INTRODUCTION: Monoallelic variants in the ALG5 gene encoding asparagine-linked glycosylation protein 5 homolog (ALG5) have been recently shown to disrupt polycystin-1 (PC1) maturation and trafficking via underglycosylation, causing an autosomal dominant polycystic kidney disease-like (ADPKD-like) phenotype and interstitial fibrosis. In this report, we present clinical, genetic, histopathologic, and protein structure and functional correlates of a new ALG5 variant, p.R79W, that we identified in 2 distant genetically related Irish families displaying an atypical late-onset ADPKD phenotype combined with tubulointerstitial damage. METHODS: Whole exome and targeted sequencing were used for segregation analysis of available relatives. This was followed by immunohistochemistry examinations of kidney biopsies, and targeted (UMOD, MUC1) and untargeted plasma proteome and N-glycomic studies. RESULTS: We identified a monoallelic ALG5 variant [GRCh37 (NM_013338.5): g.37569565G>A, c.235C>T; p.R79W] that cosegregates in 23 individuals, of whom 18 were clinically affected. We detected abnormal localization of ALG5 in the Golgi apparatus of renal tubular cells in patients' kidney specimens. Further, we detected the pathological accumulation of uromodulin, an N-glycosylated glycosylphosphatidylinositol (GPI)-anchored protein, in the endoplasmic reticulum (ER), but not mucin-1, an O- and N-glycosylated protein. Biochemical investigation revealed decreased plasma and urinary uromodulin levels in clinically affected individuals. Proteomic and glycoproteomic profiling revealed the dysregulation of chronic kidney disease (CKD)-associated proteins. CONCLUSION: ALG5 dysfunction adversely affects maturation and trafficking of N-glycosylated and GPI anchored protein uromodulin, leading to structural and functional changes in the kidney. Our findings confirm ALG5 as a cause of late-onset ADPKD and provide additional insight into the molecular mechanisms of ADPKD-ALG5.

• Keywords
• ALG5, Golgi apparatus, N-Linked protein glycosylation, UMOD, autosomal dominant tubulo-interstitial kidney disease, autosomal-dominant polycystic kidney disease,
• Publication type
• Journal Article MeSH

Glycosylation is one of the most significant and abundant post-translational modifications in cells. Glycomic and glycoproteomic analyses involve the characterization of oligosaccharides (glycans) conjugated to proteins. Glycomic and glycoproteomic analysis is highly challenging because of the large diversity of structures, low abundance, site-specific heterogeneity, and poor ionization efficiency of glycans and glycopeptides in mass spectrometry (MS). MS is a key tool for characterization of glycans and glycopeptides. However, MS alone does not always provide full structural and quantitative information for many reasons, and thus MS is combined with some separation technique. This review focuses on the role of separation techniques used in glycomic and glycoproteomic analyses, liquid chromatography and capillary electrophoresis. The most important separation conditions and results are presented and discussed.

• Keywords
• Capillary zone electrophoresis, Glycan separation, Glycopeptide separation, High-pressure liquid chromatography, Proteomics,
• Publication type
• Journal Article MeSH
• Review MeSH

Protein glycosylation analysis is challenging due to the structural variety of complex conjugates. However, chromatographically separating glycans attached to tryptic peptides enables their site-specific characterization. For this purpose, we have shown the importance of selecting a suitable hydrophilic interaction liquid chromatography (HILIC) stationary phase in the separation of glycopeptides and their isomers. Three different HILIC stationary phases, i.e., HALO® penta-HILIC, Glycan ethylene bridged hybrid (BEH) Amide, and ZIC-HILIC, were compared in the separation of complex N-glycopeptides of hemopexin and Immunoglobulin G glycoproteins. The retention time increased with the polarity of the glycans attached to the same peptide backbone in all HILIC columns tested in this study, except for the ZIC-HILIC column when adding sialic acid to the glycan moiety, which caused electrostatic repulsion with the negatively charged sulfobetaine functional group, thereby decreasing retention. The HALO® penta-HILIC column provided the best separation results, and the ZIC-HILIC column the worst. Moreover, we showed the potential of these HILIC columns for the isomeric separation of fucosylated and sialylated glycoforms. Therefore, HILIC is a useful tool for the comprehensive characterization of glycoproteins and their isomers.

• Keywords
• glycopeptide separation, glycopeptides, glycoproteomics, hydrophilic interaction liquid chromatography, separation of glycopeptide isomers,
• MeSH
• Amides chemistry   MeSH
• Chromatography, Liquid instrumentation   methods   MeSH
• Glycopeptides chemistry   isolation & purification   metabolism   MeSH
• Glycosylation MeSH
• Hemopexin chemistry   isolation & purification   MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Immunoglobulin G chemistry   isolation & purification   MeSH
• Isomerism MeSH
• Humans MeSH
• Polysaccharides chemistry   MeSH
• Temperature MeSH
• Trypsin chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Amides MeSH
• Glycopeptides MeSH
• Hemopexin MeSH
• Immunoglobulin G MeSH
• Polysaccharides MeSH
• Trypsin MeSH