Comparative Biochemistry and Physiology, Part B 169 (2014) 51–62

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Four cDNAs encoding lipoprotein receptors from shrimp (Pandalopsis japonica): Structural characterization and expression analysis during maturation Ji-Hyun Lee b, Bo Kwang Kim b, Young-il Seo c, Jung Hwa Choi c, Seung-Wan Kang d, Chang-Keun Kang e, Won-gyu Park a, Hyun-Woo Kim a,b,⁎ a

Department of Marine Biology, Pukyong National University, Busan 608–737, South Korea Interdisciplinary program of Biomedical Engineering, Pukyong National University, Busan, 608–737, South Korea c Fisheries Resources Research Division, National Fisheries Research and Development Institute, Busan, 619–902, South Korea d Gyeongsangnam-do Fisheries Resources Research Institute, South Korea e POSTECH Ocean Science and Technology Institute, Pohang University of Science and Technology (POSTECH), Pohang 790–784, South Korea b

a r t i c l e

i n f o

Article history: Received 30 October 2013 Received in revised form 18 December 2013 Accepted 19 December 2013 Available online 31 December 2013 Keywords: Decapod Pandalopsis japonica Lipophorin Reproduction Vitellogenin

a b s t r a c t As in all other oviparous animals, lipoprotein receptors play a critical role in lipid metabolism and reproduction in decapod crustaceans. Four full-length cDNAs encoding lipoprotein receptors (Paj-VgR, Paj-LpR1, Paj-LpR2A, and Paj-LpR2B) were identified from Pandalopsis japonica through a combination of EST screening and PCR-based cloning. Paj-LpR1 appears to be the first crustacean ortholog of insect lipophorin receptors, and its two paralogs, Paj-LpR2A and Paj-LpR2B, exhibited similar structural characteristics. Several transcriptional isoforms were also identified for all three Paj-LpRs. Each expression pattern was unique, suggesting different physiological roles for these proteins. Paj-VgR is an ortholog of vitellogenin (Vg) receptors from other decapod crustaceans. A phylogenetic analysis of lipoproteins and their receptors suggested that the nomenclature of Vgs from decapod crustaceans may need to be changed. A PCR-based transcriptional analysis showed that Paj-VgR and Paj-LpR2B are expressed almost exclusively in the ovary, whereas Paj-LpR1 and Paj-LpR2A are expressed in multiple tissues. The various transcriptional isoforms of the three Paj-LpRs exhibited unique tissue distribution profiles. A transcriptional analysis of each receptor using tissues with different GSI values showed that the change in transcription of Paj-VgRs, Paj-LpR2A and Paj-LpR1 was not as significant as that of Vgs during maturation. However, the transcriptional levels of Paj-LpR2B decreased in ovary at maturation, suggesting that their transcriptional regulation is involved in reproduction. © 2013 Elsevier Inc. All rights reserved.

1. Introduction As in all oviparous species, vitellogenesis in decapod crustaceans involves nutrient deposition and yolk formation to ensure an ample energy supply. Yolk proteins are lipoprotein complexes that are conjugated to carbohydrates and carotenoid pigments (Wallace et al., 1967). The major protein component of yolk protein is vitellogenin (Vg). Genes encoding Vg have been isolated from various decapods, and two copies of Vg genes have been isolated from several species, including Pandalopsis japonica, Metapenaeus ensis, and Penaeus monodon (Kung et al., 2004; Tiu et al., 2009; Jeon et al., 2010). The hepatopancreas appears to be the principal production site for Vg in the suborder Pleocyamata, which includes brachyurans, astacideans, and carideans (Okuno et al., 2002; Tsutsui et al., 2004), whereas the hepatopancreas and ovary contribute equally to Vg production in Dendrobranchiata, ⁎ Corresponding author. Department of Marine Biology, Pukyong National University, Busan 608–737, South Korea. Tel.: +82 51 629 5926; fax: +82 51 629 5930. E-mail address: [email protected] (H.-W. Kim). 1096-4959/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cbpb.2013.12.005

including penaeid shrimp (Raviv et al., 2006; Tiu et al., 2006). Interestingly, the Vg genes isolated from decapod crustaceans exhibit the highest level of similarity to lipophorins (Lps) from insects, while hemolymph clotting proteins (CPs) are clustered together with insect Vg genes (Cheng et al., 2008; Jeon et al., 2010). In insects, Lp is the main insect hemolymph lipoprotein. It is involved in lipid transport in various tissues, and in providing lipids and other yolk precursors from the fat body to the ovaries (Rodenburg and Van der Horst, 2005; Swevers et al., 2005). In insects, Vg and Lp genes are internalized into growing oocytes (Machado et al., 1996; Fan et al., 2002). Lipoproteins are internalized by membrane-bound receptors, including Vg receptors (VgRs) and Lp receptors (LpRs) (Rodenburg and Van der Horst, 2005). Genes encoding VgRs have been identified in several insect species, including Aedes aegypti (Sappington et al., 1996), Periplaneta americana (Tufail and Takeda, 2005), and Balttella germanica (Ciudad et al., 2006). Similar numbers of LpR genes have been isolated from several insect species, including Locusta migratoria (Dantuma et al., 1999), Galleria mellonella (Lee et al., 2003), and B. germanica (Ciudad et al., 2007). Although both VgR and LpRs belong to the low-

