International Journal of Molecular Sciences

Communication Differences in Recycling of E3 and E4—LDL Receptor Complexes—A Mechanistic Hypothesis

Meewhi Kim 1,* and Ilya Bezprozvanny 1,2,*

1 Department of Physiology, UT Southwestern Medical Center, Dallas, TX 75390, USA 2 Laboratory of Molecular Neurodegeneration, Peter the Great St. Petersburg State Polytechnic University, 195251 St. Petersburg, Russia * Correspondence: [email protected] (M.K.); [email protected] (I.B.)

Abstract: (ApoE) is a protein that plays an important role in the transport of fatty acids and and in cellular signaling. On the surface of the cells, ApoE lipoparticles bind to low density receptors (LDLR) that mediate the uptake of the and downstream signaling events. There are three alleles of the human ApoE . Presence of ApoE4 allele is a major risk factor for developing Alzheimer’s disease (AD) and other disorders late in life, but the mechanisms responsible for biological differences between different ApoE isoforms are not well understood. We here propose that the differences between ApoE isoforms can be explained by differences in the pH-dependence of the association between ApoE3 and ApoE4 isoforms and LDL-A repeats of LDLR. As a result, the following ApoE3-associated LDLRs are recycled back to the plasma membrane but ApoE4-containing LDLR complexes are trapped in late and targeted for degradation. The proposed mechanism is predicted to lead to a reduction in steady-  state surface levels of LDLRs and impaired cellular signaling in ApoE4-expressing cells. We hope  that this proposal will stimulate experimental research in this direction that allows the testing of Citation: Kim, M.; Bezprozvanny, I. our hypothesis. Differences in Recycling of Apolipoprotein E3 and E4—LDL Keywords: ApoE; LDL receptor; ; Alzheimer’s disease; modelling; protonation; Receptor Complexes—A Mechanistic charged interaction Hypothesis. Int. J. Mol. Sci. 2021, 22, 5030. https://doi.org/10.3390/ ijms22095030 1. Introduction Academic Editor: Michael Ugrumov Apolipoprotein E (ApoE) is a protein that plays an important role in the transport Received: 26 April 2021 of fatty acids and cholesterol and in cellular signaling [1–4]. On the surface of the cells, Accepted: 7 May 2021 ApoE lipoparticles bind to Low Density Lipoprotein receptors (LDLR) that mediate the Published: 10 May 2021 uptake of the lipids and downstream signaling events [1–4]. There are three alleles of the human ApoE gene—E2, E3, and E4—with two of these alleles co-expressed in many tissues, Publisher’s Note: MDPI stays neutral including the central nervous system [4–8]. These alleles differ in amino acids in positions with regard to jurisdictional claims in 112 and 158 of N-terminal ligand domains as follows—E2 (C112, C158), E3(C112, R158) published maps and institutional affil- and E4(R112, R158). There is well established genetic association between the presence of iations. different alleles of the ApoE gene and human diseases [9–13]. In particular, the presence of ApoE4 is a major risk factor for developing Alzheimer’s disease (AD) late in life [14–20]. The mechanisms responsible for biological differences between different ApoE isoforms are not well understood and are currently under intense investigation [21–23]. Copyright: © 2021 by the authors. The ApoE protein is composed of the amino-terminal (N) and carboxy-terminal (C) Licensee MDPI, Basel, Switzerland. domains, which are loosely hinged to each other [24–26]. The N-terminal ligand domain This article is an open access article (LD) is involved in the association with surface receptors including LDLR (Figure1A, distributed under the terms and Supplementary Figure S1); the C-terminal domain is involved in the binding of cholesterol conditions of the Creative Commons and fatty acids [27–29]. Both domains form α-helical bundles [24] with the critical allelic Attribution (CC BY) license (https:// positions C112 and C158, which are located on the side of the fourth and third α-helixes in creativecommons.org/licenses/by/ the ApoE-LD domain. 4.0/).

Int. J. Mol. Sci. 2021, 22, 5030. https://doi.org/10.3390/ijms22095030 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x Int. J. Mol. Sci. 2021, 22, 5030 2 of 10 2 of 10

