Mutation within the transmembrane domain of oxidized low-density lipoprotein receptor 1 influences oxidized low-density lipoprotein-induced signal transduction

Abstract

Objective

To investigate the important active sites within the NTFs to affect the in vitro interaction of oxidized low-density lipoprotein (ox-LDL) with its receptor, OLR1.

Methods

Simulation analysis online was performed to generate various OLR1 chimeras, truncation mutants, and site-specific mutations. They were transfected in COS-7 cells and subjected to ox-LDL stimulations to observe the different reactions. Immunoprecipitation-mass spectrometry (IP-MS) was performed to show what proteins combined with OLR1 mutants in reaction to ox-LDL. Lipid uptake in human monocytes (THP-1) originated foam cells overexpressing somatic mutant of OLR1 were also determined. Further studies focusing on these regions were conducted using truncation mutants and site-specific mutants such as G43A, V44A, L45A, C46A, and L47A.

Results

Amino acids within the TM were highly conserved, spanning amino acids 35 to 57. The induction of intracellular p-ERK1/2 in response to ox-LDL stimulation was highly promoted in Chimera 3 possessing the TM from OLR1 like OLR1/WT (p < 0.05). Sequence alignment revealed two conserved regions within the TM of OLR1, Leu45-Cys46-Leu47 and Val55-Leu56-Gly57. Western blot showed that most of the TM changes ablated ERK1/2 activation in response to ox-LDL stimulation (p < 0.05). One human somatic mutation at L45F revealed significantly lower p-ERK1/2 levels with enhanced intake of ox-LDL in THP-1-derived foam cells than the control cells (p < 0.05). L45A and C46A molecular complexes were identified. After ox-LDL stimulation, these underlined interactions with keratins, namely KRT2 and KRT6A.

Conclusion

These findings emphasize the vital role of the TM in the interactions between OLR1 and ox-LDL and point to an exciting possibility that signal transduction induced by ox-LDL through its receptor OLR1 may involve complex interactions with cytoskeletal proteins.

Keywords

Oxidized low-density lipoprotein receptor 1 transmembrane domain intracellular signal transduction ERK1/2 site mutation

Introduction

As living standards gradually improve, dietary patterns have also changed, leading to a significant increase in the incidence of cardiovascular diseases (CVD).¹ According to the World Health Organization's overview, CVD causes an estimated 17.9 million deaths each year.² These diseases have surpassed cancer as the leading cause of death, posing a substantial threat to human health.³ It is well known that the pathological basis of most cardiovascular diseases is atherosclerosis (AS), characterized by the formation of lipid deposits in the intima of medium and large arteries, which further develop into atherosclerotic plaques.⁴ Although many lipid-lowering drugs have been applied in clinical practice, impaired lipid metabolism does not improve satisfactorily, and the progress of AS is not well controlled, indicating the underlying mechanism is not yet clear.⁵

Oxidized low-density lipoprotein receptor 1 (OLR1, also known as lectin-like oxidized low-density lipoprotein receptor-1, full length 1–273 aa) primarily recognizes modified low-density lipoprotein (LDL) as its specific ligands, such as oxidized (ox)-LDL and acetylated (ac)-LDL.⁶ OLR1 consists of four structural domains, including the intracellular N terminal which contains a cytoplasmic domain (CD: 1–36 aa), a short transmembrane domain (TM: 37–57 aa), an extracellular part that involves a neck domain (NECK: 58–150 aa), and C-type lectin-like domain (CTLD: 151–273 aa).⁷ Mentrup et al. recently found that intracellular N-terminal fragments (NTFs) of OLR1, including NTF1 (N72-N92) and NTF2 (N-terminal-N72) contributed to AS.⁸ NTF mainly consists of CD and TM, with part of NECK in structure, indicating that CD-TM-NECK are also functional parts. CD-TM has been determined to be essential in OLR1 dimer formation previously. Moreover, Yamamoto et al. have found that the CD-TM also influenced the conduction of angiotensin II type 1 receptor signals.⁹ Nevertheless, how CD-TM is involved in signal conduction has not been determined. Therefore, we aimed to thoroughly investigate the important active sites within the NTFs to affect the in vitro interaction of ox-LDL with OLR1.

Materials and methods

Bioinformatic analyses

OLR1 from different species were retrieved from NCBI (https://pubmed.ncbi.nlm.nih.gov/), and the Constraint-Based Local Alignment Tool (COBALT, www.ncbi.nlm.nih.gov/tools/cobalt/) was utilized to conduct primary protein sequence alignments (with default parameter settings). Ortholog-conserved domain identification was performed among OLR1 from different species. Jalview 2.11.0 was used to visualize and format alignments. Putative physicochemical properties of OLR1 mutations were computed using ExPASy-ProtParam tools (https://web.expasy.org/protparam/). To assess the effects of mutations on proteins, we performed in silico annotation using PROVEAN (http://provean.jcvi.org/index.php) with a cutoff of −2.5. The STRING database (http://string-db.org/) was utilized to perform protein-protein interaction (PPI) analysis. PPI networks were visualized with Cytoscape version 3.8.0. The Database for Annotation, Visualization, and Integrated Discovery (DAVID, https://david.ncifcrf.gov/)¹⁰ was used to clarify the characteristics of given genes/proteins. Somatic mutations of OLR1 were obtained from the cBioPortal for Cancer Genomics database (https://www.cbioportal.org/).¹¹