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density lipoprotein (LDL) receptor (LDLR) superfamily, which harbors conserved motifs, including a ligand-binding domain (LBD), epidermal growth factor (EGF) precursor domain, O-linked sugar domain (OLSD), transmembrane domain (TMD), and cytoplasmic domain, the two types of receptors can be clearly distinguished from each other based on the conserved domain organization of each type. The phylogenetic and functional relationships among lipoproteins and their receptors have not been clearly established in decapod crustaceans. Since the first putative VgR cDNA was isolated from P. monodon (Tiu et al., 2008), three additional full-length and partial cDNA sequences encoding crustacean lipoprotein receptors have been added to the GenBank database (http://www.ncbi.nlm.nih.gov/). Based on their domain organization, all four lipoprotein receptors appear to be orthologs of insect VgRs. Homologs of LpRs have not been identified in decapod crustaceans. Pandalopsis japonica is an important species due to its commercial value and as a model system for molting and reproduction studies because it survives for long periods after eyestalk ablation (Lee et al., 2011; Jeon et al., 2012). Further, its evolutionary importance cannot be understated, as Caridea (true shrimp) can be used for comparative studies with other decapod crustaceans, including Brachyura (crabs), Astacidea (lobsters and crayfish), and Dendrobranchiata (penaeid shrimp). In the present study, we identified four distinct full-length cDNAs encoding lipoprotein receptors (Paj-LpR1, Paj-LpR2A, PajLpR2B, and Paj-VgR), and we investigated their primary structure and transcriptional characteristics, which are related to ovarian maturation. 2. Materials and methods 2.1. Experimental animals Live shrimp (P. japonica) were purchased from a local seafood market and acclimatized in circulating aerated seawater for 7 days at 4 °C before dissection. The shrimp were fed daily with squid and polychaetes. The day/night lengths were maintained at 12 h/12 h. Before dissection, the animals' wet body and gonad weights were measured. The isolated tissues were directly frozen in liquid nitrogen and stored at − 70 °C before use for total RNA extraction. The individual gonadsomatic index (GSI = [gonad weight/total body weight] × 100) was calculated as described previously (Jeon et al., 2010). 2.2. Cloning of full-length cDNAs encoding four lipoprotein receptors (Paj-LpR1, Paj-LpR2A, Paj-LpR2B, and Paj-VgR) A cDNA contig database of P. japonica was established by a commercial next-generation sequencing service (Macrogen Inc., Seoul, Korea) as described previously (Jeon et al., 2012). Using the LpR1sequence from Bombyx mori (NP_001104808) as bait, the cDNA database was screened using the Basic Local Alignment Search Tool (BLAST) program (http://ncbi.nlm.nih.gov). To isolate the full-length cDNA sequence, fragment analysis by PCR and RACE was performed as described in Supplementary data 2. Degenerate and sequence-specific primers were designed using the IDTSciTools program (http://eu.idtdna.com/analyzer/ applications/oligoanalyzer/default.aspx) targeting conserved amino acid residues to extend the sequences (Supplementary data 1). The primers used in this experiment were commercially synthesized by Bioneer Co. (Daejeon, Korea). Since it was not possible to obtain fulllength cDNA with a single reaction, fragmental sequences were also obtained by PCR with sequence-specific and degenerate primers (Supplementary data 1 and 2). 5′-RACE and walking were carried out using a CapFishingTM full-length cDNA isolation kit and DNA Walking SpeedUpTM Premix Kit according to the manufacturer's protocol (Seegene, Seoul, Korea). 3′-RACE was performed using sequencespecific forward primers and previously used 3′-RACE primers, which harbor a lab-designed linker sequence (Lee et al., 2011). The fragmental

sequences were joined together and the single transcript was reconfirmed by RT-PCR. Total RNA was isolated from the ovary and hepatopancreas, which are considered to be the major production sites for lipoprotein receptors in arthropods (Tufail and Takeda, 2009). Genomic DNA was removed by DNase Ι (Promega, Madison, WI, USA) treatment prior to cDNA synthesis. cDNAs were synthesized as described previously (Jeon et al., 2011). The reactions (20 μL) contained 1 μL of cDNA (200 ng/μL), 2 μL of sequence-specific primers (4 pM) (Supplementary data 1), 0.1 μL of Ex Taq Hot Start Version (Takara Bio Inc., Shiga, Japan), 2 μL of dNTPs (2.5 mM each), and 2 μL of 10 × buffer. The PCR conditions were 1 min at 94 °C followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. 18S rRNA primers were used as a positive control (Supplementary data 1). The reactants were separated by 1.5% agarose gel electrophoresis and stained with ethidium bromide. Those amplicons of the expected sizes were purified using an AccuPrep Gel Purification Kit (Bioneer Co.), ligated into the TA plasmid vector with a pGEM-T Easy Cloning Kit (Promega), and transformed into Escherichia coli DH5-alpha competent cells. The cDNAs were sequenced with an ABI Biosystems 3730 sequencer (Applied Biosystems, Foster City, CA, USA).

2.3. Bioinformatic analysis Nucleotide and amino acid sequence similarities were analyzed using BLAST software (http://www.ncbi.nlm.nih.gov/BLAST/). The fulllength, deduced amino acid sequences were aligned using ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and the domain organization of each lipoprotein receptor was predicted by the SMART algorithm (http://smart.embl-heidelberg.de/) (Letunic et al., 2012). Phylogenetic trees for the lipoproteins and their receptors were constructed by the minimal evolution method using Molecular Evolutionary Genetics Analysis (MEGA5) software (Tamura et al., 2011). All annotated sequences were retrieved from the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/). Analyzed cDNA sequences included the Insect LpRs of Bombyx mori (BAE71406), Aedes aegypti (AF355595), Locusta migratoria (AJ000010) and Tribolium castaneum (XP_967944); the insect VgRs of Tribolium castaneum (XM_963810), Anopheles gambiae (EAA06264), Aedes aegypti (AAK15810) and Blattella germanica (CAJ19121); the crustacean ovarian LpRs of Marsupenaeus japonica (BAH57291) and Penaeus semisulcatus (AAL79675); the crustacean VgRs of Macrobrachium rosenbergii (ADK55596) and Penaeus monodon (ABW79798); the vertebrate LDLRs of Mus musculus (CAA45759), Sus scrofa (AAC17444), Homo sapiens (AAA56833) and Bos taurus (XP_874020); the vertebrate VgRs of Oncorhynchus mykiss (CAD10640), Anguilla japonica (BAB64337) and Morone americana (AAO92396); the vertebrate VLDLRs of Homo sapiens (AAA61344), Mus musculus (AAA59384) and B. taurus (NP_776914); the insect Vg of Apis melifera (NP_001011578), Bombus ignites (ACQ91623), Nasonia vitripennis (XP_001307388) and Culex quinquefasciatus (XP_001857967); the maxillopod Vg of Tigriopus japonicus (ACJ12892) and Lepeophtheirus salmonis (ABU41134); the decopod CP of Marsupenaeus japonicus (ABK59925) and Penaeus monodon (ABW77320); the mollusk Vg of Crassostrea gigas (BAC22716) and Haliotis discus (BAF98238); the vertebrate Vg of Gallus gallus (AAA49139), Oryzias latipes 1 (BAB79696), O. latipes 2 (BAB79591), Anguilla japonica 1 (AAR82899) and A. japonica 2 (AAR82898); the vertebrate apoB of Rattus norvegicus (NP_062160) and H. sapiens (NP_000375); the insect apoLp-ΙΙ/Ι of Locusta migratoria (CAB51918), Nilaparvata lugens (BAG75121) and C. quinquefasciatus (XP_001849310); the decapods Vg of Scylla paramamosain (ACO36035), Callinectes sapidus (ABC41925), Pandalopsis japonica 1 (ACU51164), P. japonica 2 (KF731996), Litopenaeus vannamei (AAP76571), Marsupenaeus japonicus 1 (BAB01568) and M. japonicus 2 (BAD98732). The insect LDLR of B. mori (BAG12564) and the

