Identiﬁcation and Characterization of the Very-Low-Density Lipoprotein Receptor Gene from Branchiostoma belcheri : Insights into the Origin and Evolution of the Low-Density Lipoprotein Receptor Gene Family

Simple Summary: The low-density lipoprotein receptor (LDLR) family plays crucial roles in lipid metabolism, but this family has not been investigated in ancient chordates (protochordates) to date. We identiﬁed and characterized a new LDLR family member in a protochordate— Branchiostoma belcheri . Additionally, we investigated the evolutionary process and molecular function in this gene family. Our work ﬁlls a research gap in the LDLR family in protochordates and provides new insights into the origin and evolution of chordate LDLRs. Abstract: Low-density lipoprotein receptors (LDLRs) are a class of cell-surface endocytosis receptors that are mainly involved in cholesterol homeostasis and cellular signal transduction. Very-low-density lipoprotein receptors (VLDLRs), which are members of the LDLR family, have been regarded as multi-function receptors that fulﬁll diverse physiological functions. However, no VLDLR gene has been identiﬁed in protochordates to date. As a representative protochordate species, amphioxi are the best available example of vertebrate ancestors. Identifying and characterizing the VLDLR gene in amphioxi has high importance for exploring the evolutionary process of the LDLR family. With this study, a new amphioxus VLDLR gene (designated AmphiVLDLR ) was cloned and characterized using RACE-PCR. The 3217 nt transcript of the AmphiVLDLR had a 2577 nt ORF, and the deduced 858 amino acids were highly conserved within vertebrate VLDLRs according to their primary structure and three-dimensional structure, both of which contained ﬁve characteristic domains. In contrast to other vertebrate VLDLRs, which had a conserved genomic structure organization with 19 exons and 18 introns, the AmphiVLDLR had 13 exons and 12 introns. The results of a selective pressure analysis showed that the AmphiVLDLR had numerous positive selection sites. Furthermore, the tissue expression of AmphiVLDLR using RT-qPCR showed that AmphiVLDLR RNA expression levels were highest in the gills and muscles, moderate in the hepatic cecum and gonads, and lowest in the intestines. The results of the evolutionary analysis demonstrated that the AmphiVLDLR gene is a new member of the VLDLR family whose family members have experienced duplications and deletions over the evolutionary process. These results imply that the functions of LDLR family members have also undergone differentiation. In summary, we found a new VLDLR gene homolog ( AmphiVLDLR ) in amphioxi. Our results provide insight into the function and evolution of the LDLR gene family.


Introduction
Very-low-density lipoprotein receptors (VLDLRs) are classified into the superfamily of transmembrane-protein low-density lipoprotein receptors (LDLRs). The VLDLR gene was initially discovered as its homologous gene (LDLR) in rabbits [1] and was later identified in many animals including nematodes [2], fish [3,4], birds [5,6], and some other mammals [7,8]. LDLRs bind to apo-E-and apo-B-containing lipoproteins, whereas VLDLRs preferentially interact with triglyceride-rich lipoproteins and apo-E [9]. In humans, VLDLR mRNAs are expressed in various tissues such as adipose, heart, and muscle tissues but are expressed very little in the liver [8,10]. The ligand specificity and tissue distribution of the VLDLR showed that the receptor may have critical functions in fatty acid metabolism. Similar to LDLR family members, the VLDLR protein consists of four or five characteristic regions, including a C-terminal intracellular domain, a single-transmembrane-spanning segment or transmembrane domain, optional O-linked glycosylation domain, epidermal growth factor (EGF) precursor homology domain, and an N-terminal ligand-binding domain [11]. Although each structural domain of the LDLR and VLDLR proteins exhibits a unique homology to some extent, the VLDLR has eight cysteine-rich repeat regions in the ligandbinding domain, whereas LDLRs have seven. VLDLRs are classified into two subtypes: type I VLDLRs contain five functional domains and type II VLDLRs lack the O-linked sugar fragment encoded by the 16th exon and are prominent in all tissues except muscle. Type I VLDLRs are principally expressed in muscles and the heart with active lipid metabolism, whereas type II VLDLRs are mainly expressed in the kidneys, spleen, adrenal gland, and testis [12].
VLDLRs, as members of the LDLR family, are multifunctional receptors that are involved in many important physiological processes, such as regulating neuroblast migration in the cerebellum and cerebral cortex, and modulating adipose tissue inflammation [25,26]. To date, the cDNA of VLDLRs has mainly been cloned and characterized in vertebrates. Few studies have been conducted on VLDLRs in invertebrates, although some invertebrate genome annotations have been performed. The amphioxus (Branchiostoma belcheri) is a marine filter-feeding animal that generally lives in sandy, shallow habitats in tropical and temperate ocean areas [27]. Amphioxi are located at the base in the chordata, and they have been regarded as important in the study of the origin of chordates [28]. For this work, we cloned a new VLDLR gene homolog in amphioxi (designated as AmphiVLDLR). Additionally, we preliminarily investigated the evolutionary process and molecular function of the AmphiVLDLR. Identifying and characterizing the AmphiVLDLR gene in Branchiostoma belcheri is fundamental to revealing the evolutionary processes that have occurred in this family in chordates. Our results showed an LDLR family member in protochordates and provide new insights into the origin and evolution of chordate LDLRs.

