Four unreported types of glycans containing mannose-6-phosphate are heterogeneously attached at three sites (including newly found Asn 233) to recombinant human acid alpha-glucosidase that is the only approved treatment for Pompe disease

Park, Heajin; Kim, Jihye; Lee, Young Kwang; Kim, Woo Seok; You, Seung Kwan; Do, Jonghye; Jang, Yeonjoo; Oh, Doo-Byung; Kim, Jae Il; Kim, Ha Hyung

doi:10.1016/j.bbrc.2017.12.101

Cited by 25 publications

(12 citation statements)

References 28 publications

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“…The data acquisition was performed with an Xcalibur 2.1 (Thermo Fisher Scientific). Mass error was calculated as follows: ((observed mass − calculated mass)/(calculated mass)) × 10 6 [3]. All analytical experiments were repeated three times with identical LC chromatograms and MS ion chromatograms.…”

Section: N-glycan Analysis Using Lc-esi-hcd-ms/msmentioning

confidence: 99%

“…The presence of sialylated N-glycans is related to the stability and half-life of glycoproteins [2]. Phosphorylated N-glycans, including the mannose-6-phosphate group, are responsible for the efficacy of enzyme replacement therapy [3], and sulfated N-glycans are essential for electrostatic interactions and signal transduction pathways [4]. However, these N-glycans are difficult to analyze because of the labile nature of their charged moiety, their heterogeneous composition and structure, and their small proportions relative to total glycan quantities [5,6].…”

Section: Introductionmentioning

confidence: 99%

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N-Glycan Modifications with Negative Charge in a Natural Polymer Mucin from Bovine Submaxillary Glands, and Their Structural Role

Kim

Lee

et al. 2020

Polymers

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Bovine submaxillary mucin (BSM) is a natural polymer used in biomaterial applications for its viscoelasticity, lubricity, biocompatibility, and biodegradability. N-glycans are important for mucin stability and function, but their structures have not been fully characterized, unlike that of O-glycans. In this study, BSM N-glycans were investigated using liquid chromatography-tandem mass spectrometry. The microheterogeneous structures of 32 N-glycans were identified, and the quantities (%) of each N-glycan relative to total N-glycans (100%) were obtained. The terminal N-acetylgalactosamines in 12 N-glycans (sum of relative quantities; 27.9%) were modified with mono- (10 glycans) and disulfations (2 glycans). Total concentration of all sulfated N-glycans was 6.1 pmol in BSM (20 µg), corresponding to 25.3% of all negatively charged glycans (sum of present N-glycans and reported O-glycans). No N-glycans with sialylated or phosphorylated forms were identified, and sulfate modification ions were the only negative charges in BSM N-glycans. Mucin structures, including sulfated N-glycans located in the hydrophobic terminal regions, were indicated. This is the first study to identify the structures and quantities of 12 sulfated N-glycans in natural mucins. These sulfations play important structural roles in hydration, viscoelasticity control, protection from bacterial sialidases, and polymer stabilization to support the functionality of BSM via electrostatic interactions.

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Section: N-glycan Analysis Using Lc-esi-hcd-ms/msmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

N-Glycan Modifications with Negative Charge in a Natural Polymer Mucin from Bovine Submaxillary Glands, and Their Structural Role

Kim

Lee

et al. 2020

Polymers

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“…We conclude that our approach could thus be of general interest for characterization of M6P glycoproteomes as well as characterization of lysosomal enzymes used as treatment in enzyme replacement therapies targeting lysosomal storage diseases. Molecular & Cellular Proteomics 18: [16][17][18][19][20][21][22][23][24][25][26][27]2019. DOI: 10.1074/mcp.RA118.000967.…”

Section: Targeted Analysis Of Lysosomal Directedmentioning

confidence: 99%

“…However, these approaches can result in false positive identifications because of the applied removal of the M6P-tag. A few studies focused on intact M6P glycopeptides, although they were mainly focused on the analysis of a single lysosomal protein (16,17).…”

Section: En Do Pla Sm Ic Ret Icu Lummentioning

confidence: 99%

Targeted Analysis of Lysosomal Directed Proteins and Their Sites of Mannose-6-phosphate Modification

