Short communication

Cloning and characterization of 37 kDa laminin receptor precursorin pearl oyster, Pinctada fucata

Yaopeng Fu a, Liping Xie a,b,*, Rongqing Zhang a,b,*

a Institute of Marine Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, Chinab Protein Science Laboratory of the Ministry of Education, Tsinghua University, Beijing 100084, China

Received 4 April 2007; received in revised form 5 June 2007; accepted 5 June 2007

Abstract

A 1063 bp cDNA clone encoding a putative 37 kDa laminin receptor precursor (37 kDa LRP) is isolated from the mantle tissue ofpearl oyster, Pinctada fucata. The amino acid sequence predicted from the cDNA sequence is 301 residues long, with a calculated molec-ular mass of 33.5 kDa. RT-PCR analysis shows that 37 kDa LRP mRNA is especially highly expressed in the mantle while widelyexpressed in several tissues. In situ hybridization analysis reveals that 37 kDa LRP is expressed in the outer epithelial cells of the mantleedge, suggesting its involvement in cell proliferation and secretion in P. fucata. The identification and characterization of 37 kDa LRP inthe pearl oyster will help us to further understand the signal transduction in the processes of mantle epithelial cell proliferation and tissueformation.� 2007 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science inChina Press. All rights reserved.

Keywords: 37 kDa laminin receptor precursor; Mantle epithelial cell; Pearl oyster

1. Introduction

The mantle epithelial cells are directly responsible forpearl formation in pearl oyster, Pinctada fucata, by the depo-sition of aragonitic CaCO3 crystals and the secretion oforganic protein matrix to hold them [1]. Thus the cultureof outer epithelial cells of the mantle tissue is important tostudy the pearl-formation process [2]. However, theestablishment of cell lines from marine invertebrates hasencountered obstacles, mainly due to the low level of cellproliferation and viability and there is no single permanentcell line from pearl oyster to be successfully established [3–5].

The 67 kDa laminin receptor (67 kDa LR), also termedthe 67 kDa laminin binding protein, is a non-integrinreceptor of laminin. The mature 67 kDa receptor is formed

by homo- or hetero-dimerization of the acylated 37 kDaprecursor (37 kDa LRP), by non-covalent bonds [6]. Lam-inin is the main non-collagenous glycoprotein found in thebasement membrane. Various laminin isoforms areinvolved in many physiological and pathological processes[7–9]. Two laminin binding sites were identified on the37 kDa LRP [10], the first is called G peptide [11–13],and the second is at the carboxy terminal [14,15]. The67 kDa laminin receptor therefore recognizes various bind-ing sites on laminin which are different from the sites recog-nized by integrins [10,12,14,16], allowing for higher overallbinding affinity and different signal transduction options.The receptor is involved in several physiological processessuch as implantation [17], invasive phenotype of tropho-blastic tissue [18], angiogenesis [19,20], T-cell biology [16]and shear stress-dependent endothelial nitric oxide syn-thase expression [15]. It has also been found that the67 kDa laminin receptor is identical to the oncofetal anti-gen protein that is expressed by tumors [21]. The overex-

1002-0071/$ - see front matter � 2007 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited

and Science in China Press. All rights reserved.

doi:10.1016/j.pnsc.2007.06.009

* Corresponding author. Tel.: +86 10 62772900; fax: +86 10 62772899.E-mail addresses: lpxie@mail.tsinghua.edu.cn (L. Xie), rqzhang@

mail.tsinghua.edu.cn (R. Zhang).

Available online at www.sciencedirect.com

Progress in Natural Science 18 (2008) 233–238

pression of the receptor has been implicated in laminin-induced cell attachment and migration, as well as in tumorgrowth, invasion and metastasis [22–25]. In the previousattempts to establish oyster cell line and culture pearl-sacin vitro, a migration and proliferation along the extracellu-lar matrix of mantle epithelial cell was observed [26,27].Thus, an investigation on the 37 kDa LRP in P. fucata

was undertaken.In this study, a putative 37 kDa LRP was first cloned by

RT-PCR and rapid amplification of cDNA ends (RACE)from the mantle tissue of pearl oyster P. fucata. Theexpression sites of this laminin receptor were examinedby RT-PCR and in situ hybridization analysis.

