Low Density Lipoprotein Receptor Class A Repeats Are O-Glycosylated in Linker Regions

Pedersen, Nis Borbye; Wang, Shengjun; Narimatsu, Yoshiki; Yang, Zifeng; Halim, Adnan; Schjoldager, Katrine T.; Madsen, Thomas Daugbjerg; Seidah, Nabil G.; Bennett, Eric; Levery, Steven B.; Clausen, Henrik

doi:10.1074/jbc.m113.545053

Cited by 50 publications

(58 citation statements)

References 62 publications

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“…3). Note that on Western blottings (WB) the LDLR appears as a doublet consisting of an LDLR that is not O-glycosylated (ϳ110 kDa) and one that is fully mature and O-glycosylated (ϳ140 kDa) (31). Thus, WB analyses of overexpressed LDLR-WT, -R410S, and -G592E and both mutants in HEK293 cells revealed that LDLR-WT and LDLR-R410S levels were similar, whereas those of LDLR-G592E were ϳ30% of WT, and the equal combination R410S/ (Fig.…”

Section: Ldlr-r410s Is Well Expressed At the Cell Surface And The Ldlmentioning

confidence: 99%

The Proprotein Convertase Subtilisin/Kexin Type 9-resistant R410S Low Density Lipoprotein Receptor Mutation

Susan‐Resiga¹,

Girard²,

Kiss

et al. 2017

Journal of Biological Chemistry

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Edited by George M. CarmanFamilial hypercholesterolemia (FH) is characterized by severely elevated low density lipoprotein (LDL) cholesterol. Herein, we identified an FH patient presenting novel compound heterozygote mutations R410S and G592E of the LDL receptor (LDLR). The patient responded modestly to maximum rosuvastatin plus ezetimibe therapy, even in combination with a PCSK9 monoclonal antibody injection. Using cell biology and molecular dynamics simulations, we aimed to define the underlying mechanism(s) by which these LDLR mutations affect LDL metabolism and lead to hypercholesterolemia. Our data showed that the LDLR-G592E is a class 2b mutant, because it mostly failed to exit the endoplasmic reticulum and was degraded. Even though LDLR-R410S and LDLR-WT were similar in levels of cell surface and total receptor and bound equally well to LDL or extracellular PCSK9, the LDLR-R410S was resistant to exogenous PCSK9-mediated degradation in endosomes/lysosomes and showed reduced LDL internalization and degradation relative to LDLR-WT. Evidence is provided for a tighter association of LDL with LDLR-R410S at acidic pH, a reduced LDL delivery to late endosomes/ lysosomes, and an increased release in the medium of the bound/ internalized LDL, as compared with LDLR-WT. These data suggested that LDLR-R410S recycles loaded with its LDL-cargo. Our findings demonstrate that LDLR-R410S represents an LDLR loss-of-function through a novel class 8 FH-causing mechanism, thereby rationalizing the observed phenotype.

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Section: Ldlr-r410s Is Well Expressed At the Cell Surface And The Ldlmentioning

confidence: 99%

The Proprotein Convertase Subtilisin/Kexin Type 9-resistant R410S Low Density Lipoprotein Receptor Mutation

Susan‐Resiga¹,

Girard²,

Kiss

et al. 2017

Journal of Biological Chemistry

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“…4B) (42). Note that the LDLR appears as a doublet consisting of an LDLR that is not O-glycosylated (110 kDa) and that is fully mature and O-glycosylated (150 kDa) (43). Unexpectedly, in both cases, the strongest recovery of the LDLR signal was obtained in the presence of D374Y PCSK9, suggesting that P1.40 neutralized the activity of this mutant form of PCSK9 with a higher efficacy.…”

Section: Resultsmentioning

confidence: 83%

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Single Domain Antibodies Are Potent Inhibitors of Low Density Lipoprotein Receptor Degradation