Figure 1. Ligand domain (LD) of ApoE and LDL-A repeats of LDLR. (A) The secondary structure of ApoE-LD and the domainFigure structure 1. Ligand of LDLR domain are shown. (LD) The of receptor-bindingApoE and LDL interface‐A repeats is a partof LDLR. of the fourth(A) Theα-helix secondary in the ApoE-LD structure domain (shownof ApoE in blue).‐LD The and R112C the domain domain portionstructure of theof LDLR third α are-helix shown. in the The ApoE-LD receptor domain‐binding is shown interface in orange. is a part Position 112 withinof the the fourth R112C α‐ domainhelix in is the shown ApoE in purple.‐LD domain Seven amino-terminal(shown in blue). LDL-A The repeatsR112C aredomain shown portion as colored of semicircles.the (B) Primarythird α‐ sequencehelix in alignment the ApoE of‐LD R112C domain domains is shown from ApoE4 in orange. and ApoE3 Position isoforms. 112 Aminowithin acid the inR112C position domain 112 is shownis in redshown (R for ApoE4 in purple. and CSeven for ApoE3). amino (‐Cterminal) Primary LDL sequences‐A repeats of seven are LDL-Ashown repeats as colored from semicircles. the LDLR. The (B sequences) Pri‐ of RP1–RP7mary repeats sequence are color-coded alignment according of R112C to domains the panel Afrom diagram. ApoE4 and ApoE3 isoforms. Amino acid in position 112 is shown in red (R for ApoE4 and C for ApoE3). (C) Primary sequences of seven LDL‐ A repeats from the LDLR.LDLR The sequences is a single transmembraneof RP1–RP7 repeats domain are color protein‐coded that containsaccording seven to the class A repeats panel A diagram. (LDL-A) on its amino terminal (Figure1A, Supplementary Figure S1) [ 30]. These repeats are approximately 40 amino acids long and constrained by three internal disulphide bridges LDLR is a singlethat transmembrane contain conserved domain DCXDXSDE protein motifs that [31 contains] (Supplementary seven class Figure A repeats S1B). These do- (LDL‐A) on its aminomains terminal are involved (Figure in the 1A, association Supplementary with extracellular Figure S1) ligands [30]. [ 2These,6,32]. Therepeats cytosolic tail of LDLR mediates signal transduction and endocytosis [5,33–35]. Similar to other LDLR are approximatelyligands, 40 amino ApoE acids lipoparticles long and bind constrained to LDL-A amino-terminal by three internal repeats disulphide [36–41] (Figure 1A , bridges that containSupplementary conserved Figure DCXDXSDE S1). A ligand motifs binding [31] to (Supplementary the LDLR triggers cellularFigure signalingS1B). trans- These domains areduction involved and in the the internalization association ofwith the extracellular ligand–receptor ligands complex [2,6,32]. via -assisted The cy‐ en- tosolic tail of LDLRdocytosis mediates [1]. Thesignal internalized transduction complex and is sortedendocytosis to endosomal [5,33–35]. compartments Similar to where the other LDLR ligands,ligand ApoE is released lipoparticles and LDLR bind is recycled to LDL to‐A the amino plasma‐terminal membrane repeats [42–44]. [36–41] Multiple sources (Figure 1A, Supplementaryof evidence Figure demonstrate S1). A that ligand LDLR binding recycling to the requires LDLR organized triggers cellular cellular actions sig‐ includ- ing luminal endosomal acidification and calcium (Ca2+) signaling [45–51]. In particular, naling transduction and the internalization of the ligand–receptor complex via clathrin‐ endosomal acidification plays a key role in the control of LDLR recycling [40,52,53]. assisted endocytosis [1].Interestingly, The internalized experimental complex evidence is sorted suggests to thatendosomal there are compartments significant differences in where the ligand isthe released recycling and of LDLR LDLR complexed is recycled with to ApoE3the plasma or ApoE4 membrane [54–57]. However,[42–44]. Mul a mechanistic‐ tiple sources of evidenceexplanation demonstrate for the differences that LDLR in ApoE3- recycling and ApoE4-containingrequires organized complexes cellular is ac lacking.‐ To tions including luminalexplain endosomal these findings, acidification we propose thatand the calcium observed (Ca differences2+) signaling are due [45–51]. to the In differences particular, endosomalin the acidification pH-dependence plays of the a association key role betweenin the control ApoE3 andof LDLR ApoE4 recycling isoforms and LDL- [40,52,53]. A repeats of the LDLR. As a result, ApoE4-containing but not ApoE3-containing LDLR complexes are trapped in late endosomes and targeted for degradation, leading to a Interestingly,reduction experimental in the evidence steady state suggests surface that levels there of LDLRs are significant and impaired differences cellular signaling in in the recycling of LDLRApoE4-expressing complexed with cells. ApoE3 or ApoE4 [54–57]. However, a mechanistic explanation for the differences in ApoE3‐ and ApoE4‐containing complexes is lacking. To explain these findings, we propose that the observed differences are due to the differences in the pH‐dependence of the association between ApoE3 and ApoE4 isoforms and LDL‐ A repeats of the LDLR. As a result, ApoE4‐containing but not ApoE3‐containing LDLR complexes are trapped in late endosomes and targeted for degradation, leading to a re‐ duction in the steady state surface levels of LDLRs and impaired cellular signaling in ApoE4‐expressing cells.

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2. Results Estimated Association Energy between ApoE3/4 and LDL‐A Repeats of LDLR 2. Results EstimatedThe N Association‐terminal EnergyLD domain between of ApoE3/4ApoE is andfolded LDL-A into Repeats a bundle of LDLR of four α‐helixes (Figure 1A, Supplementary Figure S1). The portion of the fourth α‐helix contains Lysine‐ and Ar‐ The N-terminal LD domain of ApoE is folded into a bundle of four α-helixes (Figure1A , ginine‐rich region (labeled blue in Figure 1A and Supplementary Figure S1) which is di‐ Supplementary Figure S1). The portion of the fourth α-helix contains Lysine- and Arginine- rectlyrich region involved (labeled in the blue association in Figure with1A andLDLR Supplementary and other ApoE Figure receptors S1) which [24–26,36–41]. is directly The singleinvolved amino in the‐acid association difference with between LDLR andApoE3 other and ApoE ApoE4 receptors isoforms [24– at26 position,36–41]. The112 single(labeled purpleamino-acid in Figure difference 1A) is between located ApoE3 within and the ApoE4third α‐ isoformshelix of atthe position ApoE domain. 112 (labeled A portion purple of thein Figure third α‐1A)helix is located is shown within by the thirdorangeα-helix color ofin theFigure ApoE 1A domain. and Supplementary A portion of the Figure third S1 andα-helix the iscorresponding shown by the sequence orange color is shown in Figure in 1FigureA and 1B. Supplementary This portion Figure of the S1third and α‐ thehelix iscorresponding selected because sequence it is located is shown parallel in Figure to1 B.a portion This portion of the of fourth the third α‐helixα-helix that is selectedis directly involvedbecause it in is the located association parallel with to a LDLR portion (Figure of the fourth1A, Supplementaryα-helix that is Figure directly S1). involved We named in thisthe associationportion of the with third LDLR α‐helix (Figure the1A, “R112C Supplementary domain” Figureand its S1). sequence We named is shown this portion in Figure 1B.of theApoE3 third containsα-helix theCysteine “R112C and domain” ApoE4 andcontains its sequence Arginine is in shown position in Figure 112 (Figure1B. ApoE3 1B). As iscontains the case with ApoE3, and ApoE4 ApoE2 contains contains Arginine Cysteine in positionin position 112 112 (Figure and,1 B).for Asthe is purposes the case of ourwith study, ApoE3, we ApoE2 focused contains on ApoE3 Cysteine only. in We position reasoned 112 that and, the for Arginine the purposes is more of our likely study, to be protonatedwe focused by on the ApoE3 acidification only. We reasonedinside endosomal that the Arginine compartments is more than likely Cysteine, to be protonated leading to differencesby the acidification in the insidelocal charge endosomal of the compartments third α‐helix than between Cysteine, the leading ApoE3 to differences and ApoE4 α isoforms.in the local The charge third of α‐ thehelix third of ApoE-helix‐LD between is located the ApoE3 in close and proximity ApoE4 isoforms. to the binding The third inter‐ α-helix of ApoE-LD is located in close proximity to the binding interface between ApoE face between ApoE and LDLR (Figure 1A, Supplementary Figure 1). Thus, we reasoned and LDLR (Figure1A, Supplementary Figure S1). Thus, we reasoned that the changes that the changes in charge of the R112C domain may exert different electrostatic effects on in charge of the R112C domain may exert different electrostatic effects on the association the association between the ApoE‐LD and LDL‐A repeats of LDLR. between the ApoE-LD and LDL-A repeats of LDLR. ToTo testtest this this hypothesis, hypothesis, we we calculated calculated the localthe local charges charges of the ApoE3of the andApoE3 ApoE4 and R112C ApoE4 R112Cdomains domains as a function as a function of pH in of the pH range in the between range between 4 and 8. We4 and also 8. performed We also performed similar similarcalculations calculations for each for of the each seven of the LDL-A seven repeats LDL‐ (FigureA repeats1C). (Figure These calculations 1C). These predictedcalculations predictedgradual charge gradual shift charge from shift a negative from a charge negative at pH charge 8 to positiveat pH 8 chargeto positive at pH charge 4 for theseat pH 4 fordomains these domains (Figure2). (Figure At each 2). pH At value, each pH the value, local charge the local of thecharge R112C of the domain R112C from domain ApoE4 from ApoE4was more was positive more positive than the than local the charge local charge of the R112C of the R112C domain domain from ApoE3 from ApoE3 (Figure (Figure2A). 2A).With With some some variability variability among among LDL-A LDL repeats,‐A repeats, at each at pHeach value pH value the charge the charge of the of seventh the sev‐ enthrepeat repeat (RP7) (RP7) was thewas most the positivemost positive and the and charge the charge of the firstof the repeat first (RP1)repeat was (RP1) the was most the mostnegative negative (with (with the exception the exception of pH values of pH belowvalues 4.6, below where 4.6, the where fourth the repeat, fourth RP4, repeat, became RP4, becamethe most the negative) most negative) (Figure2 B).(Figure 2B).