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Fig. 1. Multiple deduced amino acid alignment of Paj-LpR1, Paj-LpR2A, and Paj-LpR2B with other insect LpRs. The consensus amino acid sequences are shaded black and grey. Hyphens indicate gaps in each corresponding residue. Conserved motifs are boxed. LBR, ligand-binding repeat; EGF, epidermal growth factor precursor-like repeat; β-propeller, β-propeller domain with YWTD repeats; OLSD, O-linked sugar domain; TMD, transmembrane domain. The aligned LpRs include LpR1 from D. melanogaster (NP_001262974), LpR2 from D. melanogaster (NP_001262971), LpR from B. germanica (CAL47125), and LpR from B. mori (BAE71409).

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Fig. 2. Multiple deduced amino acid alignment of Paj-VgR with other decapod and insect VgRs. The consensus amino acid sequences are shaded black and grey. Hyphens indicate gaps in each corresponding residue. Conserved motifs are boxed. LBR, ligand-binding repeat; EGF, epidermal growth factor precursor-like repeat; β-propeller, β-propeller domain with YWTD repeats; OLSD, O-linked sugar domain; TMD, transmembrane domain. The aligned VgRs include VgR from M. rosenbergii (ADK55596), VgR from P. monodon (ABW79798), and VgR from B. germanica (CAJ19121).

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Fig. 2 (continued).

chromadorea MTP of Caenorhabditis elegans (AAR27937) were used as outgroup. 2.4. Transcriptional analysis of the four lipoprotein receptors from P. japonica To identify the transcriptional profiles of the four lipoprotein receptor genes, end-point RT-PCR was performed. Two vitellogenic and mature shrimp were used for the tissue distribution experiment (Fig. 5). Total RNAs were isolated from the brain, abdominal ganglia, thoracic ganglia, hind gut, tail muscle, heart, gill, epidermis, hepatopancreas, and ovary using an RNeasy Mini Kit (Qiagen Korea Ltd., Seoul, Korea). Total RNA extraction, cDNA synthesis, and PCR were conducted in the same manner as in the cloning experiment. To measure the mRNA levels of the four lipoprotein receptors, ovaries and hepatopancreas were dissected from individuals with different GSI indices. Total 16 individuals with different GSI values were used (Fig. 6). End-point RT-PCR amplicons were visualized on an ethidium bromide-stained 1.5% agarose gel. Two Vg genes were used as a positive control for maturation (Jeon et al., 2010). qPCR was carried out to compare the expression levels of the lipoprotein receptors during maturation using the DNA Engine Chromo 4 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and SYBR Green premix Ex Taq ΙΙ (Takara Bio Inc.). Three individuals in each maturation stage were used for the qPCR (Fig. 7). The primers were the same as those used for end-point RT-PCR and qPCR (Supplementary data 1). The procedures are described elsewhere (Jeon et al., 2010). The statistical analysis of four

lipoprotein receptor expression used the one-way ANOVA test using data analysis of Excel software (Microsoft, ver. 2007). The results were considered significant at P b 0.05. 3. Results 3.1. Isolation of three lipoprotein receptors from P. japonica (Paj-LpR1, Paj-LPR2A, and Paj-LPR2B) Four full-length cDNAs encoding lipoprotein receptors were obtained from P. japonica by combining the EST database constructed previously (Jeon et al., 2012) with the traditional molecular biological strategy of 5′-RACE, 3′-RACE, and DNA walking (Matz et al., 1999). Among them, one sequence (5989 bp) turned out to be an ortholog of VgR in decapod crustaceans, whereas the other three sequences showed strong similarity to LpRs of insect species. First, two LpR cDNAs (3202 and 2698 bp, respectively) were originally identified by consecutive PCR, thereby extending the EST database sequences. Based on their similar primary structures and expression profiles, the 3202- and 2698-bp products were identified as Paj-LpR1 and Paj-LpR2, respectively. During PCR confirmation of the single Paj-LpR2 transcript, we identified another distinct cDNA sequence (2654 bp), which showed 95% nucleotide sequence identity to Paj-LpR2. Two of the Paj-LpR2 sequences were therefore renamed as Paj-LpR2A and Paj-LpR2B, respectively. Paj-LpR1 exhibited the highest degree of sequence similarity (68%) to LpR from the insect L. migratoria (GenBank accession number: CAA03855). Both

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Fig. 3. Domain organization of four lipoprotein receptors from P. japonica. The domain organization in the LpR and VgR proteins was illustrated by the SMART algorithm. Each symbol indicates a specific motif, including signal sequences (red at the N-terminus), LBD ( ), EGF ( ), β-propeller ( ), and TMD ( ).

