Ligand-binding Repeats Research Articles

LDL receptor-related protein 5 selectively transports unesterified polyunsaturated fatty acids to intracellular compartments

Polyunsaturated fatty acids (PUFAs), which cannot be synthesized by animals and must be supplied from the diet, have been strongly associated with human health. However, the mechanisms for their accretion remain poorly understood. Here, we show that LDL receptor-related protein 5 (LRP5), but not its homolog LRP6, selectively transports unesterified PUFAs into a number of cell types. The LDLa ligand-binding repeats of LRP5 directly bind to PUFAs and are required and sufficient for PUFA transport. In contrast to the known PUFA transporters Mfsd2a, CD36 and FATP2, LRP5 transports unesterified PUFAs via internalization to intracellular compartments including lysosomes, and n-3 PUFAs depend on this transport mechanism to inhibit mTORC1. This LRP5-mediated PUFA transport mechanism suppresses extracellular trap formation in neutrophils and protects mice from myocardial injury during ischemia-reperfusion. Thus, this study reveals a biologically important mechanism for unesterified PUFA transport to intracellular compartments.

Ovarian expression and localization of a low-density lipoprotein receptor related protein 13 (Lrp13) in orange-spotted grouper (Epinephelus coioides)

Low-density lipoprotein receptor (LDLR) superfamily is an ancient gene family composed of different genes encoding membrane receptors involved in ligand binding and endocytosis. These receptors typically consist of class A ligand-binding repeats (LBRs), epidermal growth factor precursor-like (EGF) domain, class b YWXD motifs, transmembrane (TM) domain, and cytoplasmic (CP) domain. Low-density lipoprotein receptor related protein 13 (Lrp13), a vitellogenin receptor with a unique domain structure, belonging to the LDLR superfamily, would play an important role in vitellogenesis in teleosts. In this study, the full-length cDNA of Ec-lrp13 was isolated from the ovary of orange-spotted grouper (Epinephelus coioides), an economically important aquaculture species in China. The open reading frame (ORF) of Ec-lrp13 is 3831 bp in length, encoding 1276 amino acids. The Ec-Lrp13 includes several typical domains of the LDLR superfamily and contains the specific structure of the Lrp13 protein. Ec-lrp13 is predominantly expressed in ovary. During ovarian development, Ec-lrp13 was highly expressed in stage II and III ovaries but its mRNA level was significantly decreased in stage IV ovary. In situ hybridization (ISH) analysis revealed that Ec-lrp13 mRNA signals were obviously observed in the ooplasm of PG and EPV oocytes, but decreased in the LPV oocytes, and weakly found in the VG oocytes. Immunohistochemistry (IHC) detection showed that Ec-Lrp13 protein signals were only observed in the LPV and VG oocytes. Our data provides preliminary evidence that Lrp13 may be involved in the Vtg uptake during ovarian development of orange-spotted grouper.

Human APOER2 Isoforms Have Differential Cleavage Events and Synaptic Properties.