FigureFigure 2. 2. LocalLocal charge charge calculations calculations for for the the R112C R112C domains domains of ApoE3/4 of ApoE3/4 and LDL and− LDLA repeats.−A repeats. (A) Local(A) Local charges charges of R112C of R112C domain domain from fromApoE3 ApoE3 (red line) (red and line) ApoE and ApoE 4 (black 4 (black line) were line) calculated were calcu- as alated function as a functionof pH in ofthe pH range in the between range between 4 and 8. 4 (B and) Local 8. (B charges) Local charges of each ofof eachseven of LDL seven−A LDL domains−A weredomains calculated were calculated as a function as a functionof pH in ofthe pH range in the between range between 4 and 8. 4 The and charge 8. The value charge curve value for curve each LDLfor each−A repeat LDL− isA color repeat‐coded is color-coded as on panel as on 1A. panel 1A.

The energy of electrostatic interactions between the R112C domain of ApoE and each of the LDL-A repeats of LDLR is governed by Coulomb’ law, which states that the

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Int. J. Mol. Sci. 2021, 22, 5030 The energy of electrostatic interactions between the R112C domain of ApoE and4 of 10each of the LDL‐A repeats of LDLR is governed by Coulomb’ law, which states that the inter‐ action energy is proportional to the product of these local charges. The positive product represents a repulsive electrostatic effect, and the negative product represents an attrac‐ interaction energy is proportional to the product of these local charges. The positive tive electrostatic effect. In the first approximation, we considered the R112C domain and product represents a repulsive electrostatic effect, and the negative product represents an eachattractive of the electrostatic LDL‐A repeats effect. Inas thepoint first charges approximation, and calculated we considered the product the R112C of these domain local chargesand each (α of) theas a LDL-A function repeats of pH. as point The calculated charges and values calculated of α thefor productthe ApoE3 of these and localApoE4 R112Ccharges domains (α) as a functionare shown of in pH. Figure The calculated3A,B. In the values case of αApoE3for the at ApoE3 each pH and value, ApoE4 the α wasR112C either domains positive are or shown close in to Figure 0 at pH3A,B. values In the near case 4.75 of (Figure ApoE3 3A). at each Only pH for value, the first the re‐ peat,α was RP1, either was positive the α slightly or close negative to 0 at pH in values the range near between 4.75 (Figure 4.253 A).and Only 4.75 (Figure for the first 3A). In contrast,repeat, RP1, for wasApoE4, the αtheslightly α was negative negative in for the most range LDL between‐A repeats 4.25 andin the 4.75 range (Figure of 3pHA). be‐ tweenIn contrast, 4.5 and for ApoE4,6 (Figure the 3B).α was To negativeestimate forcombined most LDL-A effect repeats on association in the range between of pH the R112Cbetween domains4.5 and and 6 (Figure LDL‐3AB) repeats,. To estimate we averaged combined the effectvalues on of association α across all between seven repeats. the TheR112C average domains interaction and LDL-A curves repeats, (Figure we averaged3C) suggest the strong values repulsive of α across interactions all seven repeats. between theThe R112C average domain interaction of ApoE3 curves and (Figure LDL3‐C)A repeats suggest in strong the range repulsive of pH interactions between 5 between and 8, and betweenthe R112C 4 domainand 4.5 of(Figure ApoE3 3C), and and LDL-A minimal repeats interactions in the range in of the pH range between of pH 5 and between 8, and 4.5 andbetween 5 (Figure 4 and 4.53C). (Figure In contrast,3C), and there minimal is a interactionsweak repulsive in the interaction range of pH between between the 4.5 andR112C domain5 (Figure of3C). ApoE4 In contrast, and LDL there‐A is repeats a weak in repulsive the range interaction of pH between between 7 theand R112C 8, minimal domain inter‐ actionof ApoE4 in the and pH LDL-A range repeats between in the 6 rangeand 7, of and pH attraction between 7 in and the 8, pH minimal range interaction between 4.5 in the and 6 (FigurepH range 3C). between Typical 6 andphysiological 7, and attraction pH outside in the of pH the range cells between is close 4.5to 7.5 and [44,49,58,59]. 6 (Figure3C) The. Typical physiological pH outside of the cells is close to 7.5 [44,49,58,59]. The range of range of pH values inside endocytic compartments is between 5.5 for late endosomes (LE) pH values inside endocytic compartments is between 5.5 for late endosomes (LE) and and 6.5 for early endosomes (EE) [47,51,60,61]. There is a significant difference in electro‐ 6.5 for early endosomes (EE) [47,51,60,61]. There is a significant difference in electrostatic static interaction energies between R112C domains from ApoE3 and ApoE4 and LDL‐A interaction energies between R112C domains from ApoE3 and ApoE4 and LDL-A repeats repeatsin the pH in rangethe pH between range between 4.5 and 4.5 7.5 and (Figure 7.5 3(FigureC). We 3C). propose We propose that this that difference this difference may maycontribute contribute to biological to biological differences differences between between these isoforms. these isoforms.

