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

Shengjun Wang; Yang Mao; Yoshiki Narimatsu; Zilu Ye; Weihua Tian; Christoffer K Goth; Erandi Lira-Navarrete; Nis Borbye Pedersen; Asier Benito-Vicente; Cesar Martin; Kepa B Uribe; Ramon Hurtado-Guerrero; Christina Christoffersen; Nabil G Seidah; Rikke Nielsen; Erik I Christensen; Lars Hansen; Eric P Bennett; Sergey Y Vakhrushev; Katrine T Schjoldager; Henrik Clausen

doi:10.1074/jbc.m117.817981

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

Shengjun Wang, Yang Mao, Yoshiki Narimatsu, Zilu Ye, Weihua Tian, Christoffer K Goth, Erandi Lira-Navarrete, Nis Borbye Pedersen, Asier Benito-Vicente, Cesar Martin, Kepa B Uribe, Ramon Hurtado-Guerrero, Christina Christoffersen, Nabil G Seidah, Rikke Nielsen, Erik I Christensen, Lars Hansen, Eric P Bennett, Sergey Y Vakhrushev, Katrine T SchjoldagerHenrik Clausen

30 Citationer (Scopus)

Abstract

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 O-glycan 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 receptor 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 gene-edited 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 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.

Originalsprog	Engelsk
Tidsskrift	The Journal of Biological Chemistry
Vol/bind	293
Udgave nummer	19
Sider (fra-til)	7408 –7422
ISSN	0021-9258
DOI	https://doi.org/10.1074/jbc.m117.817981
Status	Udgivet - 11 maj 2018

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10.1074/jbc.m117.817981

http://www.jbc.org/content/293/19/7408.full.pdfLicens: CC BY

Citationsformater

Wang, S., Mao, Y., Narimatsu, Y., Ye, Z., Tian, W., Goth, C. K., Lira-Navarrete, E., Pedersen, N. B., Benito-Vicente, A., Martin, C., Uribe, K. B., Hurtado-Guerrero, R., Christoffersen, C., Seidah, N. G., Nielsen, R., Christensen, E. I., Hansen, L., Bennett, E. P., Vakhrushev, S. Y., ... Clausen, H. (2018). Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions. The Journal of Biological Chemistry, 293(19), 7408 –7422. https://doi.org/10.1074/jbc.m117.817981

Wang, S, Mao, Y, Narimatsu, Y , Ye, Z, Tian, W, Goth, CK, Lira-Navarrete, E, Pedersen, NB, Benito-Vicente, A, Martin, C, Uribe, KB, Hurtado-Guerrero, R, Christoffersen, C, Seidah, NG, Nielsen, R, Christensen, EI, Hansen, L , Bennett, EP , Vakhrushev, SY , Schjoldager, KT & Clausen, H 2018, 'Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions', The Journal of Biological Chemistry, bind 293, nr. 19, s. 7408 –7422. https://doi.org/10.1074/jbc.m117.817981

@article{47e9a80705324900b74254d6720a666a,

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

abstract = "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 O-glycan 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 receptor 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 gene-edited 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 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.",

author = "Shengjun Wang and Yang Mao and Yoshiki Narimatsu and Zilu Ye and Weihua Tian and Goth, {Christoffer K} and Erandi Lira-Navarrete and Pedersen, {Nis Borbye} and Asier Benito-Vicente and Cesar Martin and Uribe, {Kepa B} and Ramon Hurtado-Guerrero and Christina Christoffersen and Seidah, {Nabil G} and Rikke Nielsen and Christensen, {Erik I} and Lars Hansen and Bennett, {Eric P} and Vakhrushev, {Sergey Y} and Schjoldager, {Katrine T} and Henrik Clausen",

note = "Copyright {\textcopyright} 2018, The American Society for Biochemistry and Molecular Biology.",

year = "2018",

month = may,

day = "11",

doi = "10.1074/jbc.m117.817981",

language = "English",

volume = "293",

pages = "7408 –7422",

journal = "Journal of Biological Chemistry",

issn = "0021-9258",

publisher = "American Society for Biochemistry and Molecular Biology, Inc.",

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TY - JOUR

T1 - Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions

AU - Wang, Shengjun

AU - Mao, Yang

AU - Narimatsu, Yoshiki

AU - Ye, Zilu

AU - Tian, Weihua

AU - Goth, Christoffer K

AU - Lira-Navarrete, Erandi

AU - Pedersen, Nis Borbye

AU - Benito-Vicente, Asier

AU - Martin, Cesar

AU - Uribe, Kepa B

AU - Hurtado-Guerrero, Ramon

AU - Christoffersen, Christina

AU - Seidah, Nabil G

AU - Nielsen, Rikke

AU - Christensen, Erik I

AU - Hansen, Lars

AU - Bennett, Eric P

AU - Vakhrushev, Sergey Y

AU - Schjoldager, Katrine T

AU - Clausen, Henrik

PY - 2018/5/11

Y1 - 2018/5/11

N2 - 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 O-glycan 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 receptor 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 gene-edited 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 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.

AB - 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 O-glycan 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 receptor 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 gene-edited 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 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.

U2 - 10.1074/jbc.m117.817981

DO - 10.1074/jbc.m117.817981

M3 - Journal article

C2 - 29559555

SN - 0021-9258

VL - 293

SP - 7408

EP - 7422

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 19

ER -

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

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