Paj-LpR2A and Paj-LpR2B showed 55% similarity to LpR from another insect, Drosophila melanogaster (Drm-LpR1; GenBank accession number: NP_001262974), indicating that all three lipoprotein receptors from P. japonica are orthologs of insect LpRs. Various transcriptional isoforms of the three Paj-LpRs were identified (Fig. 3), and two distinct alternatively spliced isotypes of Paj-LpR1 were identified. Besides the longest transcript, Paj-LpR1 (3202 bp, 864 amino acid residues), Paj-LpR1S1 lacks a 159-bp sequence within the LBD. This sequence produces a putative protein (811 residues) lacking one ligand-binding repeat (LBR) within the LBD. Paj-LpR1S2 was expressed as a transcript lacking a 306-bp sequence, which encoded a putative protein (762 residues) lacking two YWTD repeat motifs within the β-propeller domain (Fig. 3). Unlike those transcripts from insects, no transcriptional variants were found within the O-glycosylation site. Both Paj-LpR2A and Paj-LpR2B showed the same splicing pattern, leading to the production of short isoforms that were truncated from LBR5 within the LBD producing a putative soluble protein (Fig. 3). Those truncated transcriptional isoforms were named Paj-LpR2AS (1243 bp, 382 amino acids) and Paj-LpR2BS (1159 bp, 287 amino acids), respectively.

To estimate the functional similarities of the Paj-LpRs to other LpRs from insect species, a multiple amino acid sequence alignment was performed (Fig. 1). Since there was no homologous sequence from a decapod crustacean in the GenBank database, we compared three LpR sequences from P. japonica with other LpRs from representative insect species. Transcriptional variants of each LpR gene were excluded from the analysis. Paj-LpR1, Paj-LpR2A, and Paj-LpR2B are composed of 864, 867, and 864 amino acid residues, respectively, similar to LpRs from other insect species (Fig. 1). All LpRs from P. japonica exhibited the conserved domain organization of LDLR family members, including signal peptide sequences at the amino terminus, an LBD with cysteine-rich LBRs, an EGF precursor domain with three EGF-like repeats and a βpropeller domain, an OLSD, a TMD, and a cytoplasmic domain. Signal peptide sequences and cleavage sites (1–27 residues for PajLpR1, 1–23 for Paj-LpR2A, and 1–23 for Paj-LpR2B) were predicted in all three Paj-LpRs, suggesting that the obtained cDNAs encode fulllength transcripts (Fig. 1). Two different arthropod LBRs within the LBD were identified in the LpRs: eight type I and seven type II LBRs (Fig. 1). Eight LBRs were identified within the LBD of several insect

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Fig. 4. Phylogenetic trees of lipoproteins and lipoprotein receptors. The phylogenetic trees of lipoprotein receptors (A) and lipoproteins (B) were constructed on the basis of amino acid sequences by the minimal evolution method, and a bootstrapping test was carried out with 1000 replications using MEGA5 software. Each clade was shaded with a different color.

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Fig. 5. Tissue distribution of four lipoprotein receptors from P. japonica. End-point RT-PCR products were separated on 1.5% agarose gels and stained with ethidium bromide (inverted image). 18S rRNA was used as a positive control. A total of ten different tissues were analyzed, including brain (Br), abdominal ganglia (Ag), thoracic ganglia (Tg), hind gut (Hg), tail muscle (Tm), gill (Gi), epidermis (Ep), ovary (Ov). M, size marker.

LpRs, including Paj-LpR1, LpR1 from D. melanogaster, and LpR from B. mori, whereas seven were identified in others, including Paj-LpR2, LpR2 from D. melanogaster, and LpR from B. germanica (Fig. 1). Each LBR contains six conserved cysteine residues that form three disulfide bonds, except for Paj-LpR2s. In both Paj-LpR2A and Paj-LpR2B, the first and third cysteine residues in LBR5 were replaced by proline and serine, respectively. In addition, the sixth cysteine residue within LBR8 of LpR2A was changed to tyrosine (Fig. 1). Besides the LBD, all compared LpRs also showed well-conserved EGF precursor domains composed of two initial EGF-like repeats (EGF1 and EGF2), a β-propeller domain with YWXD motifs, and one EGF-like repeat (EGF3) (Fig. 1). According to Tufail and Takeda (2009), the first repeat was not recognized in the β-

propeller domain of all compared LpRs from arthropods, including the Paj-LpRs, because it lacks the YWXD motif (Tufail and Takeda, 2009). Compared to the 31% S/T residue ratio from Paj-LpR1, a ratio b 10% was identified in the OLSD of the Paj-LpR2s (Fig. 1). The length of the OLSD varies among Paj-LpRs from 23 to 65 residues, and no alternative splicing site was identified in the three Paj-LpRs, unlike the situation in insect species (Fig. 1). The TMD of all compared LpRs forms an α-helix and functions as a membrane anchor (Willnow, 1999). The cytoplasmic domain lengths were similar to each other (57–67 residues) and the NPXY motif was well-conserved in all arthropod LpRs (Fig. 1). One exception was Paj-LpR2B, in which a proline residue was replaced by leucine.

Fig. 6. Expression of two Vgs and four lipoprotein receptors in tissues with different GSI values. End-point RT-PCR products from individuals with different GSI values in the ovary (A) and hepatopancreas (B). Amplified products are shown as the inverted image of a 1.5% agarose gel stained with ethidium bromide after electrophoresis. 18S rRNA was used as a positive control. GSI values: 1 (0.54), 2 (0.65), 3 (0.68), 4 (0.98), 5 (1.00), 6 (1.22), 7 (1.38), 8 (2.36), 9 (3.03), 10 (3.46), 11 (3.64), 12 (3.95), 13 (5.07), 14 (5.38), 15 (5.48), and 16 (6.51). M, size marker.