Figure 3. The product ofof ionicionic chargescharges ((αα)) ofof electrostatic electrostatic interactions interactions between between R112C R112C domains domains of of ApoE3/4 and and LDL LDL−−AA repeats repeats (A (A,B,B).). The The product product of of ionic ionic charges charges ( (αα)) of of the the R112C R112C domain domain from from ApoE3 (A)) and ApoE4 ((BB)) andand eacheach ofof thethe sevenseven LDLLDL−−A repeats is shown as a function ofof pHpH inin the range between 44 andand 8.8. TheThe valuesvalues of ofα α forfor each each LDL-A LDL‐A repeat repeat are are color color coded coded as as on on panel panel 1A. 1A. (C) Average value value of ofα α forfor all all LDL LDL−−AA repeats repeats are are shown shown as as a functiona function of of pH pH in thein the range range between between 4 4and and 8 for8 for ApoE3 ApoE3 (red (red line) line) and and ApoE4 ApoE4 (black (black line). line).

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3. Discussion Endocytosis and Recycling of ApoE3 and ApoE4-Containing LDLR Complexes Expression of ApoE4 allele is a strong risk factor for Alzheimer’s disease (AD) and for many other disorders of aging [15,18,62]. In contrast, expression of the ApoE3 allele does not have such an effect. There is experimental evidence that defects in the recycling of ApoE4 and its receptors could contribute to brain dysfunction in AD patients [55,63–65]. Based on our modeling results (Figure3), we propose a mechanistic explanation for the differences in recycling of ApoE3-containing and ApoE4-containing receptor complexes (Figure4). Specifically, we propose that both ApoE3 and ApoE4 bind LDLR on the surface of the cell. At pH values of 7.5 in the extracellular media, we expect that the affinity of the ApoE3 isoform for LDLR will be slightly lower than the affinity of the ApoE4 isoform due to stronger electrostatic repulsion between the R112C domain and LDL-A repeats (Figure3C). This prediction is consistent with experimental studies of ApoE3 and ApoE4 association with LDLR [63,64,66,67]. Following internalization after clathrin-mediated endocytosis, ApoE3-containing and ApoE4-containing LDLR complexes move to very early endosomal (VEE) compartments (Figure4). The pH values inside these compartments are in the range between 7.0 and 7.5 [47,51,60,61], which is similar to extracellular media. From VEE compartments, ApoE-LDLR complexes proceed to early endosomal compartments (EE) with intraluminal pH in the range between 6 and 7 [47,51,60,61]. At these pH values, our model predicts significant electrostatic repulsion between the R112C domain of ApoE3 and LDL-A repeats, but minimal electrostatic interactions between the R112C domain of ApoE4 and LDL-A repeats (Figure3C). Thus, LDLRs are able to easily dissociate from the ApoE3 complex inside EE and recycle back to the plasma membrane (Figure4). In contrast, a significant fraction of LDLRs remain bound to ApoE4 and trapped in EE compartments (Figure4). Remaining complexes of LDLR and ApoE3/4 move to the late endosomal compartment (LE) (Figure4) with intraluminal pH in the range of 5.0–6.0 [ 47,51,60,61]. At these pH values, our model predicts electrostatic repulsion between the R112C domain of ApoE3 and LDL-A but electrostatic attraction between the R112C domain of ApoE4 and LDL-A repeats (Figure3C). Therefore, inside LE, remaining LDLR dissociate from ApoE3 complexes for recycling to the plasma membrane, but are trapped in ApoE4 complexes and targeted to for degradation (Figure4). Due to defects in recycling, our model predicts that steady state levels of LDLR on the plasma membrane surface are lower in aging ApoE4-expressing cells than in ApoE3-expressing cells (Figure4). Although not modelled in this study, it is likely that similar conclusions can be reached regarding other types of ApoE receptors in addition to LDLR. Even among patients that express the ApoE4 isoform, there is a significant variability in developing AD and in the rate of disease progression. It is possible that this variability can be attributed to the differences in sex and in life-long environmental stresses and factors [68,69]. Other factors that could affect ApoE-LDLR recycling are related to post- translational protein modification. For example, effects of O- or N-glycosylation of LDLR have been shown to affect its ligand affinity [70–72]. Additional modelling studies will be required to incorporate the potential effects of these modifications of LDLR on the association with ApoE3 and ApoE4. In summary, we propose that the reduction in levels of LDLR and other ApoE-binding receptors at the plasma membrane (Figure4) leads to impaired metabolism and cellular signaling and predisposes these neurons to disorders of aging and AD. We hope that this proposal will stimulate experimental research in this direction that allows the testing of our hypothesis. Int. J. Mol. Sci. 2021, 22, x 6 of 10 Int. J. Mol. Sci. 2021, 22, 5030 6 of 10