Cells and treatment

African Green Monkey Kidney Cell COS-7 cells were purchased from COBioer (Nanjing, China) and cultured in DMEM (Gibco, USA) with 10% fetal bovine serum (FBS, Thermo Fisher, Germany) and 100 U/ml penicillin-streptomycin solution (Gibco, China) at 37°C in 5% CO₂ atmosphere. THP-1 monocytes were purchased from Fubio Biological Technology Co., Ltd. (Suzhou, China) and cultured in RAPI 1640 medium (Gibco, USA) with 10% FBS and 100 U/ml penicillin-streptomycin solution.

Before the application of ox-LDL, cells were cultured in a serum-free medium to synchronize them to the G0 phase. Ox-LDL (Yiyuan Biotech, China) was then administered at a concentration of 60 μg/ml for 8 min. THP-1/L45F overexpression cells were cultured in RAPI 1640 medium containing FBS with ox-LDL stimulation because previous starvation led to no expression of ERK1/2 protein.

Foam cell induction

Stable THP-1 cells were seeded into 6-well plates at 1 × 10⁴ cells/well. At 50% confluence, a medium with phorbol 12-myristate 13-acetate (PMA, 100 nmol/L) was added and incubated for 48 h. Then the medium was changed to RPMI 1640 containing 3% FBS and ox-LDL (25 μg/mL) for 48 h to induce foam cells.

Cell transfection

Chimera 2 was a gift from Mr Koichi Yamamoto. Dectin-1 was constructed into a pcDNA vector as Chimera 2 did. Chimera 3, Chimera 4, site mutant L45F, and TM truncations were constructed into a pEGFP-N3 vector (vector 1). Other site mutants were constructed into an RFP vector (vector 2). Transient transfection of Chimeras, TM truncation mutants, and site mutants was conducted with X-tremeGENE HP Reagent (Roche, USA). TM46, L45A, and L45F plasmids (namely: TM46v, L45Av, and L45Fv) were constructed with lentiviral plasmids that were commercially available (Fubio Biological Technology Co., Ltd., Suzhou China) and transfected according to the manufacturer's protocols, and were selected using puromycin (Sangon Biotech, China) for 14 days.

qRT-PCR

Total RNA from transfected cells was extracted using TRIZol (Invitrogen, USA). At least 500 ng RNA (OD 260/280: 1.8–2.0) was reverse transcribed into cDNA using a commercial kit (FastQuant RT Kit, TIANGEN Biotech Co., Ltd. China). One Step PrimeScript III RT-PCR kit (Takara, Japan) was used for qRT-PCR analysis on the CFX96 real-time fluorescence polymerase chain reaction system (Bio-Rad). Gene expression levels were normalized to GAPDH. The relative expression of each gene was calculated using the 2^−ΔΔct method. The primers were designed by Sangon Biotech company (Shanghai, China).

Western blot

Western blot was conducted to detect OLR1 proteins from whole cell lysates. Intracellular signals induced by ox-LDL (Yiyuan Biotech, China, 60 μg/ml, incubation for 8 min) were also detected by western blot for assessing phosphorylated kinase/total kinase. Cells were collected using RIPA buffer (Biotime, Shanghai, China) supplemented with a protease inhibitor cocktail (PIC, 1:100, Biotime). Protein samples were separated in SDS-PAGE by electrophoresis and transferred to the PVDF membrane. PVDF membrane was then blocked with non-fat milk at room temperature for 1 h. Primary antibodies (p-ERK1/2, ERK 1/2, p-P38, P38, p-P65, P65, p-AKT, AKT, GAPDH) were incubated with the membrane at 4°C overnight. Secondary antibodies conjugated to horseradish peroxidase (HRP, 1:5000, Kangchen Biotech, China) were incubated with the membrane at room temperature for 1 h. A chemiluminescence detection system (OmegaLum G, Aplegen, USA) was used to detect HRP signals, and band intensities were analyzed using Quantity One software (BioRad, USA) and normalized to GADPH. Results were normalized relative to expression levels of control (non-transfected) COS-7 or THP-1 cells. pERK1/2 (relative fold) means the ratio of phosphorylated ERK1/2 (p-ERK1/2) to total ERK1/2.

