complex. Current software tools often fail to identify glycopeptides with multiple glycosylation sites, and hence we present GlycoMID, a graph-based spectral alignment algorithm that can identify glycopeptides with multiple hydroxylysine O-glycosylation sites by tandem mass spectra. GlycoMID was tested on mass spectrometry data sets of the bovine collagen α-(II) chain protein, and experimental results showed that it identified more glycopeptide-spectrum matches than other existing tools, including many glycopeptides with two glycosylation sites.
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    Mechanism of how carbamylation reduces albumin binding to FcRn contributing to increased vascular clearance
    (American Physiological Society, 2021) Yadav, Shiv Pratap S.; Sandoval, Ruben M.; Zhao, Jingfu; Huang, Yifan; Wang, Exing; Kumar, Sudhanshu; Campos-Bilderback, Silvia B.; Rhodes, George; Mechref, Yehia; Molitoris, Bruce A.; Wagner, Mark C.; Medicine, School of Medicine
    Chronic kidney disease results in high serum urea concentrations leading to excessive protein carbamylation, primarily albumin. This is associated with increased cardiovascular disease and mortality. Multiple methods were used to address whether carbamylation alters albumin metabolism. Intravital two-photon imaging of the Munich Wistar Frömter (MWF) rat kidney and liver allowed us to characterize filtration and proximal tubule uptake and liver uptake. Microscale thermophoresis enabled quantification of cubilin (CUB7,8 domain) and FcRn binding. Finally, multiple biophysical methods including dynamic light scattering, small-angle X-ray scattering, LC-MS/MS and in silico analyses were used to identify the critical structural alterations and amino acid modifications of rat albumin. Carbamylation of albumin reduced binding to CUB7,8 and FcRn in a dose-dependent fashion. Carbamylation markedly increased vascular clearance of carbamylated rat serum albumin (cRSA) and altered distribution of cRSA in both the kidney and liver at 16 h post intravenous injection. By evaluating the time course of carbamylation and associated charge, size, shape, and binding parameters in combination with in silico analysis and mass spectrometry, the critical binding interaction impacting carbamylated albumin's reduced FcRn binding was identified as K524. Carbamylation of RSA had no effect on glomerular filtration or proximal tubule uptake. These data indicate urea-mediated time-dependent carbamylation of albumin lysine K524 resulted in reduced binding to CUB7,8 and FcRn that contribute to altered albumin transport, leading to increased vascular clearance and increased liver and endothelial tissue accumulation.
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    Quantitative Serum Glycomics of Esophageal Adenocarcinoma, and Other Esophageal Disease Onsets
    (American Chemical Society, 2009-06) Mechref, Yehia; Hussein, Ahmed; Bekesova, Slavka; Pungpapong, Vitara; Zhang, Min; Dobrolecki, Lacey E.; Hickey, Robert J.; Hammoud, Zane T.; Novotny, Milos V.; Department of Medicine, IU School of Medicine
    Aberrant glycosylation has been implicated in various types of cancers and changes in glycosylation may be associated with signaling pathways during malignant transformation. Glycomic profiling of blood serum, in which cancer cell proteins or their fragments with altered glycosylation patterns are shed, could reveal the altered glycosylation. We performed glycomic profiling of serum from patients with no known disease (N=18), patients with high grade dysplasia (HGD, N=11) and Barrett’s (N=5), and patients with esophageal adenocarcinoma (EAC, N=50) in an attempt to delineate distinct differences in glycosylation between these groups. The relative intensities of 98 features were significantly different among the disease onsets; 26 of these correspond to known glycan structures. The changes in the relative intensities of three of the known glycan structures predicted esophageal adenocarcinoma with 94% sensitivity and better than 60% specificity as determined by receiver operating characteristic (ROC) analysis. We have demonstrated that comparative glycomic profiling of EAC reveals a subset of glycans that can be selected as candidate biomarkers. These markers can differentiate disease-free from HGD, disease-free from EAC, and HGD from EAC. The clinical utility of these glycan biomarkers requires further validation.