FigureFigure 4. 4. EndocytosisEndocytosis andand recycling recycling of of ApoE3 ApoE3 and and ApoE4-containing ApoE4‐containing LDLR LDLR complexes. complexes. Surface Surface LDLRLDLR (R) (R) form form complexescomplexes with with ApoE3 ApoE3 (E3) (E3) and and ApoE4 ApoE4 (E4) at (E4) plasma at plasma membrane membrane (PM). Following (PM). Following internalizationinternalization afterafter clathrin-mediated clathrin‐mediated endocytosis, endocytosis, these these complexes complexes are moved are frommoved the veryfrom early the very earlyendosomal endosomal (VEE) (VEE) compartment compartment to the endosomal to the endosomal compartment compartment (EE) and the (EE) late endosomaland the late com- endosomal compartmentpartment (LE). (LE). Dissociation Dissociation of ApoE of allows ApoE recycling allows recycling of LDLR to of the LDLR plasma to membrane. the plasma The membrane. LDLR The LDLRtrapped trapped in LE compartments in LE compartments are targeted are for targeted degradation for degradation by lysosomes. by The lysosomes. range of pH The values range for of pH valueseach endosomal for each endosomal compartment compartment is indicated. is indicated. 4. Materials and Methods EvenCrystal among structures patients of the that ApoE express N-terminal the ApoE4 LD region isoform, (pdb there 1B68 and is a 1NFN)significant [21,73 variability] inand developing the extracellular AD and domain in the of LDLRrate of (pdb disease 1N7D progression. and 1AJJ) [74, 75It ]is were possible downloaded that this from variability canPDB be bank attributed and analyzed to the using differences Coot (Crystallographic in sex and in Object-Oriented life‐long environmental Toolkit, v0.8.9.2) stresses [76] and fac‐ torsand [68,69]. Pymol Other (The PyMOL factors Molecularthat could Graphics affect ApoE System,‐LDLR Version recycling 2.0 Schrödinger, are related LLC) to posttrans‐ lationalprograms. protein A publicly modification. available For calculator example, program effects (PROTEIN of O‐ or N CALCULATOR‐glycosylation v3.4,of LDLR have http://protcalc.sourceforge.net/, accessed on 1 December 2020) was used to estimate been shown to affect its ligand affinity [70–72]. Additional modelling studies will be re‐ charges. The graphs and Figures were produced using Origin v9.0 (OriginLab Corporation, quiredNorthampton, to incorporate MA, USA). the potential effects of these modifications of LDLR on the associa‐ tion with ApoE3 and ApoE4. SupplementaryIn summary, Materials: we proposeThe following that arethe available reduction online in at levels https://www.mdpi.com/article/10 of LDLR and other ApoE‐bind‐ ing.3390/ijms22095030/s1 receptors at the plasma, Figure S1. membrane Structures of (Figure ligand domain 4) leads (LD) to of impaired ApoE and lipid LDL-A metabolism repeats and cellularof LDLR. signaling and predisposes these neurons to disorders of aging and AD. We hope thatAuthor this Contributions: proposal willConceptualization, stimulate experimental M.K.; methodology, research M.K.; formalin this analysis, direction M.K. andthat I.B.; allows the testinginvestigation, of our M.K.; hypothesis. resources, I.B.; data curation, M.K. and I.B.; writing—original draft preparation, M.K. and I.B.; writing—review and editing, M.K. and I.B.; visualization, M.K. and I.B.; supervision, I.B.; project administration, I.B.; funding acquisition, I.B. All authors have read and agreed to the 4.published Materials version and of Methods the manuscript. Funding:CrystalThis structures research was of funded the byApoE the Russian N‐terminal Science LD Foundation region Grant (pdb 19-15-00184 1B68 and (I.B.) 1NFN) and [21,73] andby the the National extracellular Institutes ofdomain Health grants of LDLR R01NS056224 (pdb 1N7D (I.B.) and and R01AG055577 1AJJ) [74,75] (I.B.). I.B.were holds downloaded the fromCarl J.PDB and Hortense bank and M. Thomsen analyzed Chair using in Alzheimer’s Coot (Crystallographic Disease Research. Object‐Oriented Toolkit, v0.8.9.2) [76] and Pymol (The PyMOL Molecular Graphics System, Version 2.0 Schrö‐ dinger, LLC) programs. A publicly available calculator program (PROTEIN CALCULA‐ TOR v3.4, http://protcalc.sourceforge.net/, accessed on 1 December 2020) was used to es‐ timate charges. The graphs and Figures were produced using Origin v9.0 (OriginLab Cor‐ poration, Northampton, MA, USA)

Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1. Structures of ligand domain (LD) of ApoE and LDL‐A repeats of LDLR.

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Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We are thankful to members of Bezprozvanny laboratory for useful discussions. Conflicts of Interest: The authors declare no conflict of interest.