Immunoprecipitation-mass spectrometry (IP-MS)

To elucidate mutation-mediated molecular complexes of OLR1 that induce intracellular signaling after ox-LDL stimulation, IP-MS was performed. Whole-cell lysates were collected as previously described, immunopurified with a mouse anti-FLAG antibody (Genescript, China) bound to Protein-G magnetic beads (MCE, USA), and separated by native-PAGE. Specific protein bands illustrated by Coomassie blue staining were excised and sent for Nano-HPLC MS/MS analysis with a NanoAquity UPLC system (Waters Corporation, USA) coupled with a quadrupole-Orbitrap mass spectrometer (Q-Exactive, Thermo Fisher Scientific, Germany) equipped with an online nano-electrospray ion source. Parameters of MS and raw data analysis were described in the Supplementary Materials.

Oil-red-o staining

The THP-1 cells were fixed with 4% paraformaldehyde at room temperature for 10 min. Then cells were stained with 0.5% oil-red-o solution for 15 min at 60°C. Then oil-red-o solution was discarded and cells were washed with 60% isopropanol according to the color darkness. Hematoxylin was used to re-stain and 0.5% ethanol was used to separate. The intracellular lipid droplets were observed under a microscope.

Statistical analysis

Statistical analyses were performed with SPSS version 26.0 (IBM, USA). All data were collected from three independent experiments. Data were presented as means ± standard deviation (SD) or percentiles (Q25, Q75). One-way ANOVA was used to compare differences among multiple groups. An independent Student's t-test was used to compare the differences between the two groups. A nonparametric (Kruskal-Wallis) test was used to compare differences among independent groups when tests of homogeneity of variances were less than 0.05. A p-value less than 0.05 indicated statistical significance.

Results

OLR1 TMs were identified as the study target

The LOX-1 receptor encoded by OLR1 is a critical transmembrane protein that mediates the binding and endocytosis of ox-LDL, and its function is closely associated with atherosclerosis. The conservation of the transmembrane domain (TM) may be directly related to its signal transduction capabilities. LOX-1 belongs to the C-type lectin family, and its TM domain plays a central role in maintaining receptor conformation and transmembrane signaling. Sequence alignments by use of COBALT showed that OLR1 sequences from 22 mammal species were aligned with hOLR1 (Homo sapiens). Further analysis showed more conserved residues within TM domains than within CD, especially in 45–48 aa (Figure 1(a) and (b)). Sequence alignment of hOLR1 and Dectin-1 also showed only two regions covered contiguous 3 conservative aa in CD-TM of OLR, site45–46–47 and 55–56–57, both within TM (Figure 1(c)). Therefore, we decided to concentrate on TM but not the CD domain. The Whole-length BLAST of 22 receptors, hOLR1/WT, and Dectin-1 were shown in Supplementary Figure 1 and Supplementary Figure 2.

Figure 1.

Orthologs of OLR1.

The MAPK pathway (ERK1/2, p38) is a central effector pathway in the ox-LDL/LOX-1 signaling cascade. Elevated phosphorylation levels of this pathway are directly associated with endothelial cell activation, inflammatory cytokine release, and the progression of atherosclerosis. The intracellular signals induced by ox-LDL were detected by western blot, and the results showed that the protein expression levels of p-ERK1/2 and p-P38 were significantly elevated in hOLR1/WT transfected COS-7 cells after ox-LDL stimulations than the control group (p < 0.05, Figure 2(a)), indicating that the MAPK pathway was mainly involved in ox-LDL signal induction. Therefore, intracellular p-ERK1/2 expression was used as an indicator for ox-LDL signals in the following experiments.

Figure 2.

hOLR1, Dectin-1 and chimeras reacted distinctly to ox-LDL stimulations.

In research from Yamamoto et al., CD-TM of OLR1 influenced intracellular signal transduction, and chimeras were constructed using Dectin-1 and OLR1. Dectin-1 is a pattern receptor for glucans, which is highly homologous to OLR1 in structure. Dectin-1 transfected COS-7 cells did not significantly affect pERK1/2 protein expression after ox-LDL stimulations as non-transfected COS-7 cells and vector-transfected cells (Figure 2(c)). After CD and/or TM were replaced by Dectin-1 in OLR1 (Chimera 2 and 4, Figure 2(b)), p-ERK1/2 expression did not increase significantly after ox-LDL stimulation (Figure 2(d)). When TM was maintained but CD replaced (Chimera 3, Figure 2(b)), pERK1/2 protein expression was recovered as OLR1/WT did (Figure 2(d)), suggesting that TM of OLR is more essential for ox-LDL signals.

Computational analysis of hOLR1 mutants out of TM region

As a typical type I transmembrane protein, OLR1 plays a crucial role in membrane anchoring and signal transduction through its N-terminal TM domain (37–53aa). We analyzed the putative characteristics of all TM-related mutants (truncations and site mutants) using the ProtParam and PROVEAN tools. Noticeable pI shifts (Table 1) and decreased hydropathicity (greater hydrophilicity) along with the discarding length in TMs were observed in truncation mutants. TM43(43–273aa)-TM45 exhibited the most similar GRAVY score to OLR1/WT. The pI and GRAVY scores of most site mutants did not change much except that C46A showed a slightly altered theoretical pI (from 6.94 to 6.95, Table 2). All TM truncations and some site mutants were “Deleterious” according to PROVEAN with C46A the lowest SCORE (−6.114). Therefore, we decided to focus on the area encompassing Leu45-Cys46 and Met54-Gly57 [i.e., truncating from L43 (discarding 1/3 of the N-terminal TM) to G48 (discarding 1/2 of the N-terminal TM)], as well as TM54–57 (containing the V55-L56-G57 area), to explore the specific site(s) responsible for signal transduction.

