The GalNAc-type O-Glycoproteome of CHO Cells Characterized by the SimpleCell Strategy

Yang, Zifeng; Halim, Adnan; Narimatsu, Yoshiki; Joshi, Hiren J.; Steentoft, Catharina; Schjoldager, Katrine T.; Schulz, Morten Alder; Sealover, Natalie R.; Kayser, Kevin J.; Bennett, Eric; Levery, Steven B.; Vakhrushev, Sergey Y.; Clausen, Henrik

doi:10.1074/mcp.m114.041541

Cited by 76 publications

(80 citation statements)

References 46 publications

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“…We previously did confirm some of the LDLR, VLDLR and LRP1 sites in WT CHO cells (18), as well as in rat liver and human plasma (19) (Fig. 1, labeled 2,3 and Suppl.…”

Section: O-glycosites In the Linker Regions Of Class A Repeats Of Ldlmentioning

confidence: 91%

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions

Wang

Mao

Narimatsu

et al. 2018

Journal of Biological Chemistry

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The low-density lipoprotein receptor (LDLR) and related receptors are important for the transport of diverse biomolecules across cell membranes and barriers. Their functions are especially relevant for cholesterol homeostasis and diseases, including neurodegenerative and kidney disorders. Members of the LDLR-related protein family share LDLR class A (LA) repeats providing binding properties for lipoproteins and other biomolecules. We previously demonstrated that short linker regions between these LA repeats contain conserved Oglycan-sites. Moreover, we found that O-glycan modifications at these sites are selectively controlled by the GalNAc-transferase isoform, GalNAc-T11. However, the effects of GalNAc-T11-mediated O-glycosylation on LDLR and related receptors localization and function are unknown. Here, we characterized O-glycosylation of LDLR-related proteins and identified conserved O-glycosylation sites in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of geneedited isogenic cell line models, we demonstrate that GalNAc-T11-mediated LDLR and VLDLR O-glycosylation is not required for transport and cell surface expression and stability of these receptors, but markedly enhances LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O-glycosylation increased affinity for LDL by approximately 5-fold. The molecular basis for this observation is currently unknown, but these findings open up new avenues for exploring the roles of LDLR-related proteins in disease.

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“…We previously did confirm some of the LDLR, VLDLR and LRP1 sites in WT CHO cells (18), as well as in rat liver and human plasma (19) (Fig. 1, labeled 2,3 and Suppl.…”

Section: O-glycosites In the Linker Regions Of Class A Repeats Of Ldlmentioning

confidence: 91%

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions

Wang

Mao

Narimatsu

et al. 2018

Journal of Biological Chemistry

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“…Sample Preparation and Lectin Affinity Chromatography-Total cell lysates (TCL) and culture medium (SEC) were processed as previously described (28,33). In brief, spent culture medium (ϳ80 ml harvested from two 175 ml T-flasks seeded at 5 ϫ 10 5 cells and cultured for 3 days) was cleared, dialyzed, and subjected to neuraminidase treatment (10 U Clostridium perfringens neuraminidase Type VI (Sigma)) before loaded on a 0.3 ml Vicia villosa agglutinin (VVA) agarose (Vector laboratories, Burlingame, CA) column.…”

Section: Methodsmentioning

confidence: 99%

Probing the O-Glycoproteome of Gastric Cancer Cell Lines for Biomarker Discovery*

Campos

Freitas

Gomes

et al. 2015

Molecular & Cellular Proteomics

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“…The highest obtained stoichiometric coefficient for UDP-Gal consumption corresponds to O-GalNAc HCP glycosylation () and is due to the high frequency of identified O-GalNAc glycosylation sites across the CHO proteome21 (Table 2) and the presence of galactose in all O-GalNAc glycans20 (Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

“…This set was then aligned with the closest homologous proteins obtained for the entire CHO proteome. Once aligned, the number of O-GalNAc glycosites per HCP () reported by Yang et al 21. was multiplied by the relative abundance of each HCP () and summed (Eq.…”

Section: Methodsmentioning

confidence: 99%

A theoretical estimate for nucleotide sugar demand towards Chinese Hamster Ovary cellular glycosylation

Val

Polizzi

Kontoravdi

2016

Sci Rep

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Glycosylation greatly influences the safety and efficacy of many of the highest-selling recombinant therapeutic proteins (rTPs). In order to define optimal cell culture feeding strategies that control rTP glycosylation, it is necessary to know how nucleotide sugars (NSs) are consumed towards host cell and rTP glycosylation. Here, we present a theoretical framework that integrates the reported glycoproteome of CHO cells, the number of N-linked and O-GalNAc glycosylation sites on individual host cell proteins (HCPs), and the carbohydrate content of CHO glycosphingolipids to estimate the demand of NSs towards CHO cell glycosylation. We have identified the most abundant N-linked and O-GalNAc CHO glycoproteins, obtained the weighted frequency of N-linked and O-GalNAc glycosites across the CHO cell proteome, and have derived stoichiometric coefficients for NS consumption towards CHO cell glycosylation. By combining the obtained stoichiometric coefficients with previously reported data for specific growth and productivity of CHO cells, we observe that the demand of NSs towards glycosylation is significant and, thus, is required to better understand the burden of glycosylation on cellular metabolism. The estimated demand of NSs towards CHO cell glycosylation can be used to rationally design feeding strategies that ensure optimal and consistent rTP glycosylation.

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The GalNAc-type O-Glycoproteome of CHO Cells Characterized by the SimpleCell Strategy

Cited by 76 publications

References 46 publications

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions

Probing the O-Glycoproteome of Gastric Cancer Cell Lines for Biomarker Discovery*

A theoretical estimate for nucleotide sugar demand towards Chinese Hamster Ovary cellular glycosylation

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