References 1. Brown, M.S.; Herz, J.; Goldstein, J.L. Calcium cages, acid baths and recycling receptors. Nat. Cell Biol. 1997, 388, 629–630. [CrossRef] 2. Holtzman, D.M.; Herz, J.; Bu, G. Apolipoprotein E and Apolipoprotein E Receptors: Normal Biology and Roles in Alzheimer Disease. Cold Spring Harb. Perspect. Med. 2012, 2, a006312. [CrossRef] 3. Mahley, R.W.; Innerarity, T.L.; Rall, S.C., Jr.; Weisgraber, K.H. Plasma : Apolipoprotein structure and function. J. Lipid Res. 1984, 25, 1277–1294. [CrossRef] 4. Braesch-Andersen, S.; Paulie, S.; Smedman, C.; Mia, S.; Kumagai-Braesch, M. ApoE Production in Human Monocytes and Its Regulation by Inflammatory Cytokines. PLoS ONE 2013, 8, e79908. [CrossRef] 5. Herz, J.; Beffert, U. Apolipoprotein E receptors: Linking brain development and Alzheimer’s disease. Nat. Rev. Neurosci. 2000, 1, 51–58. [CrossRef] 6. Rebeck, G.W.; Ladu, M.J.; Estus, S.; Bu, G.; Weeber, E.J. The generation and function of soluble apoE receptors in the CNS. Mol. Neurodegener. 2006, 1, 15. [CrossRef] 7. Davenport, P.; Tipping, P.G. The Role of Interleukin-4 and Interleukin-12 in the Progression of in Apolipoprotein E-Deficient Mice. Am. J. Pathol. 2003, 163, 1117–1125. [CrossRef] 8. Deng, M.; Gui, X.; Kim, J.; Xie, L.; Chen, W.; Li, Z.; He, L.; Chen, Y.; Chen, H.; Luo, W.; et al. LILRB4 signalling in leukaemia cells mediates T cell suppression and tumour infiltration. Nat. Cell Biol. 2018, 562, 605–609. [CrossRef] 9. Maeda, H.; Nakamura, H.; Kobori, S.; Okada, M.; Mori, H.; Niki, H.; Ogura, T.; Hiraga, S. Identification of Human Apolipoprotein E Variant Gene: Apolipoprotein E7 (Glu244,245↑Lys244,245)1. J. Biochem. 1989, 105, 51–54. [CrossRef] 10. Wardell, M.R.; Weisgraber, K.H.; Havekes, L.M.; Rall, S.C., Jr. Apolipoprotein E3-Leiden contains a seven-amino acid insertion that is a tandem repeat of residues 121–127. J. Biol. Chem. 1989, 264, 21205–21210. [CrossRef] 11. Paik, Y.K.; Chang, D.J.; Reardon, C.A.; Davies, G.E.; Mahley, R.W.; Taylor, J.M. Nucleotide sequence and structure of the humanapolipoprotein E gene. Proc. Natl. Acad. Sci. USA 1985, 82, 3445–3449. [CrossRef] 12. Nguyen, T.T.; Kruckeberg, K.E.; O’Brien, J.F.; Ji, Z.-S.; Karnes, P.S.; Crotty, T.B.; Hay, I.D.; Mahley, R.W.; O’Brien, T. Familial Splenomegaly: Hypercatabolism of Lipoproteins Associated with Apolipoprotein E [Apolipoprotein E (∆149 Leu)]. J. Clin. Endocrinol. Metab. 2000, 85, 4354–4358. [CrossRef] 13. Oikawa, S.; Matsunaga, A.; Saito, T.; Sato, H.; Seki, T.; Hoshi, K.; Hayasaka, K.; Kotake, H.; Midorikawa, H.; Sekikawa, A.; et al. Apolipoprotein E Sendai (arginine 145–>proline): A new variant associated with lipoprotein glomerulopathy. J. Am. Soc. Nephrol. 1997, 8, 820–823. [CrossRef] 14. Robinson, M.; Lee, B.Y.; Hane, F.T. Recent Progress in Alzheimer’s Disease Research, Part 2: Genetics and Epidemiology. J. Alzheimer’s Dis. 2017, 57, 317–330. [CrossRef] 15. Glorioso, C.A.; Pfenning, A.R.; Lee, S.S.; Bennett, D.A.; Sibille, E.L.; Kellis, M.; Guarente, L.P. Rate of brain aging and APOE epsilon4 are synergistic risk factors for Alzheimer’s disease. Life Sci. Alliance 2019, 2.[CrossRef] 16. Safieh, M.; Korczyn, A.D.; Michaelson, D.M. ApoE4: An emerging therapeutic target for Alzheimer’s disease. BMC Med. 2019, 17, 1–17. [CrossRef] 17. Simon, R.; Girod, M.; Fonbonne, C.; Salvador, A.; Clement, Y.; Lanteri, P.; Amouyel, P.; Lambert, J.C.; Lemoine, J. Total ApoE and ApoE4 isoform assays in an Alzheimer’s disease case-control study by targeted mass spectrometry (n = 669): A pilot assay for methionine-containing proteotypic peptides. Mol. Cell. Proteomics 2012, 11, 1389–1403. [CrossRef] 18. Yamazaki, Y.; Painter, M.M.; Bu, G.; Kanekiyo, T. Apolipoprotein E as a Therapeutic Target in Alzheimer’s Disease: A Review of Basic Research and Clinical Evidence. CNS Drugs 2016, 30, 773–789. [CrossRef] 19. Kanai, M.; Shizuka, M.; Urakami, K.; Matsubara, E.; Harigaya, Y.; Okamoto, K.; Shoji, M. Apolipoprotein E4 accelerates dementia and increases cerebrospinal fluid tau levels in Alzheimer’s disease. Neurosci. Lett. 1999, 267, 65–68. [CrossRef] 20. Wahrle, S.E.; Shah, A.R.; Fagan, A.M.; Smemo, S.; Kauwe, J.S.K.; Grupe, A.; Hinrichs, A.; Mayo, K.; Jiang, H.; Thal, L.J.; et al. Apolipoprotein E levels in cerebrospinal fluid and the effects of ABCA1 polymorphisms. Mol. Neurodegener. 2007, 2, 7–9. [CrossRef] 21. Dong, J.; Peters-Libeu, C.A.; Weisgraber, K.H.; Segelke, B.W.; Rupp, B.; Capila, I.; Hernáiz, M.J.; Lebrun, L.A.; Linhardt, R.J. Interaction of the N-Terminal Domain of Apolipoprotein E4 with Heparin. Biochemistry 2001, 40, 2826–2834. [CrossRef] 22. Segelke, B.W.; Forstner, M.; Knapp, M.; Trakhanov, S.D.; Parkin, S.; Newhouse, Y.M.; Bellamy, H.D.; Weisgraber, K.H.; Rupp, B. Conformational flexI.B.ility in the apolipoprotein E amino-terminal domain structure determined from three new crystal forms: Implications for lipid binding. Protein Sci. 2000, 9, 886–897. [CrossRef][PubMed] Int. J. Mol. Sci. 2021, 22, 5030 8 of 10