Table 1.

Predicted characteristics of 37–57 truncation mutants of OLR1 TM.

Truncation	pI	MW (kD)	GRAVY	SCORE	PROVEAN PREDICTION
OLR1/WT	6.94	30.959	−0.399	/	/
TM37	6.58	26.731	−0.320	−49.615	Deleterious
TM38	6.59	26.617	−0.337	−49.430	Deleterious
TM39	6.59	26.546	−0.346	−50.063	Deleterious
TM40	6.59	26.475	−0.356	−52.147	Deleterious
TM41	6.50	26.404	−0.365	−52.211	Deleterious
TM42	6.58	26.303	−0.363	−53.941	Deleterious
TM43	6.58	26.190	−0.381	−55.860	Deleterious
TM44	6.58	26.133	−0.381	−57.955	Deleterious
TM45	6.58	26.034	−0.401	−59.636	Deleterious
TM46	6.58	25.921	−0.420	−61.698	Deleterious
TM47	6.59	25.817	−0.433	−65.716	Deleterious
TM48	6.59	25.704	−0.451	−67.574	Deleterious
TM49	6.59	25.647	−0.452	−69.087	Deleterious
TM50	6.58	25.534	−0.471	−71.202	Deleterious
TM51	6.58	25.435	−0.491	−71.759	Deleterious
TM52	6.50	25.336	−0.513	−72.708	Deleterious
TM53	6.59	25.235	−0.512	−74.024	Deleterious
TM54	6.52	25.122	−0.535	−75.320	Deleterious
TM55	6.58	24.990	−0.546	−75.828	Deleterious
TM56	6.59	24.891	−0.567	−76.496	Deleterious
TM57	6.59	24.778	−0.588	−78.387	Deleterious

pI: Isoelectric point; MW: molecular weight; GRAVY: Grand average of hydropathicity; PROVEAN: Protein Variation Effect Analyzer.

Table 2.

Predicted characteristics of 37–57 site mutants of OLR1 TM.

SITE MUTATION	pI	MW (kD)	GRAVY	SCORE	PROVEAN PREDICTION
OLR1/WT	6.94	30.959	−0.399	/	/
L37A	6.94	30.917	−0.406	−0.683	Neutral
A38-40*	/		/	/	/
T41A	6.94	30.929	−0.39	−2.115	Neutral
L42A	6.94	30.917	−0.406	−2.867	Deleterious
G43A	6.94	30.973	−0.391	−2.084	Neutral
V44A	6.94	30.931	−0.408	−2.567	Deleterious
L45A	6.94	30.917	−0.406	−3.279	Deleterious
L45F	6.94	30.993	−0.403	NA	Deleterious
C46A	6.95	30.927	−0.401	−6.114	Deleterious
L47A	6.94	30.917	−0.406	−2.818	Deleterious
G48A	6.94	30.973	−0.391	−1.276	Neutral
L49A	6.94	30.917	−0.406	−2.566	Deleterious
V50A	6.94	30.931	−0.408	−1.478	Neutral
V51A	6.94	30.931	−0.408	−1.759	Neutral
T52A	6.94	30.929	−0.39	−1.224	Neutral
I53A	6.94	30.917	−0.409	−0.136	Neutral
M54A	6.94	30.899	−0.399	−0.89	Neutral
V55A	6.94	30.931	−0.408	−1.667	Neutral
L56A	6.94	30.917	−0.406	−2.845	Deleterious
G57A	6.94	30.973	−0.391	−1.7	Neutral

*: Because site 38–40 carries A, we did not apply site-directed mutagenesis.

We further explored the somatic mutation profile of OLR1 using the cBioPortal website (Table 3). There were six main mutations of OLR1, of which one was located in the transmembrane domain (L45F), two in the NECK domain (E101K, K103E), and three in CTLD (X227-splice, R231Q, T242N). Since L45F was found in human tissues, and also located in TM of OLR1, we carried out some other tests about L45F later. Expressions of TM in COS-7 cells were shown in Supplementary Figure 3.

Table 3.

The somatic mutation profile of OLR1.

Protein changes	Mutation types	Variant types	HGVSg	Allele freq (T)	dbSNP
L45F	Missense_Mutation	SNP	12: g.10321718G > A	0.19	rs768512963
R231Q	Missense_Mutation	SNP	12: g.10312609C > T	0.46	rs986422401
E101K	Missense_Mutation	SNP	12: g.10319434C > T	0.28	rs1189099806
T242N	Missense_Mutation	SNP	12: g.10312576G > T	0.23
X227_splice	Splice_Site	SNP	12: g.10312621C > T	0.46
K103E	Missense_Mutation	SNP	12: g.10319428T > C	0.42

Table 4.