Human apolipoprotein E receptor 2 (APOER2) is a type I transmembrane protein with a large extracellular domain (ECD) and a short cytoplasmic tail. APOER2-ECD contains several ligand-binding domains (LBDs) that are organized into exons with aligning phase junctions, which allows for in-frame exon cassette splicing events. We have identified 25 human APOER2 isoforms from cerebral cortex using gene-specific APOER2 primers, where the majority are exon-skipping events within the N-terminal LBD regions compared with six identified in the heart. APOER2 undergoes proteolytic cleavage in response to ligand binding that releases a C-terminal fragment (CTF) and transcriptionally active intracellular domain (ICD). We tested whether the diversity of human brain-specific APOER2 variants affects APOER2 cleavage. We found isoforms with differing numbers of ligand-binding repeats generated different amounts of CTFs compared with full-length APOER2 (APOER2-FL). Specifically, APOER2 isoforms lacking exons 5-8 (Δex5-8) and lacking exons 4-6 (Δex4-6) generated the highest and lowest amounts of CTF generation, respectively, in response to APOE peptide compared with APOER2-FL. The differential CTF generation of Δex5-8 and Δex4-6 coincides with the proteolytic release of the ICD, which mediates transcriptional activation facilitated by the Mint1 adaptor protein. Functionally, we demonstrated loss of mouse Apoer2 decreased miniature event frequency in excitatory synapses, which may be because of a decrease in the total number of synapses and/or VAMP2 positive neurons. Lentiviral infection with human APOER2-FL or Δex4-6 isoform in Apoer2 knockout neurons restored the miniature event frequency but not Δex5-8 isoform. These results suggest that human APOER2 isoforms have differential cleavage events and synaptic properties.SIGNIFICANCE STATEMENT Humans and mice share virtually the same number of protein-coding genes. However, humans have greater complexity of any higher eukaryotic organisms by encoding multiple protein forms through alternative splicing modifications. Alternative splicing allows pre-mRNAs transcribed from genes to be spliced in different arrangements, producing structurally and functionally distinct protein variants that increase proteomic diversity and are particularly prevalent in the human brain. Here, we identified 25 distinct human APOER2 splice variants from the cerebral cortex using gene-specific APOER2 primers, where the majority are exon-skipping events that exclude N-terminal ligand-binding regions of APOER2. We show that some of the APOER2 variants have differential proteolytic properties in response to APOE ligand and exhibit distinct synaptic properties.

Examination of a Low-density Lipoprotein Receptor Relative with 11 Ligand-binding Repeats (LR11) as a Biomarker in Esophageal Cancer.

Some previous studies reported that the levels of a low-density lipoprotein receptor relative with 11 ligand-binding repeats (LR11) was a prognostic marker in some malignant tumors; however, whether LR11 is related to survival in patients with esophageal cancer remains unclear. In this study, we measured LR11 in the preoperative serum of 46 patients of esophageal cancer who undergoing surgery using a sandwich enzyme-linked immunosorbent assay (ELISA) method with anti-LR11 monoclonal antibodies. We investigated the correlation between the level of LR11 and survival of patients with esophageal cancer. Clinicopathological data were retrospectively retrieved from our institution's database. The patients were divided into two groups (low LR11 and high LR11) based on the level of LR11. There was no statistical difference in clinicopathological factors between these two groups. The low LR11 group had a significantly longer overall survival than the high LR11 group. LR11 can be measured with a relatively simple ELISA and is potentially a new prognostic marker for esophageal cancer.

Circulating sLR11 levels predict severity of pulmonary hypertension due to left heart disease.

Heart failure is a severe condition often involving pulmonary hypertension (PH). Soluble low-density lipoprotein receptor with 11 ligand-binding repeats (sLR11) has been associated with pulmonary artery hypertension. We examined whether sLR11 correlates with PH in left heart disease and can be used as a predictive marker. We retrospectively analyzed patients with severe mitral regurgitation who underwent right heart catheterization before surgery for valve replacement or valvuloplasty from November 2005 to October 2012 at Juntendo University. We measured sLR11 levels before right heart catheterization and analyzed correlations with pulmonary hemodynamics. We compared prognoses between a group with normal sLR11 (≤9.4 ng/ml) and a group with high sLR11 (>9.4 ng/ml). Follow-up was continued for 5 years, with end points of hospitalization due to HF and death due to cardiovascular disease. Among 34 patients who met the inclusion criteria, sLR11 correlated with mean pulmonary artery pressure (r = 0.54, p<0.001), transpulmonary pressure gradient (r = 0.42, p = 0.012), pulmonary vascular resistance (r = 0.36, p<0.05), and log brain natriuretic peptide (BNP). However, logBNP did not correlate with pulmonary vascular resistance (p = 0.6). Levels of sLR11 were significantly higher in the 10 patients with PH (14.4±4.3 ng/ml) than in patients without PH (9.9±3.9 ng/ml; p = 0.002). At 5 years, the event rate was higher in the high-sLR11 group than in the normal-sLR11 group. The high-sLR11 group showed 5 hospitalizations due to HF (25.0%) and 2 deaths (10.0%), whereas the normal-sLR11 group showed no hospitalizations or deaths. Analyses using receiver operating characteristic curves showed a higher area under the concentration-time curve (AUC) for sLR11 level (AUC = 0.85; 95% confidence interval (CI) = 0.72-0.98) than for BNP (AUC = 0.80, 95%CI = 0.62-0.99) in the diagnosis of PH in left heart disease. Concentration of sLR11 is associated with severity of PH and offers a strong predictor of severe mitral regurgitation in patients after surgery.