23. Weers, P.M.; Narayanaswami, V.; Choy, N.; Luty, R.; Hicks, L.; Kay, C.M.; Ryan, R.O. Lipid binding ability of human apolipoprotein E N-terminal domain isoforms: Correlation with protein stability? Biophys. Chem. 2002, 100, 481–492. [CrossRef] 24. Hatters, D.M.; Peters-Libeu, C.A.; Weisgraber, K.H. Apolipoprotein E structure: Insights into function. Trends Biochem. Sci. 2006, 31, 445–454. [CrossRef][PubMed] 25. Van Giau, V.; Bagyinszky, E.; An, S.S.; Kim, S. Role of apolipoprotein E in neurodegenerative diseases. Neuropsychiatr. Dis. Treat. 2015, 11, 1723–1737. [CrossRef] 26. Hsieh, Y.-H.; Chou, C.-Y. Structural and functional characterization of human apolipoprotein E 72-166 peptides in both aqueous and lipid environments. J. Biomed. Sci. 2011, 18, 4. [CrossRef] 27. Yamazaki, Y.; Zhao, N.; Caulfield, T.R.; Liu, C.-C.; Bu, G. Apolipoprotein E and Alzheimer disease: Pathobiology and targeting strategies. Nat. Rev. Neurol. 2019, 15, 501–518. [CrossRef][PubMed] 28. Kanekiyo, T.; Xu, H.; Bu, G. ApoE and Abeta in Alzheimer’s disease: Accidental encounters or partners? Neuron 2014, 81, 740–754. [CrossRef] 29. Iurescia, S.; Fioretti, D.; Mangialasche, F.; Rinaldi, M. The pathological cross talk between apolipoprotein E and amyloid-beta peptide in Alzheimer’s disease: Emerging gene-based therapeutic approaches. J. Alzheimers Dis. 2010, 21, 35–48. [CrossRef] 30. Südhof, T.C.; Goldstein, J.L.; Brown, M.S.; Russell, D.W. The LDL receptor gene: A mosaic of shared with different proteins. Science 1985, 228, 815–822. [CrossRef] 31. Jeon, H.; Blacklow, S.C. Structure and physiologic function of the low-density lipoprotein receptor. Annu. Rev. Biochem. 2005, 74, 535–562. [CrossRef] 32. Mahley, R.W.; Huang, Y. Atherogenic remnant lipoproteins: Role for proteoglycans in trapping, transferring, and internalizing. J. Clin. Investig. 2007, 117, 1–5. [CrossRef] 33. Berriman, M.; Hall, N.; Sheader, K.; Bringaud, F.; Tiwari, B.; Isobe, T.; Bowman, S.; Corton, C.; Clark, L.; Cross, G.A.; et al. The architecture of variant surface glycoprotein sites in Trypanosoma brucei. Mol. Biochem. Parasitol. 2002, 122, 131–140. [CrossRef] 34. Poirier, J.; Miron, J.; Picard, C.; Gormley, P.; Theroux, L.; Breitner, J.; Dea, D. Apolipoprotein E and lipid in the etiology and treatment of sporadic Alzheimer’s disease. Neurobiol. Aging 2014, 35, S3–S10. [CrossRef][PubMed] 35. Dose, J.; Huebbe, P.; Nebel, A.; Rimbach, G. APOE genotype and stress response-a mini review. Lipids Heal. Dis. 2016, 15, 121. [CrossRef] 36. Ruiz, J.; Kouiavskaia, D.; Migliorini, M.; Robinson, S.; Saenko, E.L.; Gorlatova, N.; Li, D.; Lawrence, D.; Hyman, B.T.; Weisgraber, K.H.; et al. The apoE isoform binding properties of the VLDL receptor reveal marked differences from LRP and the LDL receptor. J. Lipid Res. 2005, 46, 1721–1731. [CrossRef] 37. Altenburg, M.; Arbones-Mainar, J.M.; Johnson, L.; Wilder, J.; Maeda, N. Human LDL receptor enhances sequestration of ApoE4 and VLDL remnants on the surface of hepatocytes but not their internalization in mice. Arter. Thromb. Vasc. Biol. 2008, 28, 1104–1110. [CrossRef] 38. Bres, E.E.; Faissner, A. Low Density Receptor-Related Protein 1 Interactions With the : More Than Meets the Eye. Front. Cell Dev. Biol. 2019, 7, 31. [CrossRef][PubMed] 39. Simmons, T.; Newhouse, Y.M.; Arnold, K.S.; Innerarity, T.L.; Weisgraber, K.H. Human low-density lipoprotein receptor fragment. Successful refolding of a functionally active ligand-binding domain produced in Escherichia coli. J. Biol. Chem. 1997, 272, 25531–25536. [CrossRef] 40. Herz, J. Deconstructing the LDL receptor—A rhapsody in pieces. Nat. Genet. 2001, 8, 476–478. [CrossRef] 41. Gabcova-Balaziova, D.; Stanikova, D.; Vohnout, B.; Huckova, M.; Stanik, J.; Klimes, I.; Raslova, K.; Gasperikova, D. Molecular- genetic aspects of familial . Endocr. Regul. 2015, 49, 164–181. [CrossRef] 42. Noel, Z.R.; Beavers, C.J. Proprotein Convertase Subtilisin/Kexin Type 9 InhI.B.itors: A Brief Overview. Am. J. Med. 2017, 130, 229.e1–229.e4. [CrossRef] 43. Yang, H.-X.; Zhang, M.; Long, S.-Y.; Tuo, Q.-H.; Tian, Y.; Chen, J.-X.; Zhang, C.-P.; Liao, D.-F. Cholesterol in LDL receptor recycling and degradation. Clin. Chim. Acta 2020, 500, 81–86. [CrossRef] 44. Arias-Moreno, X.; Velazquez-Campoy, A.; Rodriguez, J.C.; Pocovi, M.; Sancho, J. Mechanism of low density lipoprotein (LDL) release in the endosome: Implications of the stability and Ca2+ affinity of the fifth binding module of the LDL receptor. J. Biol. Chem. 2008, 283, 22670–22679. [CrossRef] 45. Murphy, R.F.; Powers, S.; Cantor, C.R. Endosome pH measured in single cells by dual fluorescence flow cytometry: Rapid acidification of to pH 6. J. Cell Biol. 1984, 98, 1757–1762. [CrossRef] 46. Cuesta-Geijo, M.A.; Galindo, I.; Hernáez, B.; Quetglas, J.I.; Dalmau-Mena, I.; Alonso, C. Endosomal Maturation, Rab7 GTPase and Phosphoinositides in African Swine Fever Entry. PLoS ONE 2012, 7, e48853. [CrossRef][PubMed] 47. Albrecht, T.; Zhao, Y.; Nguyen, T.H.; Campbell, R.E.; Johnson, J.D. Fluorescent biosensors illuminate calcium levels within defined beta-cell endosome subpopulations. Cell Calcium 2015, 57, 263–274. [CrossRef] 48. Hu, Y.-B.; Dammer, E.B.; Ren, R.-J.; Wang, G. The endosomal-lysosomal system: From acidification and cargo sorting to neurodegeneration. Transl. Neurodegener. 2015, 4, 1–10. [CrossRef] Int. J. Mol. Sci. 2021, 22, 5030 9 of 10