Predicted characteristics of different mutations of TM35-37, W35A, C36A and L47A.

MUTATION	pI	MW (kD)	GRAVY	SCORE	PROVEAN PREDICTION
TM35	6.58	27.020	−0.310	−40.115	Deleterious
TM36	6.58	26.834	−0.308	−5.044	Deleterious
W35A	6.94	30.844	−0.389	−5.934	Deleterious
C36A	6.95	30.927	−0.401	−2.766	Deleterious
L37A	6.94	30.917	−0.406	−0.683	Neutral

Majority of mutations within the OLR1 Tm influenced pERK1/2 after ox-LDL stimulation

ERK1/2 phosphorylation (pERK1/2) represents a pivotal signaling event that occurs following LOX-1 activation, playing a key role in inflammatory responses and cellular phenotype switching. Through a systematic approach involving truncation and site-directed mutagenesis, we comprehensively evaluated the impact of the OLR1 transmembrane domain on ox-LDL signal transduction. In each transfected cell subset, most TM truncations abolished p-ERK1/2 after ox-LDL stimulation except TM46 (occupied only 46–273 aa with 1–45aa removed) (Figure 3(a) and (b)). Because the transfection efficiency was 30 to 50% (Supplementary Materials), we also constructed a lentivirus-derived TM46v. Indeed, TM46v manifested the same rise in p-ERK1/2 as TM46 did. This result indicated that COS-7 cells over-expressing OLR1 with TM deficiencies cannot transduce ox-LDL-induced signals successfully. Furthermore, we constructed site mutations into Alanine near site 45 (G43A, V44A, L45A, C46A, and L47A), then transfected them into COS-7 cells and subjected these cells to the same ox-LDL stimulations. Most site mutations except L45A, which was confirmed by results from lentivirus-derived L45Av transfection and stimulation experiments, caused similar inhibitions in pERK1/2 (Figure 3(a) and (b)). The expressions of OLR1 in transfected cells were shown in Supplementary Figure 4.

Figure 3.

Densitometric analysis of pERK1/2 induced by ox-LDL in TM43-48, TM54-57 truncations and site mutants.

Lipidation modification sites were not involved in altered p-ERK1/2 induced by ox-LDL

According to the above data, site Leu45-Cys46 within the OLR1 TM was revealed to be unique in its reactions to ox-LDL stimulation. The C46 site within OLR1 is reported to be one of the receptor's two lipidation sites, so we also tested the other one, site 36 (cysteine). TM35, TM36, TM37, W35A, and C36A mutation plasmids were constructed, and their characteristics were predicted similarly (Table 4). Next, ox-LDL reactions were tested as described above. However, only C36A (not TM36 or W35A) evoked p-ERK1/2 as observed in TM46-transfected cells after the introduction of ox-LDL (Figure 4).

Figure 4.

Densitometric analysis of pERK1/2 induced by ox-LDL in TM35-37 truncations and site mutants.

Molecular complex analysis of L45A and C46A mutations in response to ox-LDL stimulations

Site 45 and 46 are both conserved amino acids by previous analysis, but only mutation of Leu45 into alanine retained activity after ox-LDL stimulations. There must be something different that happened to L45A compared with C46A (or other adjacent aa within TM) after ox-LDL stimulations. IP showed that anti-FLAG (tag of OLR1) antibodies captured an extremely large band of approximately 150 kD in both L45A and C46A transfected cells after ox-LDL stimulations by native-PAGE (Figure 5(a)). However, OLR1 fragments from WCL/input samples were detected at about 60 kD, and fusion proteins OLR1 with GFP or RFP were predicted as MW 60 kD (Figure 5(a)), suggesting that OLR1 mutants were able to form molecular complexes. IP-MS analysis was used to identify proteins in different groups (Table 5). Compared with the C46A-ox-LDL group, the L45A-ox-LDL group gained two additional proteins, including Keratin 2 (KRT2) and Keratin 6A (KRT6A) (Figure 5(b)). Expressions of OLR1 in transfected cells were shown in Supplementary Figure 5.

Figure 5.

Complex formed by L45A and C46A after ox-LDL stimulation.

Table 5.

Proteins identified by MS from different mutants and stimulation conditions.