Knock out of a major vitellogenin receptor gene with eight ligand binding repeats in medaka (Oryzias latipes) using the CRISPR/Cas9 system

Recent studies of vitellogenesis engendered a novel model of teleost yolk formation in which multiple yolk precursors, vitellogenins (Vtgs), and their receptors (Vtgrs) interact to ensure proper yolk composition for embryonic development and larval growth. As a step toward verification of this concept, we examined the role of one candidate Vtgr, termed low-density lipoprotein receptor relative with eight ligand-binding repeat (Lr8), in the medaka, a representative teleost and established laboratory model. A homozygous lr8 knock out (lr8-KO) medaka was produced to perform reverse-genetic functional analyses. In ovaries of wild type (WT) medaka, Western blotting detected a putative Lr8 protein band at ~130 kDa, while immunohistochemistry detected the putative Lr8 signal at the periphery of the oocyte underneath the zona radiata. These signals disappeared in ovaries of the lr8-KO group. Offspring of lr8-KO medaka exhibited decreased survival rate compared to WT fish, but KO of lr8 was not 100% lethal. There was no significant difference in total yolk protein content or size of eggs between WT and lr8-KO fish. However, LC-MS/MS analyses revealed a remarkable decrease in the relative abundance of yolk proteins derived from VtgAb in lr8-KO eggs, in conjunction with a compensatory increase in proteins derived from VtgAa1. These findings strongly support the conclusion that Lr8 is an important receptor for VtgAb in medaka. The disruption of proper yolk composition by lr8-KO is possibly one cause of the low offspring survival.

Computational approach to explore the inhibitory potential of biologically derived compounds against Spodoptera litura vitellogenin receptor (VgR) using structure based virtual screening and molecular dynamics

In the everlasting combat against the pests of agricultural importance, it is essential to come up with a novel strategy for pest control. This can be achieved through understanding the insect pest at cellular and molecular levels. Vitellogenin (Vg) and vitellogenin receptor (VgR) are essential for successful reproduction in insect. The N-terminal Ligand Binding Domain (LBD) of VgR is responsible for the transportation of vital protein (Vg) to the developing oocyte through receptor mediated endocytosis pathway. This can be implemented to various predicaments and betterments for exploitation in pest management, in a target specific and eco-friendly manner. For this, natural metabolites isolated from various biological sources were used as ligand. The purpose of this study was to analyze the inhibitory potential of 14 biologically derived compounds against N-terminal (Ligand Binding Repeats) LBRs of S. litura by computational docking. 3 D structure of LBRs of S. litura was generated by Raptor X software and the structure was refined and validated. The validated structure was docked using autodock vina. Docking results revealed that spinosyn A and milbemycin A4 have inhibitory activity against VgR. Molecular dynamics (MD) simulation confirms the stable binding of the ligand. From the above results, spinosyn A and milbemycin A4 may be exploited for the pest management. Communicated by Ramaswamy H. Sarma

High-Affinity Binding of LDL Receptor-Related Protein 1 to Matrix Metalloprotease 1 Requires Protease:Inhibitor Complex Formation.