Cloning the Full-Length cDNA of the AmphiVLDLR Gene
Total Branchiostoma belcheri RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The cDNA template was Animals 2023, 13, 2193 3 of 13 synthesized using Moloney murine leukemia virus reverse transcriptase (TaKaRa, Dalian, China). According to the expressed sequence tag of a potential Branchiostoma floridae partial VLDLR sequence obtained from a library that was constructed in our previous studies [32], two primers were designed to amplify the middle region of AmphiVLDLR using PCR ( Figure S1). Subsequently, several specific primers were designed to execute 5 and 3 RACE experiments ( Figure S1). The 5 and 3 RACE experiments were conducted using a First Choice ® RLM-RACE Kit (Ambion, Austin, TX, USA), according to the manufacturer's instructions. The cloned fragments were ligated into T-Vector pMD™ 19 plasmids (TaKaRa, Dalian, China) and sequenced. Sequencing results were assembled via DNAMAN to acquire the full-length AmphiVLDLR cDNA. Finally, several specific primers were designed to further test the complete CDS, 5 -UTR, ORF, and 3 -UTR regions ( Figure S1).

Data Collection, Sequence Alignment, Phylogenetic Analysis, Selective Pressure Analysis, and Gene Annotation
The predicted AmphiVLDLR protein was analyzed by using BLAST programs (http:// www.ncbi.nlm.nih.gov/blast/Blast.cgi, accessed on 2 June 2022) [33]. The functional region of the AmphiVLDLR was predicted using the SMART database (http://smart.embl.de/, accessed on 10 June 2022) [34]. The similarity or identity of the known VLDLR sequences were calculated using MatGAT software with the default parameters [35].
To explore the degree of conservation of ten LDLRs, human proteins, including LRP1, LRP1b, LRP2 (megalin), LRP5, LRP6, MegF7, LDLR, VLDLR, ApoER2, and SorLA/LR11, were acquired from the NCBI website (https://www.ncbi.nlm.nih.gov/, accessed on 12 August 2019). These homologous LDLR family proteins were found by using tBLASTn and BLASTp on the genomes of the following species: Mus musculus, Gallus gallus, Xenopus laevis, Danio rerio, Petromyzon marinus, Ciona intestinalis, Branchiostoma belcheri, Strongylocentrotus purpuratus, Drosophila melanogaster, and Caenorhabditis elegans (Table S1). All the Homo sapiens LDLRs were searched against the Refseq protein data of the above species (except lamprey and amphioxi) (Table S1). These LDLR family sequences in lamprey were searched against the Ensembl database, and those in the amphioxi were searched against the Chinese Lancelet database (http://mosas.sysu.edu.cn/genome/, accessed on 12 August 2019) (Table S1). We filtered all the blast hits, and only eligible sequences (blast score > 150 and length > 50) were examined. Then, all the sequences were searched by using reciprocal blast methods. Additionally, the sequences from the previous step were considered to be homologous genes if the best hit of the initial blast results matched the best hit results of the reciprocal blast. The multiple alignments of LDLR family members were conducted by using the MUSCLE program with default parameters [36]. The phylogenetic tree of the LDLR family proteins was constructed by using phyML software [37]. The signal peptide of the AmphiVLDLR was predicted using SignalP-4.1 (https://services.healthtech.dtu. dk/service.php?SignalP-4.1, accessed on 6 November 2022) [38]. The three-dimensional structures of the Branchiostoma belcheri and Homo sapiens VLDLR were predicted by using the alphafold2 method in ColabFold (https://colab.research.google.com/github/sokrypton/ ColabFold/blob/main/AlphaFold2.ipynb, accessed on 6 November 2022)) [39,40]. The root mean square deviation (RMSD) value was calculated by using VMD to compare the spatial differences in the proteins [41,42]. The genomic structure (exons and introns) analysis was performed by using the Splign platform (https://www.ncbi.nlm.nih.gov/ sutils/splign/splign.cgi, accessed on 20 November 2022) [43]. A selective pressure analysis was conducted by using PAML 4.9j [44]. A GO annotation of these VLDLRs was executed by using the InterPro website (https://www.ebi.ac.uk/interpro/, accessed on 2 December 2022), and the KEGG annotation was executed by using eggnog-mapper v2 [45,46].