Weider

Susan‐Resiga

Essalmani

et al. 2016

Journal of Biological Chemistry

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Single domain antibodies (sdAbs) correspond to the antigenbinding domains of camelid antibodies. They have the same antigen-binding properties and specificity as monoclonal antibodies (mAbs) but are easier and cheaper to produce. We report here the development of sdAbs targeting human PCSK9 (proprotein convertase subtilisin/kexin type 9) as an alternative to anti-PCSK9 mAbs. After immunizing a llama with human PCSK9, we selected four sdAbs that bind PCSK9 with a high affinity and produced them as fusion proteins with a mouse Fc. All four sdAb-Fcs recognize the C-terminal Cys-His-rich domain of PCSK9. We performed multiple cellular assays and demonstrated that the selected sdAbs efficiently blocked PCSK9-mediated low density lipoprotein receptor (LDLR) degradation in cell lines, in human hepatocytes, and in mouse primary hepatocytes. We further showed that the sdAb-Fcs do not affect binding of PCSK9 to the LDLR but rather block its induced cellular LDLR degradation. Pcsk9 knock-out mice expressing a human bacterial artificial chromosome (BAC) transgene were generated, resulting in plasma levels of ϳ300 ng/ml human PCSK9. Mice were singly or doubly injected with the best sdAb-Fc and analyzed at day 4 or 11, respectively. After 4 days, mice exhibited a 32 and 44% decrease in the levels of total cholesterol and apolipoprotein B and ϳ1.8-fold higher liver LDLR protein levels. At 11 days, the equivalent values were 24 and 46% and ϳ2.3-fold higher LDLR proteins. These data constitute a proof-of-principle for the future usage of sdAbs as PCSK9-targeting drugs that can efficiently reduce LDL-cholesterol, and as tools to study the Cys-His-rich domain-dependent sorting the PCSK9-LDLR complex to lysosomes.For over 30 years, a large number of clinical trials have firmly consolidated the importance of lowering LDL-cholesterol (LDLc) 3 in the prevention of cardiovascular diseases (CVD) and its associated devastating sequelae (1). Healthy diets and exercise are highly recommended to lower LDLc in patients with high baseline levels. However, many individuals, including those suffering from familial hypercholesterolemia (FH), cannot reach the recommended LDLc levels to prevent cardiovascular complications. With an overall incidence of ϳ1:200, FH is a common inherited disease that affects at least 30 million people, of whom Յ1% have been diagnosed. It is characterized by plasma LDLc levels greater that the 95th percentile, which result in tendon xanthomas, xanthelasmas, corneal arcus, and premature atherosclerosis, leading to premature ischemic vascular disease and mortality if left untreated. In most cases, FH subjects exhibit mutations in the LDL receptor (LDLR; 67%) and its ligand apolipoprotein B (apoB; 14%), hampering LDL clearance from the circulation (2). In 2003, merging biological studies with human genetics led to the discovery of PCSK9, the 9th and last member of the family of proprotein convertases related to subtilisin and kexin (3), and the demonstration that the PCSK9 gene represents the 3rd locus of autosomal domin...

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“…We were, however, unable to demonstrate activity with short peptides covering the LDLR linker regions (Pedersen et al 2014;Kong et al 2015), suggesting that for some protein substrates, proper folding may be required for substrate recognition. Thus, it appears that identification of unique substrates at least for some GalNAc-T isoforms will require analysis of actual protein substrates.…”

Section: Discussionmentioning

confidence: 97%

“…Human GalNAc-T11 is the only isoform capable of glycosylating the low-density lipoprotein receptor (LDLR) in its ligand-binding region, where the short linker regions between the LDLR class A repeats contain a highly conserved O-glycosite (Steentoft et al 2013;Pedersen et al 2014). This unique function was verified ex vivo in an Cartoon representation of the X-ray structure of GalNAcT-1 (PDB id:1XHB (Fritz et al 2004)).…”

Section: Discussionmentioning

confidence: 99%

“…isogenic cell model system with and without expression of GalNAc-T11 as well as with in vitro glycosylation assays with a recombinant reporter protein expressed in E. coli containing the ligand-binding region of LDLR with multiple folded LDLR class A repeats (Pedersen et al 2014). We were, however, unable to demonstrate activity with short peptides covering the LDLR linker regions (Pedersen et al 2014;Kong et al 2015), suggesting that for some protein substrates, proper folding may be required for substrate recognition.…”

Section: Discussionmentioning

confidence: 99%

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Revisiting the human polypeptide GalNAc-T1 and T13 paralogs

Festari

Trajtenberg²,

Berois

et al. 2016

Glycobiology

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Polypeptide GalNAc-transferases (GalNAc-Ts) constitute a family of 20 human glycosyltransferases (comprising 9 subfamilies), which initiate mucin-type O-glycosylation. The O-glycoproteome is thought to be differentially regulated via the different substrate specificities and expression patterns of each GalNAc-T isoforms. Here, we present a comprehensive in vitro analysis of the peptide substrate specificity of GalNAc-T13, showing that it essentially overlaps with the ubiquitous expressed GalNAc-T1 isoform found in the same subfamily as T13. We have also identified and partially characterized nine splice variants of GalNAc-T13, which add further complexity to the GalNAc-T family. Two variants with changes in their lectin domains were characterized by in vitro glycosylation assays, and one (Δ39Ex9) was inactive while the second one (Ex10b) had essentially unaltered activity. We used reverse transcription-polymerase chain reaction analysis of human neuroblastoma cell lines, normal brain and a small panel of neuroblastoma tumors to demonstrate that several splice variants (Ex10b, ΔEx9, ΔEx2-7 and ΔEx6/8-39bpEx9) were highly expressed in tumor cell lines compared with normal brain, although the functional implications remain to be unveiled. In summary, the GalNAc-T13 isoform is predicted to function similarly to GalNAc-T1 against peptide substrates in vivo, in contrast to a prior report, but is unique by being selectively expressed in the brain.

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Low Density Lipoprotein Receptor Class A Repeats Are O-Glycosylated in Linker Regions

Cited by 50 publications

References 62 publications

The Proprotein Convertase Subtilisin/Kexin Type 9-resistant R410S Low Density Lipoprotein Receptor Mutation

The Proprotein Convertase Subtilisin/Kexin Type 9-resistant R410S Low Density Lipoprotein Receptor Mutation

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Single Domain Antibodies Are Potent Inhibitors of Low Density Lipoprotein Receptor Degradation

Revisiting the human polypeptide GalNAc-T1 and T13 paralogs

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