Mutant	Stimulation	Numbers of Proteins	Name of Protein
L45A	(-)	9	Keratin 14, Keratin 16, Keratin 1, Keratin 5, Keratin 10, Keratin 6A, Keratin 9, Keratin 2, Albumin
L45A	ox-LDL	8	Keratin 10, Keratin 6A, Keratin 1, Keratin 14, Keratin 9, Albumin, Keratin 2, Keratin 5
C46A	(-)	8	Keratin 2, Keratin 6A, Keratin 1, Keratin 14, Keratin 9, Albumin, Keratin 10, Keratin 16
C46A	ox-LDL	6	Keratin 10, Keratin 1, Keratin 14, Keratin 9, Keratin 5, Albumin

We queried KRT2 and KRT6A in STRING and found 15 different proteins in the network (Figure 6). GO function enrichment analysis of these proteins was performed, and two clusters were distinguished by DAVID. One is mainly composed of KRTs and one with only three proteins (SOAT2, CERS3, and PCSK5) (Table 6). The annotation Cluster 1 with Enrichment Score of 11.90672661596519 was shown in Supplementary Table 1. We further investigated functions related to these three proteins (Table 7) and found that SOAT2 was involved in cholesterol homeostasis. Though PCSK5 and CERS3 were not reportedly involved in this process, they facilitated peptidase activity and bound DNA, both of which were essential for intracellular signal transduction.

Figure 6.

Predicted protein-protein interactions of Keratin 2 (KRT2) and Keratin 6A (KRT6A).

Table 6.

Annotation cluster 2 (enrichment score: 0.041464607559271144).

Category	Term	p values
UP_KEYWORDS	Transmembrane helix	0.879588
UP_KEYWORDS	Transmembrane	0.879894
UP_KEYWORDS	Membrane	0.888769
GOTERM_CC_DIRECT	GO:0016021∼integral component of membrane	0.9923

Table 7.

Functional annotations of SOAT2, CERS3, and PCSK5.

ID	Gene Name	GOTERM_BP	GOTERM_CC	GOTERM_MF
SOAT2	Sterol O-acyltransferase 2	Cholesterol esterification (GO:0034435); Cholesterol homeostasis (GO:0042632)	Endoplasmic reticulum membrane (GO:0005789); Integral component of membrane (GO:0016021)	Cholesterol O-acyltransferase activity (GO:0034736)
PCSK5	Proprotein convertase subtilisin/kexin type 5	/	Integral component of membrane (GO:0016021)	Serine-type endopeptidase activity (GO:0004252)
CERS3	Ceramide synthase 3	/	Nucleus (GO:0005634); endoplasmic reticulum (GO:0005783); Integral component of membrane (GO:0016021)	DNA binding (GO:0003677)

Human somatic mutation in Leu45 of OLR1 alleviated p-ERK1/2 but contributed to lipid uptake after ox-LDL stimulations

Using the cBioPorta website, we revealed one somatic mutation located in the transmembrane domain (L45F, rs768512963, SNP, Figure 7(a)). L45F also changed site Leu45, so we constructed L45F mutation plasmids. Interestingly, p-ERK1/2 expression was decreased in L45F-transfected COS-7 cells after the induction of ox-LDL (Figure 7(b) and (c)). Since it is a human somatic mutant, we further tested lipid uptake capability in THP-1 (WT or overexpressing L45F) induced foam cell models, which indicated that THP-1/L45F took in more ox-LDL compared to THP-1/WT (Figure 7(d)).

Figure 7.

Somatic mutant L45F attenuated pERK1/2 and contributed to lipid uptake in foam cells models.

Discussion

The OLR1 gene is located on chromosome 12p13.2 and consists of six exons, encoding a protein of 273 amino acids.¹² Although several crystal structures of this receptor are available, the longest structure only describes the region from amino acids 133 to 272, excluding the intracellular and transmembrane (TM) domains (PDB entry: 3VLG).¹³ However, our understanding of the cytoplasmic domain (CD) and TM regions of this receptor remains limited.

Twenty-four mutations in the OLR1 gene have been previously reported, most of which are located in the extracellular domain.^14,15 Among these, two mutations are situated near the cytoplasmic region (22–25 aa [KKAKEEAE], E70K). Mutations within the cytoplasmic domain (Lys-22, Lys-23, and L25Et) impair the transport of the receptor's C-terminal region to the extracellular surface,¹⁶ indicating that interference with amino acids in the N-terminal fragments (NTFs) may affect receptor expression. Our data demonstrate that truncated versions of OLR1 are successfully expressed in COS-7 cells, as evidenced by GFP/RFP expression. Both qRT-PCR and Western blot analyses confirmed the presence of the OLR1 target in the transfected cell lines. These findings collectively suggest that defects in the cytoplasmic domain and transmembrane regions have minimal impact on receptor expression, which contrasts with the results of previous studies.