Matrix metalloprotease (MMP) activation contributes to the degradation of the extracellular matrix (ECM), resulting in a multitude of pathologies. Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifaceted endocytic and signaling receptor that is responsible for internalization and lysosomal degradation of diverse proteases, protease inhibitors, and lipoproteins along with numerous other proteins. In this study, we identified MMP-1 as a novel LRP1 ligand. Binding studies employing surface plasmon resonance revealed that both proMMP-1 and active MMP-1 bind to purified LRP1 with equilibrium dissociation constants (KD) of 19 and 25 nM, respectively. We observed that human aortic smooth muscle cells readily internalize and degrade 125I-labeled proMMP-1 in an LRP1-mediated process. Our binding data also revealed that all tissue inhibitors of metalloproteases (TIMPs) bind to LRP1 with KD values ranging from 23 to 33 nM. Interestingly, the MMP-1/TIMP-1 complex bound to LRP1 with an affinity (KD = 0.6 nM) that was 30-fold higher than that of either component alone, revealing that LRP1 prefers the protease:inhibitor complex as a ligand. Of note, modification of lysine residues on either proMMP-1 or TIMP-1 ablated the ability of the MMP-1/TIMP-1 complex to bind to LRP1. LRP1's preferential binding to enzyme:inhibitor complexes was further supported by the higher binding affinity for proMMP-9/TIMP-1 complexes than for either of these two components alone. LRP1 has four clusters of ligand-binding repeats, and MMP-1, TIMP-1, and MMP-1/TIMP-1 complexes bound to cluster III most avidly. Our results reveal an important role for LRP1 in controlling ECM homeostasis by regulating MMP-1 and MMP-9 levels.

Prognostic value of soluble ST2 and soluble LR11 on mortality and cardiovascular events in peritoneal dialysis patients

BackgroundAlthough the soluble form of suppression of tumorigenicity 2 (sST2) and soluble low-density lipoprotein receptor relative with 11 ligand-binding repeats (sLR11) have emerged as novel cardiovascular biomarkers in patients with cardiovascular disease, their prognostic value has not been fully investigated in peritoneal dialysis (PD) patients.MethodsWe included 74 prevalent PD patients from a prospective cohort and measured serum sST2 and sLR11 concentrations by an enzyme-linked immunosorbent assay. The association of these biomarkers and all-cause mortality and major adverse cardiac and cerebrovascular events (MACCEs) was evaluated.ResultsDuring a follow-up of 38.5 months, all-cause deaths and MACCEs were observed in 13 (17.6%) patients and 23 (31.3%) patients. Multivariable Cox analyses demonstrated that greater sST2 was independently associated with higher risk of all-cause mortality (≥75.8 ng/mL; hazard ratio [HR] = 5.551; 95% confidence interval [CI] = 1.360–22.660) and MACCEs (≥72.5 ng/mL; HR = 4.609; 95% CI = 1.608–13.208). Furthermore, sST2 showed additive predictive value for mortality to the base model including traditional risk factors (net reclassification index = 0.598, P = 0.04). sLR11 was not significantly associated with all-cause mortality or MACCE.ConclusionssST2, but not sLR11, indicated a significant prognostic value for all-cause mortality and cardiovascular events in PD patients. Further research is needed to validate emerging biomarkers in these populations.

Bone morphogenetic protein 1 cleaves the linker region between ligand-binding repeats 4 and 5 of the LDL receptor and makes the LDL receptor non-functional.

The cell-surface low-density lipoprotein receptor (LDLR) internalizes low-density lipoprotein (LDL) by receptor-mediated endocytosis and plays a key role in the regulation of plasma cholesterol levels. The ligand-binding domain of the LDLR contains seven ligand-binding repeats of approximately 40 residues each. Between ligand-binding repeats 4 and 5, there is a 10-residue linker region that is subject to enzymatic cleavage. The cleaved LDLR is unable to bind LDL. In this study, we have screened a series of enzyme inhibitors in order to identify the enzyme that cleaves the linker region. These studies have identified bone morphogenetic protein 1 (BMP1) as being the cleavage enzyme. This conclusion is based upon the use of the specific BMP1 inhibitor UK 383367, silencing of the BMP1 gene by the use of siRNA or CRISPR/Cas9 technology and overexpression of wild-type BMP1 or the loss-of-function mutant E214A-BMP1. We have also shown that the propeptide of BMP1 has to be cleaved at RSRR120↓ by furin-like proprotein convertases for BMP1 to have an activity towards the LDLR. Targeting BMP1 could represent a novel strategy to increase the number of functioning LDLRs in order to lower plasma LDL cholesterol levels. However, a concern by using BMP1 inhibitors as cholesterol-lowering drugs could be the risk of side effects based on the important role of BMP1 in collagen assembly.