Real-Time Quantitative PCR Analysis of AmphiVLDLR
To investigate the spatial expression pattern of the AmphiVLDLR, the total RNA was extracted from the amphioxi gonads, hepatic cecum, intestines, gills, and muscles (Branchiostoma belcheri), and the cDNA was synthesized according to the methods outlined in Section 2.2. The specific VLDLR gene primers (5 -GCACAGGACGAACCGCTATC-3 ; 5 -GCCACGAGGACGACCAGAG-3 ) were designed to execute a quantitative real-time PCR. Additionally, the β-actin gene (5 -GCCTCCCTGTCCACCTTCC-3 ; 5 -AACTTGCCATCCT-TAGCCACTG-3 ) of Branchiostoma belcheri was used as the internal control. The RT-qPCR was performed using an SYBR ® Premix Ex Taq TM kit (TaKaRa, Dalian, China) following the manufacturer's instructions. All the experiments were conducted in triplicate and were shown in terms of the relative mRNA expression level mean ± SE (n = 4). All the data were processed by using the 2 −∆∆CT method, and the gene expression levels in each experiment were visualized in terms of gonads [47].

Cloning and Characterization of the AmphiVLDLR Gene
A 1639 bp middle fragment of the AmphiVLDLR gene was amplified from Branchiostoma belcheri using RT-PCR. Based on the 1639 bp region, 203 bp (via 5 RACE) and 1567 bp (via 3 RACE) fragments were amplified ( Figure 1A-C). The full-length AmphiVLDLR cDNA (3217 bp) was assembled by overlapping the three cloned fragments. The complete Am-phiVLDLR cDNA sequence was further tested with an end-to-end PCR reaction ( Figure 1D). Additionally, the 5 -UTR, ORF, and 3 -UTR regions were verified using PCR ( Figure 1E     The results of a genomic sequence analysis showed that the AmphiVLDLR gene had 13 exons and 12 introns. The VLDLR gene had a conserved exon-intron organization with 19 exons and 18 introns across the vertebrates, even when considering the intron phases and positions ( Figure 5). In addition, both the LDLRs and VLDLRs also had a similar gene structure in vertebrates. The only difference was that the VLDLR had 19 exons, whereas the LDLR had 18 exons ( Figure 5). The extra exon exactly corresponded to a cysteine-rich repeat sequence in the ligand-binding domain. The intron insertion sites and phases were almost identical in both the VLDLRs and LDLRs, except for the last intron in Danio rerio (which was 1 instead of 0). However, in the amphioxi, the insertion positions and phases of the intron underwent large changes, except in the EGFPD region.
The primary AmphiVLDLR structure was compared with other species' VLDLRs ( Figure 3, Table 1). The AmphiVLDLR protein was highly similar to the VLDLR protein from the vertebrates, and the identity degree was different in diverse protein domains ( Table 1). The identities of the five characteristic domains were variable. Among them, the identity of LBD, EGFPD, and CPD was relatively high, and that of the other two domains was relatively low (Table 1).  Figure 3. AmphiVLDLR alignment with homologous protein sequences from vertebrates. The eight cysteine-rich repeat domains are numbered I to VIII. The asterisks represent conserved cysteine amino acid residues in the EGFPD (EGF A, B, and C) and cysteine-rich repeat domains. The six cysteine repeats in the LBD may contribute to the formation of a rigid structure in this domain with negatively charged residue clusters (underlined). The YW(T)D sequence was found in multiple tandem repeats. Additionally, the sequences that were predicted to form beta propeller structures are underlined. The OLSD is labeled with double underlining. The conserved FDNPVY motif involved in the receptor endocytosis process is underlined with dots.