The analysis of pI (isoelectric point) and GRAVY (grand average of hydropathicity) scoring parameters did not reveal a clear association between the isoelectric, hydrophilic, and hydrophobic characteristics of these truncated proteins. However, compelling insights were gained from the PROVEAN analysis. Specifically, the C46A mutation was classified as “deleterious,” exhibiting a significantly lower score of −6.114 compared to all other mutants. The W35A mutation had the second-lowest score of −5.934. This may explain the strong intracellular response to ligand stimulation observed in our in vitro experiments for mutations near W35 and C46 (C36A and L45A). Focusing on the mutations at the two lipidation sites, C36A and C46A, a slight increase in hydrophilicity was observed (GRAVY = −0.401), with an isoelectric point of 6.95. Notably, these mutations did not exhibit the same effects. This discrepancy can be attributed to the microenvironment surrounding Cys36, which includes hydrophilic amino acids such as tryptophan (W) and transmembrane hydrophobic amino acids like leucine (L), whereas C46 is surrounded by two transmembrane hydrophobic amino acids (L), and Cys36 is not located within a conserved region. Furthermore, when modifying the amino acids preceding site 46 (V44A and L45A), the resulting domains displayed increased hydrophilicity (scores of −0.408 and −0.406, respectively) and lower deleterious effects (PROVEAN scores of −2.567 and −3.279) compared to C46A (−6.114), aligning more closely with C36A (−2.766). Therefore, discerning the complex relationships among the characteristics of these mutants appears to be a challenging task.

Intracellular signaling does not always exhibit uniformity in response. Phylogenetic analysis across different species reveals a high density of conserved amino acids within the transmembrane (TM) domain, particularly at positions 42–47 and 55–57. Our ligand stimulation experiments further underscore the importance of most conserved amino acid changes in mediating ox-LDL signaling through OLR1 into the cells. Adjacent to Leu45 is a cysteine residue (Cys46), which plays a ubiquitous role in numerous cellular signaling events as well as in kinase and protease functions. Both Cys46 and Cys36 are identified as lipidation sites on OLR1. Protein lipidation is involved in various critical cellular functions and signaling pathways. Cysteine residues can undergo modification through the addition of a 16-carbon S-palmitoyl group, a process known as S-palmitoylation, which represents one of the most common mechanisms of protein lipidation. In our experiments, mutations at the Cys36 site did not exhibit characteristics similar to those at Cys46; consequently, the relationship with lipidation was not further investigated.

Our IP-MS analysis indicated that compared to COS-7 cells transfected with C46A, cells transfected with L45A exhibited affinity not only for OLR1 but also for two additional cytoskeletal proteins. According to our protein-protein interaction (PPI) results, the proteins interacting with these cytoskeletal components can be categorized into two functional groups. One cluster primarily consists of keratins, which possess relatively non-specific functions, while the other cluster is mainly involved in cellular signaling, with a particular emphasis on the regulation of cholesterol homeostasis. Given that the receptor under investigation plays a crucial role in lipid metabolism, and our experiments primarily involve stimulating cells with lipoproteins, the second group is noteworthy despite its p-value exceeding 0.05. This may be attributed to the limited number of proteins available for analysis. Within this cluster, SOAT2 encodes sterol O-acyltransferase 2, an enzyme known for its role in catalyzing the formation of fatty acid cholesterol esters.^17–19 This enzyme is essential for lipoprotein assembly and dietary cholesterol absorption.¹⁹ Conversely, PCSK5 is a serine endopeptidase that plays a critical role in processing various pro-proteins by cleaving at paired basic amino acid sites, which is particularly significant in the context of pregnancy.²⁰ Finally, ceramide synthase CERS3 is responsible for catalyzing the synthesis of ceramide from sphingosine and acyl-CoA substrates.²¹ It is a key factor in maintaining epidermal lipid homeostasis and promoting terminal differentiation.²¹

A recent study has demonstrated that the targeted inhibition of Thr188 on mouse ERK2 results in the inhibition of cardiac hypertrophy and pressure-overload-induced heart failure, while notably avoiding cardiotoxic side effects.²² This finding underscores the potential of selectively disrupting specific protein sites as a precise therapeutic strategy for treating diseases. In line with this notion, we propose that most site within TM of OLR1 holds promise as a target for intervention, aiming to obstruct interactions that drive the development of AS.

Numerous studies have acknowledged the association between oxidized low-density lipoprotein (ox-LDL) and various types of cancer, including breast and colorectal cancers. Additionally, OLR1 has been identified as a potential link between atherosclerosis and cancer,⁸ with dyslipidemia shown to increase the risk of certain cancers.²³ In our research, somatic mutations in OLR1 were observed in lung cancer, particularly in lung adenocarcinoma (LUAD), with the L45F (SNP) mutation having an allele frequency (T) of 0.19. Currently, information regarding this mutation is limited, although the somatic mutation frequency of OLR1 is noted to be less than 0.1%. According to the GEPIA database, OLR1 demonstrated significant downregulation in tumor tissues, including LUAD, compared to adjacent normal tissues (Supplementary Figure 6a (1)). Interestingly, high expression of OLR1 was associated with overall survival in LUAD patients (Supplementary Figure 6a (2)), suggesting that OLR1 may play a protective role in lung adenocarcinoma endothelium. However, we were unable to provide a clear assessment of the mutation spectrum associated with the L45F variant in LUAD about survival outcomes. It is noteworthy that in our experiments, foam cells overexpressing the L45F variant exhibited reduced levels of pERK1/2, contributing to increased lipid uptake. This suggests that specific modifications at Leu45 may have dual effects on both atherosclerosis and cancer. Nevertheless, further in-depth investigations are required to elucidate the underlying mechanisms in this context.