Proteolysis of the low density lipoprotein receptor by bone morphogenetic protein-1 regulates cellular cholesterol uptake

The development of cardiovascular disease is intimately linked to elevated levels of low-density lipoprotein (LDL) cholesterol in the blood. Hepatic LDL receptor (LDLR) levels regulate the amount of plasma LDL. We identified the secreted zinc metalloproteinase, bone morphogenetic protein 1 (BMP1), as responsible for the cleavage of human LDLR within its extracellular ligand-binding repeats at Gly171↓Asp172. The resulting 120 kDa membrane-bound C-terminal fragment (CTF) of LDLR had reduced capacity to bind LDL and when expressed in LDLR null cells had compromised LDL uptake as compared to the full length receptor. Pharmacological inhibition of BMP1 or siRNA-mediated knockdown prevented the generation of the 120 kDa CTF and resulted in an increase in LDL uptake into cells. The 120 kDa CTF was detected in the livers from humans and mice expressing human LDLR. Collectively, these results identify that BMP1 regulates cellular LDL uptake and may provide a target to modulate plasma LDL cholesterol.

Strategies to prevent cleavage of the linker region between ligand-binding repeats 4 and 5 of the LDL receptor

A main strategy for lowering plasma low-density lipoprotein (LDL) cholesterol levels is to increase the number of cell-surface LDL receptors (LDLRs). This can be achieved by increasing the synthesis or preventing the degradation of the LDLR. One mechanism by which an LDLR becomes non-functional is enzymatic cleavage within the 10 residue linker region between ligand-binding repeats 4 and 5. The cleaved LDLR has only three ligand-binding repeats and is unable to bind LDL. In this study, we have performed cell culture experiments to identify strategies to prevent this cleavage. As a part of these studies, we found that Asp193 within the linker region is critical for cleavage to occur. Moreover, both 14-mer synthetic peptides and antibodies directed against the linker region prevented cleavage. As a consequence, more functional LDLRs were observed on the cell surface. The observation that the cleaved LDLR was present in extracts from the human adrenal gland indicates that cleavage of the linker region takes place in vivo. Thus, preventing cleavage of the LDLR by pharmacological measures could represent a novel lipid-lowering strategy.

Exploring Regulation of Protein O-Glycosylation in Isogenic Human HEK293 Cells by Differential O-Glycoproteomics

Most proteins trafficking the secretory pathway of metazoan cells will acquire GalNAc-type O-glycosylation. GalNAc-type O-glycosylation is differentially regulated in cells by the expression of a repertoire of up to twenty genes encoding polypeptide GalNAc-transferase isoforms (GalNAc-Ts) that initiate O-glycosylation. These GalNAc-Ts orchestrate the positions and patterns of O-glycans on proteins in coordinated, but poorly understood ways - guided partly by the kinetic properties and substrate specificities of their catalytic domains, as well as by modulatory effects of their unique GalNAc-binding lectin domains. Here, we provide the hereto most comprehensive characterization of nonredundant contributions of individual GalNAc-T isoforms to the O-glycoproteome of the human HEK293 cell using quantitative differential O-glycoproteomics on a panel of isogenic HEK293 cells with knockout of GalNAc-T genes (GALNT1, T2, T3, T7, T10, or T11). We confirm that a major part of the O-glycoproteome is covered by redundancy, whereas distinct O-glycosite subsets are covered by nonredundant GalNAc-T isoform-specific functions. We demonstrate that the GalNAc-T7 and T10 isoforms function in follow-up of high-density O-glycosylated regions, and that GalNAc-T11 has highly restricted functions and essentially only serves the low-density lipoprotein-related receptors in linker regions (C6XXXTC1) between the ligand-binding repeats.