cysteine-rich repeat domains are numbered I to VIII. The asterisks represent conserved cyst amino acid residues in the EGFPD (EGF A, B, and C) and cysteine-rich repeat domains. The cysteine repeats in the LBD may contribute to the formation of a rigid structure in this domain w negatively charged residue clusters (underlined). The YW(T)D sequence was found in mult tandem repeats. Additionally, the sequences that were predicted to form beta propeller struct are underlined. The OLSD is labeled with double underlining. The conserved FDNPVY m involved in the receptor endocytosis process is underlined with dots.  The results of a genomic sequence analysis showed that the AmphiVLDLR gene had 13 exons and 12 introns. The VLDLR gene had a conserved exon-intron organization with 19 exons and 18 introns across the vertebrates, even when considering the intron phases and positions ( Figure 5). In addition, both the LDLRs and VLDLRs also had a similar gene structure in vertebrates. The only difference was that the VLDLR had 19 exons, whereas the LDLR had 18 exons ( Figure 5). The extra exon exactly corresponded to a cysteine-rich repeat sequence in the ligand-binding domain. The intron insertion sites and phases were almost identical in both the VLDLRs and LDLRs, except for the last intron in Danio rerio (which was 1 instead of 0). However, in the amphioxi, the insertion positions and phases of the intron underwent large changes, except in the EGFPD region. The primary AmphiVLDLR structure was compared with other species' VLDLRs ( Figure 3, Table 1). The AmphiVLDLR protein was highly similar to the VLDLR protein from the vertebrates, and the identity degree was different in diverse protein domains ( Table 1). The identities of the five characteristic domains were variable. Among them, the identity of LBD, EGFPD, and CPD was relatively high, and that of the other two domains was relatively low (Table 1).

Alignments and Phylogenetic Analysis of AmphiVLDLR
To explore the relationship between the AmphiVLDLR and other LDLR family members, representative invertebrate and vertebrate LDLR family sequences (Table S1) were used to construct the evolutionary tree with a maximum likelihood algorithm. All the LDLRs were classified into two clades (Figure 7). Clade I included megalin, LRP1, and LRP1b. Before Strongylocentrotus purpuratus appeared, the LRP1 gene appeared. Along with the evolution of the species, the duplication of the LRP1 gene occurred before vertebrates emerged. The LRP1b gene was found in jawed vertebrates. Clade II included VLDLR, LDLR, ApoER2, SorLA, MEGF7, LRP5, and LRP6. Clade II was divided into two subclades. Subclade I included VLDLR, LDLR, ApoER2, and SorLA. Before jawed vertebrates emerged, both LDLR and ApoER2 were generated from the VLDLR gene replication. Subclade II