Protein mutations are widely recognized as key factors in the pathogenesis of various diseases, such as ESR1 fusion proteins in breast cancer.²⁴ Additionally, genetic variations among individuals have been demonstrated to play a critical role in human diseases. For instance, the missense variant K26R of ACE2 can reduce receptor affinity, thereby affecting susceptibility to SARS-CoV-2 infection. Currently, most targeted therapies focus primarily on specific signaling pathways or the modulation of various kinases. However, nearly all signaling pathways or kinases produce numerous downstream effects, often leading to excessive side reactions that significantly limit their clinical applicability.²⁵ Therefore, there is an urgent need for more precise therapeutics. In this context, emerging research includes investigations into novel COVID-19 treatment strategies, such as the use of engineered ACE2 variants (e.g., Asp427Arg, Pro451Met, and Gly448Trp).²⁶ Furthermore, there is growing interest in the potential modifications of OLR1.²⁷ In the future, specific modifications at the Leu45 site within the transmembrane domain of OLR1 may become a unique focus for the treatment of atherosclerosis, cardiovascular disease (CVD), and various types of cancer.

Alanine scanning is a site-directed mutagenesis technique that serves as a valuable tool for identifying the specific contributions of individual amino acid residues to the stability and function of a given protein.²⁸ Alanine is the preferred choice for this method due to its small size and chemically inert properties, characterized by a methyl functional group, while still mimicking the secondary structure preferences of many other amino acids.²⁹ In our study, we introduced mutations within the transmembrane (TM) domain of OLR1 by substituting various amino acids with alanine. The results indicated that, except for Leu45, these substitutions interfered with ox-LDL-induced signaling. Notably, when Leu45 was mutated to alanine, there was no significant impact on pERK1/2 levels, suggesting that this residue is relatively inconsequential in this context. However, substituting Leu45 with phenylalanine resulted in markedly different downstream signaling responses, indicating that the TM46 region may adopt a unique spatial conformation that potentially shares functional similarities with OLR1. In contrast, the mutation of Leu45 to phenylalanine disrupted this spatial conformation. Overall, these findings strongly suggest that Leu45 within the TM domain of OLR1 plays a critical role in the context of drug intervention, and further research in this area holds great promise for understanding and manipulating the function of this protein.

However, there are also some limitations in this study. It is important to acknowledge that our experiments were conducted in vitro, primarily utilizing non-human cell lines. Consequently, there may be discrepancies between the findings of this study and the results obtained from in vivo perturbations involving OLR1. Given these considerations, further research is warranted to elucidate the potential mechanisms of signal transduction mediated by OLR1 in response to ox-LDL. Additionally, targeted functional exploration of specific sites within OLR1 is necessary for potential therapeutic applications in cardiovascular disease (CVD) and cancer.

Conclusion

Supplemental Material

sj-docx-1-ini-10.1177_17534259251350447 - Supplemental material for Mutation within the transmembrane domain of oxidized low-density lipoprotein receptor 1 influences oxidized low-density lipoprotein-induced signal transduction

Supplemental material, sj-docx-1-ini-10.1177_17534259251350447 for Mutation within the transmembrane domain of oxidized low-density lipoprotein receptor 1 influences oxidized low-density lipoprotein-induced signal transduction by Zhen Ma, Ran Xu, Jing Lu, Xiong Huang, Hao Jia, Zhiwen Ding, Jie Yuan and Yunzeng Zou in Innate Immunity

Footnotes

Acknowledgements

The authors thank Mr. Koichi Yamamoto from Osaka University, Japan, for his kind gift of chimera plasmid, and Mr. Xie Weijian from Fubio Biological Technology Co., Ltd China, for his strong support in cell line establishments. We thank Yang Chunjie and Jia Jianguo from our laboratory for their help in experimental skill pieces of training. We also would like to thank anonymous reviewers who gave valuable suggestions that have helped to improve the manuscript.

ORCID iD

Yunzeng Zou

Author contributions

Conceptualization: Zhen Ma, Zhiwen Ding, Jie Yuan, and Yunzeng Zou; Data curation: Zhen Ma, Ran Xu, Jing Lu, Xiong Huang and Hao Jia; Formal analysis: Xiong Huang and Zhen Ma; Funding acquisition: Yunzeng Zou; Investigation: Zhen Ma, Ran Xu, Jing Lu, and Hao Jia; Project administration: Zhen Ma and Yunzeng Zou; Writing-original draft: Zhen Ma; Writing-review & editing: Ran Xu, Jing Lu, Xiong Huang, Hao Jia, Jie Yuan, Zhiwen Ding and Yunzeng Zou. All authors reviewed the manuscript.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China, (grant number 82230009).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability statements

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Supplemental material

Supplemental material for this article is available online.

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