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A

Ovary 300

Relative copy number (x104)

Paj-VgR Paj-LpR1

250

Paj-LpR2A Paj-LpR2B

200

*

150 100 50 0 Immature (0.54 - 0.68)

B

Vitellogenic (1.38 - 3.03)

Mature (5.38 - 6.51)

Hepatopancreas 16

Relative copy number (x104)

Paj-LpR1

14

Paj-LpR2A

12 10 8 6 4 2 0 Immature (0.54 - 0.68)

Vitellogenic (1.38 - 3.03)

Mature (5.38 - 6.51)

Fig. 7. Relative copy number of Paj-LpR1, Paj-LpR2A, Pa-LpR2B, and Paj-VgR in ovarian and hepatopancreatic tissues with different GSI values. The relative copy numbers were normalized against the copy number of 18S rRNA. The three maturation stages considered were the immature stage (0.54–0.68), vitellogenic stage (1.38–3.03), and mature stage (5.38–6.51). One-way ANOVA was used to determine significant effects. Statistical significance was accepted only when P b 0.05 (*).

A phylogenetic analysis showed that the LpRs from arthropods were clustered together (Fig. 4A). Mammalian lipoprotein receptors, including VgRs, VLDLRs, and LDLR, were closely related to each other, but were distinct from invertebrate lipoprotein receptors. The VgRs between invertebrates and vertebrates did not form monophyletic relationships, suggesting that they evolved differently (Fig. 4A). 3.2. A VgR (Paj-VgR) from P. japonica Besides three Paj-LpRs, a full-length cDNA (5989 bp) encoding a putative protein with 1927 residues, which showed a different primary structure, was identified (Fig. 2). Four full-length and partial decapod crustacean VgR sequences were identified in the GenBank database. The isolated sequence showed the strongest similarity to VgR (70%) from Macrobrachium rosenbergii and was named Paj-VgR. The MLLRAFISLLAFVHLFSFVDG sequence was predicted to be a signal peptide cleaved between the DG and VL residues, suggesting that the isolated Paj-VgR encoded a full-length receptor sequence (Fig. 2). Among the four VgR sequences currently identified in the GenBank database (three from Dendrobranchiata and one from Pleocyemata), only the Pem-VgRs from P. monodon (GenBank accession number: ABW79798) and M. rosenbergii (GenBank accession number: ADK55596) are full-length receptor sequences (Tiu et al., 2008; Roth and Khalaila, 2012). Two

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other VgR sequences from the Dendrobranchiata Penaeus semisulcatus (GenBank accession number: AAL79675) and Marsupenaeus japonicus (GenBank accession number: BAH57291) appear to be partial sequences because they were smaller in size and a signal peptide sequence was not predicted at the amino terminus of the two sequences (data not shown). As three VgR sequences were collected from different decapod crustaceans, including Paj-VgR, we compared their primary structure with those from insects (Fig. 2). The major difference between the VgRs from LpRs of arthropods is that the VgR harbors two LBDs and EGF precursor domains, whereas only a single LBD and EGF precursor domain was found in the LpRs. Although the five LBRs were well conserved within the first LBD of all compared VgRs, much higher variation was identified in the second LBD (Fig. 2). In Pleocyemata, including Paj-VgR, only seven LBRs were identified in the second LBD, which lacked LBR5. Interestingly, eight LBRs were found in the VgR of Dendrobranchiata. Pem-VgR is present in insects (Fig. 2). In addition, the second cysteine residue in the LBR4 and sixth cysteine residue in the LBR6 of Pem-VgR were replaced by glycine and aspartic acid, respectively (Fig. 2). The consensus organization of the first EGF precursor domain in the arthropod VgRs was EGF1, EGF2, β-propeller domain1, EGF3, β-propeller domain2, and EGF4 from the amino terminus (Fig. 2). The number of YWXD motifs in the β-propeller domain was not conserved in all of the VgRs compared. Only five YWXD motifs in the first β-propeller domain and three in the second β-propeller domain were identified in the VgRs compared (Fig. 2). In addition, the YWXD motif itself was not conserved among EGF precursor domains, but various analogous sequences were identified, including YFXD, YWXN, and FWXD. It is unclear whether folding into a functional propeller domain with those highly variable motifs is possible. The second EGF precursor domain was composed of EGF1, EGF2, β-propeller domain3, EGF3, and EGF4. An OLSD was identified only in Blg-VgR (not in Paj-VgR, MarVgR, or Pem-VgR). Interestingly, EGF4 was not found in Blg-VgR, but was identified in all other VgRs (Fig. 2). Our phylogenetic analysis showed that the VgRs from decapod crustaceans were clustered together with VgRs from insects (Fig. 4A). Two sequences from Pleocyemata, including the Paj-VgRs and VgR from M. rosenbergii, were grouped together with three other VgRs of Dendrobranchiata, corresponding to their evolutionary relationship (Porter et al., 2005). As described previously (Jeon et al., 2010), the Vgs from decapod crustaceans were clustered together with the Lps of insects and apoBs of mammals, suggesting that their nomenclature is incorrect (Fig. 4B).

3.3. Transcriptional analysis of four lipoprotein receptors from P. japonica To estimate the physiological implications of the four lipoprotein receptors from P. japonica, a distribution profile was analyzed by endpoint RT-PCR using tissue samples. Paj-VgR was expressed almost exclusively in the ovary (Fig. 5). Trace expression of Paj-VgR was also detected in neuronal tissues, including the brain, abdominal and thoracic ganglia, and epidermis. In contrast to Paj-VgR, Paj-LpR1 was ubiquitously expressed (Fig. 5). Two transcriptional isoforms, Paj-LpR1S1 and Paj-LpR1S2, exhibited different expression profiles; Paj-LpR1S2 was expressed in all tissues examined, while Paj-LpR1S1 transcription was detected only in the hindgut and ovary (Fig. 5). Two paralogs, Paj-LpR2A and Paj-LpR2B, also showed a different tissue distribution profile. Paj-LpR2A was expressed in all the tissues except for the tail muscle, whereas Paj-LpR2B transcription was detected only in the ovary. Truncated isoforms of two paralogs, Paj-LpR2AS and PajLpR2BS, exhibited different expression patterns. Paj-LpR2AS was expressed in the brain and thoracic ganglia, whereas Paj-LpR2BS was expressed only in the thoracic ganglia (Fig. 5). All four lipoprotein receptors from shrimp were weakly expressed in the hepatopancreas, the crustacean organ that corresponds to the liver of vertebrates.