Alignments and Phylogenetic Analysis of AmphiVLDLR
To explore the relationship between the AmphiVLDLR and other LDLR family members, representative invertebrate and vertebrate LDLR family sequences (Table S1) were used to construct the evolutionary tree with a maximum likelihood algorithm. All the LDLRs were classified into two clades (Figure 7). Clade I included megalin, LRP1, and LRP1b. Before Strongylocentrotus purpuratus appeared, the LRP1 gene appeared. Along with the evolution of the species, the duplication of the LRP1 gene occurred before vertebrates emerged. The LRP1b gene was found in jawed vertebrates. Clade II included VLDLR, LDLR, ApoER2, SorLA, MEGF7, LRP5, and LRP6. Clade II was divided into two subclades. Subclade I included VLDLR, LDLR, ApoER2, and SorLA. Before jawed vertebrates emerged, both LDLR and ApoER2 were generated from the VLDLR gene replication. Subclade II included MEGF7, LRP5, and LRP6. The LRP5 gene was generated via LPR6 gene replication before jawed vertebrates emerged. The high bootstrap values supported the precision of the topology. Abstract: This study addresses the importance of focal nodes in understanding the structural 12 composition of networks. To identify these crucial nodes, a novel technique based on parallel Fuzzy 13 Cognitive Maps (FCMs) is proposed. By utilising the focal nodes produced by the parallel FCMs, 14 the algorithm efficiently creates initial clusters within the population. The community discovery 15 process is accelerated through a distributed genetic algorithm that leverages the focal nodes 16 obtained from the parallel FCM. This approach mitigates the randomness of the algorithm, 17 addressing the limitations of random population selection commonly found in genetic algorithms. 18 The proposed algorithm improves the performance of the genetic algorithm by enabling informed 19 decision-making and forming a better initial population. This enhancement leads to improved 20 convergence and overall algorithm performance. Furthermore, as graph sizes grow, traditional 21 algorithms struggle to handle the increased complexity. To address this challenge, distributed 22 algorithms are necessary for effectively managing larger data sizes and complexity. The proposed 23 method is evaluated on diverse benchmark networks, encompassing both weighted and 24 unweighted networks. The results demonstrate the superior scalability and performance of the 25 proposed approach compared to existing state-of-the-art methods.

AmphiVLDLR Expression in Different Tissues
The spatial expression pattern of the AmphiVLDLR in Branchiostoma belcheri was explored via RT-qPCR on five different tissues (gonads, hepatic cecum, intestines, gills, and muscles). The results indicated that the AmphiVLDLR was ubiquitously expressed in all these tissues (Figure 8). The AmphiVLDLR expression level was high in the gills and muscles, moderate in the gonads and hepatic cecum, and low in the intestines (Figure 8).
The spatial expression pattern of the AmphiVLDLR in Branchiostoma belch explored via RT-qPCR on five different tissues (gonads, hepatic cecum, intestine and muscles). The results indicated that the AmphiVLDLR was ubiquitously expre all these tissues (Figure 8). The AmphiVLDLR expression level was high in the gi muscles, moderate in the gonads and hepatic cecum, and low in the intestines (Fig   Figure 8. The AmphiVLDLR expression level in different tissues.

Discussion
The LDLR family consists of a group of related receptor proteins with similar structures and functions. The main members are LDLR, VLDLR, ApoER2, LRP1, megalin (LRP2), LRP1b, LRP5, LRP6, MegF7, and SorLA/LR11 [13,14]. Studies on VLDLR/VLDLR-like genes are ongoing. Until now, VLDLRs have been cloned in mammals, birds, fish, and nematodes, but not in protochordates [1][2][3][4][5][6][7][8]. Amphioxi are considered a species that transformed from invertebrates to vertebrates and represent a basic model for studying the origin and evolution of chordates. Therefore, identifying and characterizing the AmphiVLDLR will have high importance for investigating the evolution and function of animal VLDLR genes.

AmphiVLDLR Is a New Member of the VLDLR Family
In Branchiostoma belcheri, the AmphiVLDLR, with an encoding 3217 bp transcript, was highly homologous to other VLDLR proteins (Figures 3 and 4 and Table 1). Similar to other LDLR family members, the AmphiVLDLR also contained five characteristic domains (Figure 3), and the five domains had high similarities to those for other vertebrates (Figures 3 and 4 and Table 1). The large difference between the Homo sapiens and Branchiostoma belcheri CPDs (RMSD CPD = 20.345) was due to the high percentage of unstable regions ( Figure 4). In the LBD, eight cysteine-rich repeat sequences existed, which was consistent with the VLDLR protein. As shown by the phylogenetic tree, the AmphiVLDLR clustered with the VLDLR + LDLR + ApoER2 invertebrate and vertebrate family members, which was closer to the VLDLRs in invertebrates (Figure 7). This is consistent with the classic evolutionary theory of species. These results support the idea that AmphiVLDLR is a type of VLDLR.