SUN-072 PROGNOSTIC VALUE OF SOLUBLE ST2 AND SOLUBLE LR11 ON MORTALITY AND CARDIOVASCULAR EVENT IN PERITONEAL DIALYSIS PATIENTS

Because end-stage renal disease (ESRD) patients are at high risk of cardiovascular (CV) disease, developing a biomarker for CV risk stratification has clinical importance. Soluble form of suppression of tumorigenicity 2 (sST2) and soluble low-density lipoprotein receptor relative with 11 ligand-binding repeats (sLR11) are emerging CV biomarkers, however, to date, the prognostic value of those has not been investigated in patients undergoing peritoneal dialysis (PD). We determined serum sST2 and sLR11 concentrations using enzyme-linked immunosorbent assay, and evaluated the association of those biomarkers with all-cause mortality and major adverse cardiac and cerebrovascular event (MACCE) in 74 prevalent PD patients. The median (interquartile range) concentrations of sST2 and sLR11 were 70.9 (57.8-89.8) ng/mL and 15.2 (12.3-19.6) ng/mL, respectively. During a median follow-up of 38.5 months, 13 (17.6%) patients died and MACCE was observed in 23 (31.3%) patients. When patients were dichotomized by the median value of sST2 and sLR11, Kaplan-Meier analyses showed that higher sST2 group was significantly associated with lower event-free survival rates (log-rank test; P=0.002 for all-cause death; P=0.01 for MACCE). In multivariable Cox analyses, higher sST2 was independent risk factor for all-cause mortality (per 1 standard deviation [SD] increase; hazard ratio [HR]=1.947; 95% confidence interval [CI]=1.124-3.371) and MACCE (per 1 SD increase; HR=1.647; 95% CI=1.079-2.516). In contrast, sLR11 did not have a significant association with all-cause mortality or MACCE. Furthermore, only sST2 provided a significant predictive value for all-cause mortality (AUC=0.699; P=0.03). sST2, not sLR11, was independently associated with greater risk of all-cause mortality and CV outcome in prevalent PD patients. Additional studies are needed to confirm these findings and examine underlying mechanism between new biomarkers and CV disease in ESRD populations

Identification of amino acid residues in the ligand binding repeats of LDL receptor important for PCSK9 binding

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes LDL receptor (LDLR) degradation, increasing plasma levels of LDL cholesterol and the risk of cardiovascular disease. We have previously shown that, in addition to the epidermal growth factor precursor homology repeat-A of LDLR, at least three ligand-binding repeats (LRs) of LDLR are required for PCSK9-promoted LDLR degradation. However, how exactly the LRs contribute to PCSK9’s action on the receptor is not completely understood. Here, we found that substitution of Asp at position 172 in the linker between the LR4 and LR5 of full-length LDLR with Asn (D172N) reduced PCSK9 binding at pH 7.4 (mimic cell surface), but not at pH 6.0 (mimic endosomal environment). On the other hand, mutation of Asp at position 203 in the LR5 of full-length LDLR to Asn (D203N) significantly reduced PCSK9 binding at both pH 7.4 and pH 6.0. D203N also significantly reduced the ability of LDLR to mediate cellular LDL uptake, whereas D172N had no detectable effect. These findings indicate that amino acid residues in the LRs of LDLR play an important role in PCSK9 binding to the receptor.

Identification of full-length cDNA sequences for three development-relevant genes from yellow catfish Pelteobagrus fulvidraco and their transcriptional responses to high fat diet.