2. Materials and methods

2.1. Preparation of the total RNA

Live individuals of adult oyster P. fucata were obtainedfrom Guofa Pearl Farm in Beihai, Guangxi Province,China. Total RNA was extracted from mantle tissue withRNAzol RNA isolation kit (Biotex, USA) following theuser’s manual. RNA integrity was determined by separa-tion on a 1.2% formaldehyde-denatured agarose gel andstained with ethidium bromide. The quantity of RNAwas determined by measuring OD260nm with an Ultrospec3000 UV/Visible Spectrophotometer (Amersham, USA).

2.2. RT-PCR and RACE

Single-stranded cDNA for all rapid amplification ofcDNA end reactions were prepared from the mantle totalRNA. 5 0-RACE and 3 0-RACE were conducted accordingto the manufacturer’s instructions, using the SMARTRACE cDNA Amplification Kit (Clontech) and Advan-tage 2 cDNA Polymerase Mix (Clontech). The gene-spe-cific primers 5RP (5 0-GGT CAC AAC CAA CAG ACGAGG CTC-3 0) for 5 0-RACE and 3RP (5 0-GAY TTYCAR ATG GAR CAG TAT G-3 0) for 3 0-RACE, respec-tively, were prepared based on the conserved regions of37 kDa LRP. The RT reaction and following PCR wereperformed according to the manufacturer’s instructionson a T gradient Thermocylcer (Biometra, Germany).PCR cycles were conducted at the following setting: dena-turation at 95 �C for 5 min, followed by 30 cycles at 95 �Cfor 30 s, 46 �C for 30 s, 72 �C for 1 min, and then elonga-tion at 72 �C for 10 min.

PCR products were then subjected to electrophoresisand bands of the expected size were excised and purifiedwith Wizard PCR prep DNA Purification System (Pro-mega, USA). The purified PCR products were subclonedinto pMD 19-T vector (TaKaRa, China) and sequenced.

To confirm cloning and sequencing accuracy, the entirecDNA from the start codon to stop codon was re-amplifiedwith high fidelity polymerase (TaKaRa), using a pair ofgene-specific primers FP1 (5 0-GTC TCG ACT CCA TTTGGC ATT AC-3 0) and FP2 (5 0-CTT TGA ACA GAG

AGA GTG TCC CA-3 0). The purified PCR products weresubcloned into pMD 19-T vector followed byre-sequencing.

2.3. Sequence and phylogenetic analyses

Sequence similarity searches were performed with theBLAST program in GenBank, National Center for Bio-technology Information (http://www.ncbi.nlm.nih.gov/).Multiple alignments were created using the Clustal W pro-gram. The phylogenetic tree was constructed using ClustalW program with the Neighbor-Joining (NJ) algorithm.

2.4. Tissue expression analysis by RT-PCR

Tissue expression of the oyster 37 kDa LRP mRNA wasinvestigated by RT-PCR. Total RNA was isolated frommantle, foot, gill, gonad, digestive gland, and adductormuscle tissues of the adult individual of P. fucata. Equalquantities (2 lg) of total RNA from different tissues werereverse transcribed into cDNA as described above. Thegenerated cDNA was used as the template for PCR withprimers of RP1 (5 0-GAC TTT CAG ATG GAG CAGTAT G-3 0) and RP2 (5 0-GGT CAC AAC CAA CAGACG AGG CTC-3 0). The reaction was performed with adenaturation at 95 �C for 5 min, 20 cycles of 95 �C for30 s, 47 �C for 30 s, 72 �C for 45 s, and then elongation at72 �C for 10 min. Amplification of GADPH was as theinternal reference in PCR with primers GADPH1(5 0-GAT GGT GCC GAG TAT GTG GTA-3 0) and GAD-PH2 (5 0-CG TTG ATT ATC TTG GCG AGT G-3 0), andnegative controls were carried out in the absence of cDNAtemplate to examine the cross contamination of thesamples.