Evolution of LDLR Family Genes
Based on conservative analysis results, the LDLR family members gradually increased from invertebrate to vertebrate ( Figure 6). These new family members may perform different functions. Compared with Strongylocentrotus purpuratus and Branchiostoma belcheri, Ciona intestinalis may have secondarily lost the VLDLR and LRP1 genes, whereas Petromyzon marinus may have secondarily lost the VLDLR and MEGF7 genes ( Figure 6). The megalin gene from Strongylocentrotus purpuratus, Branchiostoma belcheri, and Ciona intestinalis did not cluster with other megalin family members. This may be due to the three sequences having extra molecular functions compared with the sequences in the megalin family of other species, or the genome annotation was incomplete in these three species.
Combined with the results of the conservative analysis and phylogenetic tree (Figures 6 and 7), we found that the LDLR family members experienced duplication and deletion events during evolution. When transitioning from invertebrates to vertebrates, the LDLR family members experienced duplications three times, i.e., LRP1b was duplicated from LRP1, ApoER2 and LDLR were duplicated from VLDLR, and LRP5 was duplicated from LRP6. These gene-generating events may be related to two rounds of whole-genome duplication in vertebrates [48]. The VLDLR, as one of the ancient LDLR family members, must have important biological functions; thus, identifying the AmphiVLDLR gene will be helpful in uncovering the function and evolution of the LDLR family in different species.
Based on the phylogenetic tree, we further compared the AmphiVLDLR with the LDLRs and VLDLRs of vertebrates. Among all the analyzed LDLR family proteins, the LDLR and VLDLR gene structures were very similar in vertebrates except for the insertion phase of the last intron from the LDLR in Danio rerio. The sequence similarities and the gene structures indicated that the LDLRs and VLDLRs may have originated from a common ancestral gene. The differences in the intron numbers of the invertebrate VLDLR homolog genes suggest that some introns may have been inserted into the VLDLR homologues during invertebrate evolution [49,50]. Compared with the LDLR and VLDLR in vertebrates, the AmphiVLDLR gene structure is very different. Therefore, we speculate that intron insertion events also occurred in vertebrates. The intron gain in vertebrates may be related to the functional elements of introns. Intron insertion is probably relevant for multiple functions, including encoding untranslated RNA, alternative splicing, and enhancing a normal or necessary mRNA transcription level and function at the DNA level [51][52][53][54][55][56]. An unknown mechanism whereby introns are appropriately added needs to be further investigated in the future to clarify this phenomenon.

VLDLR Tissue Expression Pattern
To investigate the function of the AmphiVLDLR, we detected its expression level in various tissues. VLDLR mRNA is mainly present in the heart, skeletal muscles, and adipose tissues in human [10]. Mouse VLDLRs are most abundant in the brain and are especially highly expressed in skeletal muscles and the heart. The existence of high-level free lipids in these tissues supports the idea that VLDLRs play vital roles in fatty acid and cholesterol metabolism [57]. The AmphiVLDLR mRNA was present in all tissues and was abundant in the gills and muscles and deficient in the gonads and intestines (Figure 8). The relatively abundant AmphiVLDLR mRNA distribution in the muscles was similar to that in mice and humans, which may also be related to cholesterol and fatty acid metabolism. The GO annotation indicated that a major function of the AmphiVLDLR was calcium-ion binding (Table S2). Calcium ions perform multiple metabolic functions in muscles, and gills are the major organ for active calcium ion uptake, which has a high importance for the maintenance of calcium ion homeostasis [58]. The high expression level of the Branchiostoma belcheri VLDLR gene in the muscles and gills demonstrates that the AmphiVLDLR has similar conserved functions. Although both the GO and KEGG annotations showed that the functions of the VLDLR family were conserved among different species (Tables S2 and  S3), the results of the selection pressure analysis demonstrated that the VLDLR gene in amphioxi had numerous positive selection sites (Table S4). These results implied that the AmphiVLDLR not only performs conserved functions compared with other VLDLR genes, but it may also have unique biological functions after undergoing adaptive evolution.

Conclusions
In summary, we identified the AmphiVLDLR as a new member of the VLDLR family and provided insight into the evolution of the LDLR family members from invertebrates to vertebrates.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ani13132193/s1, Figure S1: The schematic diagram of PCR primers and corresponding regions; Table S1: The information of LDLR family genes and proteins; Table S2: GO annotations of the VLDLR family members; Table S3: KEGG annotations of the VLDLR family members; Table S4: Selective pressure analysis (branch-site model) of AmphiVLDLR.