The goal of this study was to clone and characterize complete cDNA sequences of three important development-relevant genes of yellow catfish Pelteobagrus fulvidraco, including lrp6, sox9a1 and fgfr2c, and explore their transcriptional responses in several tissues of P. fulvidraco to high fat diet. The predicted amino acid sequences of P. fulvidraco Lrp6, Sox9a1 and Fgfr2c contained all of the conserved structural features that were characteristic of these genes in other species, including YWTD domains, EGF-like repeats, LDLR ligand binding repeats, PPSP repeats motifs, HMG box, TA-binding functional domain, Ig I-III and PTK I-II. The mRNAs of the three genes were expressed in various tissues, but their mRNA levels varied among tissues. Compared to the control, high fat diet tended to down-regulate the mRNA expression of sox9a1 and fgfr2c in mesenteric fat, liver and ovary, and up-regulate their mRNA levels in muscle and kidney; in contrast, high fat diet down-regulated lrp6 mRNA levels in the ovary and muscle, but had no significant effects on lrp6 mRNA expression in mesenteric fat, liver and kidney. Our findings provide the first data about their expression responses to dietary lipid in teleosts and reinforce the multiple functions at the molecular level.

Serum Levels of Soluble LR11 Are Associated with Insulin Resistance in the Patients with Type 2 Diabetes Mellitus

Obesity is now a worldwide epidemic, with overweight, obesity, and morbid obesity all increasing. Recently, there is increasing support that obesity is a major risk factor for developing insulin resistance and type 2 diabetes mellitus (T2D). Plasma soluble low-density lipoprotein receptor-relative with 11 ligand-binding repeats (sLR11) plays a role in the development of atherosclerosis, and has also been linked to the metabolism of triglyceride-rich lipoproteins and adiposity. To assess the relationship between sLR11 levels and T2D, we analyzed the sLR11 levels in T2D patients. Serum sLR11 levels were measured in 70 patients with T2D (mean age 60.8 ± 7.0, 68.6% males, and BMI 24.7 ± 3.2) and 76 healthy control subjects (mean age 58.5 ± 6.5, 65.8% males, and BMI 24.1 ± 2.5) by ELISA. Circulating sLR11 levels were significantly increased in T2D patients compared with healthy control subjects (10.2 ± 4.2 vs. 8.3 ± 2.1, P = 1.4 × 10-3). Stepwise multivariate regression analysis showed that serum sLR11 levels were significantly associated with HOMA-IR (β = 0.26, P = 4.8 × 10-3), BMI (β = 0.24, P = 1.0 × 10-2) and age (β = 0.20, P = 2.7 × 10-2). Logistic regression analyses indicated that sLR11 was independently associated with HOMA-IR (odds ratio [OR], sLR11 tertile increment: 6.81; 95% confidence interval [CI]: 1.71—27.13) and BMI (OR, sLR11 tertile increment: 1.86; 95% CI: 1.03—3.35). We then analyzed a gene-dosage effect that links LR11 expression to insulin resistance and obesity using db/db mice with genetic loss of LR11 (LR11-KO db/db). LR11-KO db/db mice displayed significantly lower fasting plasma glucose and insulin levels, and improved glucose tolerance compared with age-matched db/db mice, although there was no significant difference in body weight between them. Our findings imply that LR11 may be mechanistically involved in the prevalence and development of insulin resistance and T2D. Disclosure H. Unoki-Kubota: None. M. Jiang: None. H. Kajio: None. R. Yamamoto-Honda: None. K. Tobe: Research Support; Self; Bristol-Myers Squibb Company, Takeda Pharmaceutical Company, Teijin Pharma Limited, Japan Diabetes Society, Eli Lilly and Company, MSD K.K., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Pfizer Inc., Astellas Parma Inc, Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Tsumura &amp; Co., Ono Pharmaceutical Co., Ltd., Novartis Pharma K.K., Sumitomo Dainippon Pharma Co. M. Noda: None. H. Bujo: None. Y. Kaburagi: None.

Extracellular LDLR repeats modulate Wnt signaling activity by promoting LRP6 receptor endocytosis mediated by the Itch E3 ubiquitin ligase.