2.5. In situ hybridization of 37 kDa LRP

The mantle was removed from the adult P. fucata andimmediately fixed in 4% paraformaldehyde containing0.1% DEPC (Sigma, USA) overnight. In situ hybridizationof oyster 37 kDa LRP mRNA was carried out on frozensections of the mantle. Digoxigenin-labeled DNA probeswere generated from the plasmid with the insertion frag-ment encoding partial sequence of LRP using a DIG HighPrime DNA Labeling and Detection Starter kit I (Roche,Germany). In situ hybridization was performed asdescribed previously with some modification [28]. Thehybridization temperature was 45 �C.

3. Results

3.1. Cloning and sequence analysis of cDNA sequence

encoding 37 kDa LRP

A cDNA product of 883 bp was obtained by 3 0-RACEusing total mantle RNA as the template. Based on thesequence, a gene-specific primer was synthesized for

234 Y. Fu et al. / Progress in Natural Science 18 (2008) 233–238

5 0-RACE (5RP). To confirm the sequence obtained byRACE, two-specific primers (FP1 and FP2) correspondingto 5 0- and 3 0-UTR sequences of putative 37 kDa LRPmRNA were designed and RT-PCR was performed. ThePCR products were cloned and sequenced, which matchedwell the sequence expected from the results of 5 0- and 3 0-RACE.

As shown in Fig. 1, the complete cDNA sequenceincluding the poly-A tail is 1063 bp. It contains a 67 bp5 0-untranslated sequence, an ORF consisting of 903 bp, aTGA stop codon, a 77 bp 3 0-untranslated sequence, anda poly-A tail of 13 nucleotides. A putative polyadenylationsignal (AATAAA) was recognized at position 1042 on theupstream of the poly-A tail. This cDNA sequence has beendeposited in GenBank with Accession No. EF427937.

The ORF of this cDNA clone encoded a protein consist-ing of 301 amino acid residues, which shared high similar-ity with 37 kDa LRP in other species. Comparison of twoconserved laminin binding sites of 37 kDa LRP with thosefrom the other organisms is shown in Fig. 2.

A phylogenetic tree was then constructed based on thealignment of the full length amino acid sequences of37 kDa LRP inP. fucata and the other known 37 kDaLRPs by Clustal W program with NJ algorithm (Fig. 3).

3.2. Gene expression analyses of oyster 37 kDa LRP

To investigate the functions of oyster 37 kDa LRPin vivo, tissue-specific expression of 37 kDa LRP mRNAwas examined by RT-PCR with primers RP1 and RP2.Samples of total RNA were prepared separately from man-tle, foot, gill, gonad, digestive gland, and adductor muscletissues of P. fucata. The results revealed that 37 kDa LRPtranscript was especially highly expressed in mantle whilewidely expressed in several tissues, suggesting its multifunc-tional influence on physiological and pathological pro-cesses in P. fucata (Fig. 4).

To determine the more precise expression site of 37 kDaLRP mRNA in mantle, in situ hybridization was per-formed on frozen sections of the mantle tissue. Stronghybridization signals for 37 kDa LRP mRNA weredetected in the outer epithelial cells along the mantle edge(Fig. 5).

4. Discussion

The complete cDNA sequence of 37 kDa LRP includingthe poly-A tail is 1063 bp, encoding a protein consisting of301 amino acid residues. The protein has a calculated

Fig. 1. The cDNA sequence and deduced amino acid sequence of 37 kDa LRP from pearl oyster, Pinctada fucata. The start codon, the stop codon, andthe putative polyadenylation signal (AATAAA) are boxed. Two putative laminin binding sites are underlined, and the putative transmembrane domain isin shadow.