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To estimate the contribution of the four lipoprotein receptors to ovarian maturation, the transcriptional levels of each receptor were measured from individuals with different GSI values (Fig. 6). 18S rRNA was used as a positive control of cDNA quality, and the two previously identified Vg (Paj-Vg1 and Paj-Vg2) cDNAs from P. japonica were used as positive controls of ovarian maturation (Jeon et al., 2010). The basal level of Paj-Vg2 transcription was b1.00 in GSI value and subsequently increased (Fig. 6A), similar to our previous results (Jeon et al., 2011). The level of Paj-LpR1 transcription was found to be high throughout different maturation stages (GSI values: 0.54–6.51). Two paralogs, PajLpR2A and Paj-LpR2B, showed highly similar expression patterns, in which PCR amplicons were detected in the ovaries of three mature individuals (GSI: 5.38–6.51). As in the ovary, Paj-Vg1 was used as a positive control in the hepatopancreas (Fig. 6B). Since its transcription level was at least 100-fold higher than ovarian Paj-Vg2 regardless of the maturation stage (Jeon et al., 2011), the number of PCR cycles was reduced to 25. This is ten cycles less than that used for ovarian Paj-Vg2 and other lipoprotein receptors. Unlike in ovary, Paj-VgR and Paj-LpR2B PCR products were not detected in hepatopancreas. High levels of Paj-LpR1 transcription were constantly shown regardless of the maturation stage, similar to the transcription level in ovary (Fig. 6). Conversely, the expression pattern of Paj-LpR2A was similar in both the hepatopancreas and the ovary, in which weaker PCR bands were identified only in the mature stage (GSI value: 5.38–6.51). These findings suggest that PajLpR2A expression is regulated by the same physiological conditions. The alternatively spliced Paj-LpR isoforms were highly variable in individuals, and did not exhibit any significant expression patterns. To confirm the expression levels at different stages of maturation, the relative transcript numbers of each lipoprotein receptor were measured by qPCR from individuals at the following stages: immature (GSI value: 0.54–0.68), vitellogenic (GSI value: 1.38–3.03), and mature (GSI value: 5.38–6.51). As evaluated by end-point RT-PCR, two lipoprotein receptors, Paj-VgR and Paj-LpR2B, were below the detection level in hepatopancreas (Fig. 7B). In ovary, the transcript levels of Paj-VgR, PajLpR1, and Paj-LpR2A were unchanged, regardless of the stage (Fig. 7A). Conversely, the ovarian expression level of Paj-LpR2B decreased significantly in the mature stage (Fig. 7A). Collectively, we identified Paj-VgR as an ovary-specific receptor, whereas Paj-LpR1 was ubiquitously expressed. Neither expression level changed with maturation. 4. Discussion 4.1. Similarities and differences of lipoprotein receptors in arthropods We identified four sequences encoding lipoprotein receptors from decapod crustaceans. Based on the primary structure of isolated lipoprotein receptors from P. japonica, all four characterized lipoprotein receptors are classified as LpRs or VgRs. They can be easily distinguished by their domain organizations (Fig. 3). Representative LpRs contain several conserved domain combinations as LDLR family members, including signal peptide sequences, LBDs, EGF precursor domains, OLSDs, TMDs, and cytoplasmic domains (Tufail and Takeda, 2009). Although all isolated Paj-LpRs exhibited a well-conserved arthropod LpR domain organization, several unique characteristics were detected. The first and third cysteine residues of LBR5 in the LBD were replaced by proline and serine in both Paj-LpR2s. In addition, the cysteine residue within LBR8 of Paj-LpR2A was changed to tyrosine (Fig. 1). We also identified eight LBRs within the LBD of Paj-LpR1 and seven LBRs from those of PajLpR2s (Fig. 1). Seven and eight LBRs have been identified within the LBD of insect species (Lee et al., 2003; Seo et al., 2003; Ciudad et al., 2007). Each LBR is composed of 40 residues with a triple disulfide bond and negatively charged residues that specify ligand interactions. Negatively charged (S/T)DE residues of each LBR are involved in binding with positively charged ligands (Schneider, 1996). The interaction between LBR4/5 in the LBD and β-propeller domain is involved in ligand