Čaval

Zhu

Tian

et al. 2019

Molecular & Cellular Proteomics

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Correspondence a.j.r.heck@uu.nl In BriefHere a Fe 3ϩ -IMAC-based enrichment method is presented targeting mannose-6-phosphate (M6P) modified glycopeptides enabling the detailed profiling of these in both HeLa and CHO cells. A gene engineering strategy was used to remove the Acp2/5 phosphatases, which increased the abundance and coverage of the M6P-modified glycoproteome. We demonstrate that such a gene engineering strategy can be beneficial for the expression of alpha-galactoside and other enzymes used in the treatment of lysosomal storage diseases. Graphical AbstractP P P P P P P P P Fe 3+ -IMAC P P P P P P P P P P P P P P P P P P Peptide mixture HCD P P P P P P P P P P EThcD P P P P P P P P P P LVQAEYWHDPIKEDVYRNHSIFLADINQER P P Highlights• Fe 3ϩ -IMAC enables co-enrichment of mannose-6-phosphate (M6P) modified glycopeptides.• Detailed characterization of intact M6P modified glycopeptides from HeLa and CHO cell lines.• EThcD results in a cleavage between 2 core GlcNAc residues enabling unambiguous glycoform identification.• Double knock out of the phosphatases Acp2/5 results in a 20-fold increase in abundance of M6P modified glycopeptides.• Recombinant expression of alpha-galactoside in an Acp2/5 KO CHO cell drastically increases its M6P content.

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“…Bisphosphorylated N-glycans are known to have the highest binding affinity of all known carbohydrate structures for the CI-MPR and enable binding at low nanomolar concentrations ( 12 , 14 ). Approximately only 1% of N-glycans on the standard-of-care rhGAA ERT bear bis -M6P ( 13 , 15 ). This ERT contains predominantly monophosphorylated glycans that are known to have approximately 3000-fold lower affinity for CI-MPR than bisphosphorylated N-glycans ( 14 , 15 ).…”

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confidence: 99%

Endolysosomal N-glycan processing is critical to attain the most active form of the enzyme acid alpha-glucosidase

Selvan

Mehta

Venkateswaran

et al. 2021

Journal of Biological Chemistry

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Acid alpha-glucosidase (GAA) is a lysosomal glycogen-catabolizing enzyme, the deficiency of which leads to Pompe disease. Pompe disease can be treated with systemic recombinant human GAA (rhGAA) enzyme replacement therapy (ERT), but the current standard of care exhibits poor uptake in skeletal muscles, limiting its clinical efficacy. Furthermore, it is unclear how the specific cellular processing steps of GAA after delivery to lysosomes impact its efficacy. GAA undergoes both proteolytic cleavage and glycan trimming within the endolysosomal pathway, yielding an enzyme that is more efficient in hydrolyzing its natural substrate, glycogen. Here, we developed a tool kit of modified rhGAAs that allowed us to dissect the individual contributions of glycan trimming and proteolysis on maturation-associated increases in glycogen hydrolysis using in vitro and in cellulo enzyme processing, glycopeptide analysis by MS, and high-pH anion-exchange chromatography with pulsed amperometric detection for enzyme kinetics. Chemical modifications of terminal sialic acids on N-glycans blocked sialidase activity in vitro and in cellulo , thereby preventing downstream glycan trimming without affecting proteolysis. This sialidase-resistant rhGAA displayed only partial activation after endolysosomal processing, as evidenced by reduced catalytic efficiency. We also generated enzymatically deglycosylated rhGAA that was shown to be partially activated despite not undergoing proteolytic processing. Taken together, these data suggest that an optimal rhGAA ERT would require both N-glycan and proteolytic processing to attain the most efficient enzyme for glycogen hydrolysis and treatment of Pompe disease. Future studies should examine the amenability of next-generation ERTs to both types of cellular processing.

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Four unreported types of glycans containing mannose-6-phosphate are heterogeneously attached at three sites (including newly found Asn 233) to recombinant human acid alpha-glucosidase that is the only approved treatment for Pompe disease

Cited by 25 publications

References 28 publications

N-Glycan Modifications with Negative Charge in a Natural Polymer Mucin from Bovine Submaxillary Glands, and Their Structural Role

N-Glycan Modifications with Negative Charge in a Natural Polymer Mucin from Bovine Submaxillary Glands, and Their Structural Role

Targeted Analysis of Lysosomal Directed Proteins and Their Sites of Mannose-6-phosphate Modification

Endolysosomal N-glycan processing is critical to attain the most active form of the enzyme acid alpha-glucosidase

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