49. Xu, S.; Olenyuk, B.Z.; Okamoto, C.T.; Hamm-Alvarez, S.F. Targeting receptor-mediated endocytotic pathways with nanoparticles: Rationale and advances. Adv. Drug Deliv. Rev. 2013, 65, 121–138. [CrossRef] 50. Lloyd-Evans, E.; Waller-Evans, H.; Peterneva, K.; Platt, F.M. Endolysosomal calcium regulation and disease. Biochem. Soc. Trans. 2010, 38, 1458–1464. [CrossRef][PubMed] 51. Van der Kant, R.; Neefjes, J. Small regulators, major consequences-Ca(2)(+) and cholesterol at the endosome-ER interface. J. Cell. Sci. 2014, 127, 929–938. [CrossRef][PubMed] 52. Martínez-Oliván, J.; Arias-Moreno, X.; Hurtado-Guerrero, R.; Carrodeguas, J.A.; Miguel-Romero, L.; Marina, A.; Bruscolini, P.; Sancho, J. The closed conformation of the LDL receptor is destabilized by the low Ca++ concentration but favored by the high Mg++ concentration in the endosome. FEBS Lett. 2015, 589, 3534–3540. [CrossRef] 53. Beglova, N.; Blacklow, S.C. The LDL receptor: How acid pulls the trigger. Trends Biochem. Sci. 2005, 30, 309–317. [CrossRef] 54. Chen, Y.; Durakoglugil, M.S.; Xian, X.; Herz, J. ApoE4 reduces glutamate receptor function and synaptic plasticity by selectively impairing ApoE receptor recycling. Proc. Natl. Acad. Sci. USA 2010, 107, 12011–12016. [CrossRef][PubMed] 55. Xian, X.; Pohlkamp, T.; Durakoglugil, M.S.; Wong, C.H.; Beck, J.K.; Lane-Donovan, C.; Plattner, F.; Herz, J. Reversal of ApoE4- induced recycling block as a novel prevention approach for Alzheimer’s disease. eLife 2018, 7, 7. [CrossRef] 56. Braun, N.A.; Mohler, P.J.; Weisgraber, K.H.; Hasty, A.H.; Linton, M.F.; Yancey, P.G.; Su, Y.R.; Fazio, S.; Swift, L.L. Intracellular trafficking of recycling apolipoprotein E in Chinese hamster ovary cells. J. Lipid Res. 2006, 47, 1176–1186. [CrossRef][PubMed] 57. Asaro, A.; Carlo-Spiewok, A.; Malik, A.R.; Rothe, M.; Schipke, C.G.; Peters, O.; Heeren, J.; Willnow, T.E. Apolipoprotein E4 disrupts the neuroprotective action of sortilin in neuronal lipid metabolism and endocannabinoid signaling. Alzheimer’s Dement. 2020, 16, 1248–1258. [CrossRef] 58. Zhang, D.-W.; Garuti, R.; Tang, W.-J.; Cohen, J.C.; Hobbs, H.H. Structural requirements for PCSK9-mediated degradation of the low-density lipoprotein receptor. Proc. Natl. Acad. Sci. USA 2008, 105, 13045–13050. [CrossRef] 59. Martínez-Oliván, J.; Rozado-Aguirre, Z.; Arias-Moreno, X.; Angarica, V.E.; Velázquez-Campoy, A.; Sancho, J. Low-density lipoprotein receptor is a calcium/magnesium sensor-role of LR4 and LR5 ion interaction kinetics in low-density lipoprotein release in the endosome. FEBS J. 2014, 281, 2638–2658. [CrossRef] 60. Somiya, M. Where does the cargo go?: Solutions to provide experimental support for the “extracellular vesicle cargo transfer hypothesis”. J. Cell Commun. Signal. 2020, 14, 135–146. [CrossRef] 61. Weddell, J.C.; Imoukhuede, P.I. Integrative meta-modeling identifies endocytic vesicles, late endosome and the nucleus as the cellular compartments primarily directing RTK signaling. Integr. Biol. 2017, 9, 464–484. [CrossRef][PubMed] 62. Neu, S.C.; Pa, J.; Kukull, W.; Beekly, D.; Kuzma, A.; Gangadharan, P.; Wang, L.S.; Romero, K.; Arneric, S.P.; Redolfi, A.; et al. Apolipoprotein E Genotype and Sex Risk Factors for Alzheimer Disease: A Meta-analysis. JAMA Neurol. 2017, 74, 1178–1189. [CrossRef] 63. Riddell, D.R.; Zhou, H.; Atchison, K.; Warwick, H.K.; Atkinson, P.J.; Jefferson, J.; Xu, L.; Aschmies, S.; Kirksey, Y.; Hu, Y.; et al. Impact of Apolipoprotein E (ApoE) Polymorphism on Brain ApoE Levels. J. Neurosci. 2008, 28, 11445–11453. [CrossRef] 64. Minta, K.; Brinkmalm, G.; Janelidze, S.; Sjödin, S.; Portelius, E.; Stomrud, E.; Zetterberg, H.; Blennow, K.; Hansson, O.; Andreasson, U. Quantification of total apolipoprotein E and its isoforms in cerebrospinal fluid from patients with neurodegenerative diseases. Alzheimer’s Res. Ther. 2020, 12, 1–11. [CrossRef] 65. Van Acker, Z.P.; Bretou, M.; Annaert, W. Endo-lysosomal dysregulations and late-onset Alzheimer’s disease: Impact of genetic risk factors. Mol. Neurodegener. 2019, 14, 1–20. [CrossRef] 66. Cash, J.G.; Kuhel, D.G.; Basford, J.E.; Jaeschke, A.; Chatterjee, T.K.; Weintraub, N.L.; Hui, D.Y. Apolipoprotein E4 Impairs Macrophage Efferocytosis and Potentiates Apoptosis by Accelerating Stress. J. Biol. Chem. 2012, 287, 27876–27884. [CrossRef] 67. Huang, Y.A.; Zhou, B.; Nabet, A.M.; Wernig, M.; Sudhof, T.C. Differential Signaling Mediated by ApoE2, ApoE3, and ApoE4 in Human Neurons Parallels Alzheimer’s Disease Risk. J. Neurosci. 2019, 39, 7408–7427. [CrossRef][PubMed] 68. Yan, Y.; Dominguez, S.; Fisher, D.W.; Dong, H. Sex differences in chronic stress responses and Alzheimer’s disease. Neurobiol. Stress 2018, 8, 120–126. [CrossRef] 69. Canet, G.; Hernandez, C.; Zussy, C.; Chevallier, N.; Desrumaux, C.; Givalois, L. Is AD a Stress-Related Disorder? Focus on the HPA Axis and Its Promising Therapeutic Targets. Front. Aging Neurosci. 2019, 11, 269. [CrossRef][PubMed] 70. Pedersen, N.B.; Wang, S.; Narimatsu, Y.; Yang, Z.; Halim, A.; Schjoldager, K.T.-B.G.; Madsen, T.D.; Seidah, N.G.; Bennett, E.P.; Levery, S.B.; et al. Low Density Lipoprotein Receptor Class A Repeats Are O-Glycosylated in Linker Regions. J. Biol. Chem. 2014, 289, 17312–17324. [CrossRef][PubMed] 71. Wang, S.; Mao, Y.; Narimatsu, Y.; Ye, Z.; Tian, W.; Goth, C.K.; Lira-Navarrete, E.; Pedersen, N.B.; Benito-Vicente, A.; Martin, C.; et al. Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions. J. Biol. Chem. 2019, 294, 8349. [CrossRef][PubMed] 72. Boogert, M.A.V.D.; Larsen, L.E.; Ali, L.; Kuil, S.D.; Chong, P.L.; Loregger, A.; Kroon, J.; Schnitzler, J.G.; Schimmel, A.W.; Peter, J.; et al. N-Glycosylation Defects in Humans Lower Low-Density Lipoprotein Cholesterol Through Increased Low-Density Lipoprotein Receptor Expression. Circulation 2019, 140, 280–292. [CrossRef][PubMed] Int. J. Mol. Sci. 2021, 22, 5030 10 of 10

73. Dong, L.-M.; Parkin, S.; Trakhanov, S.D.; Rupp, B.; Simmons, T.; Arnold, K.S.; Newhouse, Y.M.; Innerarity, T.L.; Weisgraber, K.H. Novel mechanism for defective receptor binding of apolipoprotein E2 in type III hyperlipoproteinemia. Nat. Struct. Mol. Biol. 1996, 3, 718–722. [CrossRef][PubMed] 74. Fass, D.; Blacklow, S.; Kim, P.S.; Berger, J.M. Molecular basis of familial hypercholesterolaemia from structure of LDL receptor module. Nat. Cell Biol. 1997, 388, 691–693. [CrossRef][PubMed] 75. Rudenko, G.; Henry, L.; Henderson, K.; Ichtchenko, K.; Brown, M.S.; Goldstein, J.L.; Deisenhofer, J. Structure of the LDL Receptor Extracellular Domain at Endosomal pH. Science 2002, 298, 2353–2358. [CrossRef][PubMed] 76. Emsley, P.; Lohkamp, B.; Scott, W.G.; Cowtan, K. Features and development ofCoot. Acta Crystallogr. Sect. D Biol. Crystallogr. 2010, 66, 486–501. [CrossRef]