displacement (Rudenko et al., 2002), and this pH-dependent conformational change may explain the dissociation of LDL from the LBD of LDLP in acidified endosomal vesicles (Innerarity, 2002). In addition, the first repeat in the β-propeller domain is usually difficult to recognize in LpRs from arthropods, including Paj-LpRs (Tufail and Takeda, 2009). The biological implications of the variations in the LBD and EGF precursor domain of Paj-LpRs are unclear. The length of and S/T ratios in the OLSD also varied for each Paj-LpR (Fig. 1). The OLSD of Paj-LpR1 showed similarity in length and S/T ratio to insect LpRs (~30%), whereas a much smaller S/T ratio (b 10%) was identified in both Paj-LpR2s. Generally, the abundance of S/T residues in insect species is 30% (Cheon et al., 2001; Tufail and Takeda, 2007). Although there are several proposed functions of the OLSD, it does not appear to involve ligand binding, endocytosis, and degradation, but how its variation in length and S/T ratio affects LpR function is unclear (Tycko and Maxfield, 1982; Davis et al., 1986; Jentoft, 1990). In contrast to the OLSD, all compared LpRs contained a well-conserved TMD and cytoplasmic domain with an NPXY motif (Fig. 1). The TMD of LDLR family members forms a transmembrane αhelix and functions as a membrane anchor (Willnow, 1999; Herz and Bock, 2002). It is noteworthy that the NPXY motif in the cytoplasmic domain of Paj-LpR2B was mutated to NLVY. The NPXY motif is involved in cellular internalization, which serves as a signal for receptor-mediated endocytosis (Tufail and Takeda, 2009). Although the two paralogs exhibited strong sequence similarities, the motif in the cytoplasmic domain is critical for cellular trafficking, suggesting that Paj-LpR2A and Paj-LpR2B have different biological functions. Although three LpR genes were identified for the first time in decapod crustaceans, Paj-VgR turned out to be the VgR ortholog reported previously in decapod crustaceans. Four VgR sequences were identified, including those from P. monodon, M. japonicus, P. semisulcatus (Dendrobranchiata), and M. rosenbergii (Pleocymata). Two sequences, Pes-VgR from P. semisulcatus and Maj-VgR from M. japonicus, appeared to be partial sequences and were excluded from the analysis. Although they exhibited the highest sequence similarity compared with the other two full-length VgR sequences, the sequences lacked signal sequences and were thus smaller in size (1081 and 1120 amino acid residues, respectively). The domain organization of VgRs in decapod crustaceans includes signal peptide sequences, an LBD, EGF precursor domain 1, a second LBD, EGF precursor domain 2, an OLSD, a TMD, and a cytoplasmic domain (Fig. 2). According to a previous study, based on the domain organization there are two types of VgRs in insects (Tufail and Takeda, 2009). Major differences between the type I and II VgRs include the number of LBRs in the LBD (five LBRs for type I and four for type II) and the presence of an OLSD in type I (Tufail and Takeda, 2009). Not all isolated VgRs from decapod crustaceans belong to this classification, which is identified by five LBRs in the first LBD and the absence of an OLSD (Fig. 2). In addition, the number of LBRs in the second LBD varies in decapod crustaceans (Fig. 2). Eight LBRs were identified in the second LBD of Pem-VgR from P. monodon, like those of insects. In contrast, seven LBRs exist within the second LBD of the VgRs from Pleocyemata, including P. japonica and M. rosenbergii, which are missing LBR5 (Fig. 2). Further, several cysteine residues of LBRs within the second LBD were mutated in both shrimp (VgR from P. monodon) and insects (VgR from Anopheles gambiae) (Schonbaum et al., 1995). Regardless of the species, most variation was identified only in the second LBD, whereas the first LDB was well-conserved. The biological effects of the variation in the second LBD of arthropods are unclear. Although trace expression was detected in several tissues, including nerves, the heart, and epidermis, the major production site for Paj-VgR was the ovary (Fig. 5), in accordance with previous studies (Amdam et al., 2003; Guidugli-Lazzarini et al., 2008). These results suggest that the primary function of VgR is in oocyte growth and development, and that it is well-conserved in arthropods. We failed to identify transcriptional changes during ovarian development and maturation, indicating that the transcriptional regulation of VgR is weak during oocyte maturation.

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4.2. Multiple copies of LpR genes from P. japonica In the present study, we identified multiple LpR genes from P. japonica. Until now, only a single copy of the LpR gene had been identified in the insect (Seo et al., 2003; Ciudad et al., 2007; Tufail et al., 2009). Different isoforms are usually produced by alternative splicing in insects. In B. mori, four isoforms (LpR1–LpR4) have been reported (Gopalapillai et al., 2006), and two transcriptional variants have been identified in A. aegypti, B. germanica, and G. mellonella (Lee et al., 2003; Seo et al., 2003; Ciudad et al., 2007). Indeed, LpR from A. aegypti showed a tissue-specific expression pattern with a different LBD, suggesting different biological roles between the fat body and ovary (Seo et al., 2003). In contrast to the other insect LpRs, two distinct LpR genes (Drm-LpR1 and Drm-LpR2) were identified from D. melanogaster (Parra-Peralbo and Culi, 2011). A common domain organization in each gene was identified between the two species, in which both Paj-LpR1 and Drm-LpR1 harbor eight LBRs within the LBD, whereas seven were identified within the LBD of Paj-LpR2 and Drm-LpR2. Previous studies showed that LpRs can be divided into type I (8 LBRs) and type II (7 LBRs) in insects (Tufail and Takeda, 2009). Currently, the functional differences between the two LpR types are unclear. Moreover, how the two different LpR genes evolved in D. melanogaster and P. japonica is unknown. Additionally, based on the strong sequence and domain organization similarity, two paralogs, Paj-LpR2A and Paj-LpR2B, were generated by a recent gene duplication event. It is unclear whether having three or more copies of LpR genes is common among crustacean LpRs. Multiple copies of LpR genes are uncommon in insects; thus, further study should be conducted in decapod crustaceans. In addition to multiple copies of LpR genes, various isoforms of PajLpRs were identified (Fig. 3). In insects, splicing progresses characteristically in three distinct regions (Parra-Peralbo and Culi, 2011). First, splicing occurs within the OLSD of the LpR in A. aegypti, B. mori, G. mellonella, and B. germanica (Lee et al., 2003; Seo et al., 2003; Gopalapillai et al., 2006; Ciudad et al., 2007). Second, a common site for splicing is within the LBD. In B. mori, an additional splice site was identified within the LBD (LpR3), in which exon 4 was replaced by exon 4a (Gopalapillai et al., 2006). Similar isoforms were also detected in D. melanogaster (Parra-Peralbo and Culi, 2011). A recent comprehensive study of LpRs in D. melanogaster identified a third splice site in the promoter region that produced different N-terminal sequences (ParraPeralbo and Culi, 2011). Unlike the splicing in insect species, splicing occurs at different sites in Paj-LpRs (Fig. 3). Although transcript variants of Paj-LpR1S1 were produced in the second case, another splicing event in the Paj-LpR1 gene occurred within the EGF precursor domain that produced Paj-LpR1S2. In addition, no truncated isoforms were detected in insect LpRs (Fig. 3). The multiple copies of LpR genes and their transcript variants produce much more diverse LpR proteins compared with those from insects. The expression level of Paj-VgR did not change significantly at different vitellogenic stages (Fig. 6). These results are in contrast to previous results, wherein the VgR mRNA level was elevated during vitellogenesis (Mekuchi et al., 2008; Tiu et al., 2008; Roth and Khalaila, 2012). This is mainly due to more variable Paj-VgR mRNA levels compared to the VgR of M. rosenbergii. Considering that very little difference (~1.3 fold) in mean values separated the Mar-VgR mRNA level at the immature (GSI: 0.85 ± 0.15) and mid-vitellogenic stages (GSI: 5.2 ± 1.1), it was hard to determine the statistical significance for those with high individual variability. In contrast, increased levels of Paj-Vg transcription were commonly detected due to a significant difference (up to 100 fold) between the immature and mid-vitellogenic stages in both species (Jeon et al., 2011). Collectively, our findings are consistent with those described previously. One new finding of our study is the decreased ovarian Paj-LpR2B transcript levels and their potential roles in decapod crustacean reproduction (Fig. 7). Although Paj-LpR2A was highly expressed in the hindgut, gill, and ovary, Paj-LpR2B was exclusively expressed in the ovary. Further, the transcriptional levels of Paj-LpR2B