The LOW-density lipoprotein related protein 6 (LRP6) receptor is an important effector of canonical Wnt signaling, a developmental pathway, whose dysregulation has been implicated in various diseases including cancer. The membrane proximal low-density lipoprotein (LDL) receptor repeats in LRP6 exhibit homology to ligand binding repeats in the LDL receptor (LDLR), but lack known function. We generated single amino acid substitutions of LRP6-LDLR repeat residues, which are highly conserved in the human LDLR and mutated in patients with Familial Hypercholesteremia (FH). These substitutions negatively impacted LRP6 internalization and activation of Wnt signaling. By mass spectrometry, we observed that the Itch E3 ubiquitin ligase associated with and ubiquitinated wild type LRP6 but not the LDLR repeat mutants. These findings establish the involvement of LRP6-LDLR repeats in the regulation of canonical Wnt signaling.

Soluble LR11 associates with aortic root calcification in asymptomatic treated male patients with familial hypercholesterolemia

Background and aimsDespite statin treatment, a high prevalence of severe vascular calcification is found in patients with familial hypercholesterolemia (FH). We assessed the relation between the circulating soluble form of low-density lipoprotein receptor relative with 11 ligand-binding repeats (sLR11), a risk factor for cardiovascular disease, and vascular calcification in asymptomatic statin-treated heterozygous FH patients. MethodsIn 123 asymptomatic heterozygous FH patients (age 40–69 years), aortic root (ARC), aortic valve (AVC) and coronary artery calcification (CAC) were determined with CT-based calcium scoring expressed in Agatston units. Plasma sLR11 levels were measured by sandwich ELISA. ResultsSeventy-three patients displayed ARC, 48 had AVC and 96 CAC. Plasma sLR11 levels were positively correlated with the presence of ARC (r = 0.2, p = 0.03), but not with AVC or CAC. The correlation between sLR11 levels and ARC was restricted to male FH patients (r = 0.31, p = 0.006). Multivariate logistic analyses showed that the association of plasma sLR11 with the presence of ARC was independent of other determinants (Adjusted Odds Ratio, 2.01 (95% CI = 1.28–3.16) p = 0.002). ConclusionsPlasma sLR11 is associated with ARC in male FH patients and may be mechanistically involved in the differential distribution of atherosclerotic lesions in the vasculature.

Levels of the soluble LDL receptor-relative LR11 decrease in overweight individuals with type 2 diabetes upon diet-induced weight loss

Background and aimsCardiovascular disease (CVD) is a major complication in patients with type 2 diabetes (T2D), especially in those with obesity. Plasma soluble low density lipoprotein receptor-relative with 11 ligand-binding repeats (sLR11) plays a role in the development of atherosclerosis and has been linked to the metabolism of triglyceride-rich lipoproteins, adiposity, and vascular complications in T2D. We aimed to determine the effect of diet-induced weight loss on plasma sLR11 levels in overweight and obese individuals with T2D. MethodsPlasma sLR11 levels were determined in 64 individuals with T2D and BMI >27 kg/m2 before and after a 20-week weight loss diet. As a reference, sLR11 levels were also determined in 64 healthy, non-obese controls, matched as a group for age and sex. ResultsMedian plasma sLR11 levels of the T2D study-group at baseline (15.4 ng/mL (IQR 12.9–19.5)) were higher than in controls (10.2 (IQR: 8.7–12.2) ng/mL; p = 0.001). The diet resulted in a weight loss of 9.7 ± 5.2% (p = 0.001) and improved CVD risk factors. sLR11 levels were reduced to 13.3 ng/mL (IQR 11.0–17.1; p = 0.001). Changes in sLR11 levels positively associated with changes in non-HDL cholesterol (B = 1.54, R2 = 0.17, p = 0.001) and HbA1c (B = 0.07, R2 = 0.11, p = 0.007), but not with weight loss (B = 0.04, R2 = 0.05, p = 0.076). The changes in non-HDL cholesterol and HbA1c together explained 24% of the variance of sLR11 reduction (p = 0.001). ConclusionsWeight loss dieting in overweight and obese individuals with T2D resulted in a reduction in plasma sLR11 levels that was associated with improvements in lipid-profile and glycemic state.