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were significantly decreased at the mature stage, suggesting that PajLpR2B may be involved in reproduction (Fig. 7). Interestingly, DrmLpR2 from D. melanogaster has a similar structure, while its knockout mutants are sterile due to impaired lipid trafficking into growing oocytes. These findings lend further support to the involvement of PajLpR2B in reproduction (Parra-Peralbo and Culi, 2011). Based on expression analyses, mutation studies in D. melanogaster, and the current study, LpR1s appear to play a role in overall lipid trafficking in most tissues, whereas LpR2B is likely involved in reproduction by transporting lipids into growing oocytes. In addition, two LpR2 paralogs, Paj-LpR2A and Paj-LpR2B suggest that regulation of LpRs in decapod crustaceans is more complicated than in D. melanogaster. 4.3. Functional relationship between Lp/Vg and LpR/VgR in decapod crustaceans As shown in Fig. 4, the relationship of lipoproteins is different from their receptors. The Vgs from decapod crustaceans are more closely related to insect Lps and vertebrate apoBs, rather than to the Vgs from insects and maxillopods. Our phylogenetic tree showed that the Vgs and Lps were clearly divided (Fig. 4B). The Vgs from insects, vertebrates, and mollusks were clustered together with CPs from decapod crustaceans. The Vgs from decapod crustaceans were clustered together with apolipoproteins (apoLp) from vertebrates and insects (Fig. 4B). This suggests that decapod crustacean Vgs are orthologs of apoLp in vertebrates, while the CPs from decapod crustaceans are orthologs of Vgs from other species. Considering the evolutionary distance between insects and decapod crustaceans, and the consensus function of lipoproteins throughout all taxa, it is interesting that CP may play a totally different role in reproduction from insect Vgs. Until now, CPs have been identified only from Dendrobranchiata and no ortholog was found in Pleocyemata; moreover, it is still unknown whether CPs are involved in decapod crustacean reproduction. These questions are worthy of further study in different species. Recently, Avarre et al. (2007) suggested a new nomenclature for Vgs, apolipocrustacein, in decapod crustaceans, which shows an orthologous relationship to insect apoLp-II/I and vertebrate apoB. Although that nomenclature indicates the unique evolutionary relationship among currently known Vgs in decapod crustaceans, it may misrepresent the evolutionary relationship between decapod crustaceans and insects if we consider new taxonomic classifications in arthropods. The pancrustacea taxon, which comprises all crustaceans and hexapods, is now widely accepted (Regier et al., 2010). This suggests that there is no reason to separate decapod crustaceans from insects in protein nomenclature. In present study, we showed that lipoproteins and their receptors were well conserved in all arthropod and Vgs in decapod crustacean is the ortholog of hexapod Lps. Considering the multiple roles of Lps in Pancrustacean species, the ortholog of hexapod Lp, Vg is not the proper name in decapod crustaceans. Although both LpR and VgR play a role in reproduction, those receptors may be differently involved in arthropod reproduction. VgRs appear to be oocyte-specific receptors and play a role in transporting yolk proteins. Drosophila mutant yl- females are genetically deficient in VgR, and their oocytes show a significant reduction in the number of coated vesicles and very little proteinaceous yolk (DiMario and Mahowald, 1987; Schonbaum et al., 1995). In contrast to VgRs, various LpR isoforms are produced with different expression profiles, and some of them play a role in insect reproduction. LpRs appear to be responsible for lipid accumulation in growing oocytes. Studies of mutants have shown that LpR2 plays a major role in transporting lipids into growing oocytes in D. melanogaster, and that it is required for normal oogenesis (ParraPeralbo and Culi, 2011). It is noteworthy that lipid uptake in oocytes is mainly dependent on Lp, not Vg; in M. sexta, Vg contributes about 5% to egg lipids (Kawooya and Law, 1988). In conclusion, we isolated four full-length cDNAs encoding lipoprotein receptors (three LpRs and a VgR). The transcriptional patterns of

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Paj-VgR were ovary-specific, whereas the three Paj-LpRs and their alternatively spliced isoforms showed different tissue distribution profiles and transcriptional changes during maturation. These findings suggest multiple biological functions, including a role for Paj-LpR2s in reproduction. Further study is required to better understand the roles of lipoprotein receptors in decapod crustacean lipid metabolism and reproduction. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cbpb.2013.12.005. Acknowledgment This work was supported by a grant from the National Fisheries Research and Development Institute (RP-2013-FR-065). References Amdam, G.V., Norberg, K., Hagen, A., Omholt, S.W., 2003. Social exploitation of vitellogenin. Proc. Natl. Acad. Sci. U. S. A. 100, 1799–1802. Avarre, J.C., Lubzens, E., Babin, P.J., 2007. 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