Y. Fu et al. / Progress in Natural Science 18 (2008) 233–238 235

molecular mass of 33.5 kDa, which is consistent with thosein other species, and a little smaller than the identical pro-tein ribosomal p40 [29–31]. Phylogenetic analysis on therelationship between the 37 kDa LRP and p40 indicatedthat all of these protein sequences were derived fromorthologous genes and that the 37 kDa LRP is indeed aribosomal protein that acquired laminin-binding capabilitywith the appearance of the palindromic sequenceLMWWML during evolution [32].

Two laminin binding sites were identified on 37 kDaLRP. One is called G peptide (Fig. 2a), which binds specifi-cally with high affinity (Kd = 51.8 nM) to laminin. It is ofinterest to note that G peptide contains a palindromicsequence of six amino acids, LMWWML, as the actual bind-ing site. Another is at the carboxyl terminal (amino acids205–229) (Fig. 2b), which binds to the peptide YIGSR onb1 chain of laminin [14,15]. Comparison of these two con-

Fig. 2. Comparison of amino acid sequences at two putative laminin binding sites in 37 kDa LRPs. The numbers indicate positions of the amino acidresidues in each sequence. The identical residues are shaded. The species included: pearl oyster (Pinctada fucata, ABO10190); African clawed frog (Xenopus

laevis, AAW62261); fruit fly (Drosophila melanogaster, AAA28667); hydroid (Hydra viridis, P38984); echinus (Strongylocentrotus purpuratus, P46771);amoeba (Acanthamoeba healyi, AAQ63482); human (Homo sapiens, AAB22299); monkey (Chlorocebus aethiops, ABC69238); cattle (Bos taurus,NP_776804); pig (Sus scrofa, Q4GWZ2); house mouse (Mus musculus, AAA39413).

Fig. 3. Phylogenetic tree of 37 kDa LRP sequences. The tree was deducedby a neighbor-joining analysis based on amino acid sequence alignment offull length 37 kDa LRP by Clustal W program. The species are referred toFig. 2.

Fig. 4. RT-PCR for analysis of 37 kDa LRP tissue expression in P. fucata. Lane 1, gonad; lane 2, mantle; lane 3, foot; lane 4, adductor muscle; lane 5,digestive glade; lane 6, gill; lane 7, negative control. The housekeeping gene GADPH was included as a positive control.

Fig. 5. Detection of 37 kDa LRP mRNA in the mantle of P. fucata by in situ hybridization. (a and b) show the hybridization signals which were stained inpurple and indicated by arrows. (a) is a zoom-in result for the area of the box in (b). A control section stained with a sense probe is shown in (c). PG,periostracal groove; OF, outer fold; MF, middle fold; IF, inner fold; PM, Pallial mantle.

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served laminin binding sites with those from other organismsshowed that these two laminin binding sites were similar, butnot the same, with those of other species. There are still sev-eral amino acid residues different from the consensussequence, compared with the high conserved sequences invertebrates. The palindromic sequence LMWWML wasobserved as LMWWLL with a mutation of M to L in P.

fucata, Drosophila melanogaster, and Acanthamoeba healyi,suggesting that they are genetically close in relationship dur-ing evolution. Comparison of full length of these 37 kDaLRPs revealed that all vertebrate proteins are nearly identi-cal, including the C-terminal region, which is the most diver-gent in other invertebrates. A phylogenetic tree based on itshown in Fig. 3 also illustrates the high identity of vertebrate37 kDa LRPs with invertebrate 37 kDa LRPs.

According to the previous reports, a membrane-associ-ated domain resides within the region of the 37 kDa LRPfrom residue 86 to 101. These data defined the two-thirdof carboxyl terminal of the 37 kDa LRP to be extracellular.Analysis of signal peptide cleavage site carried out usingthe SignalP v3.0 program showed the absence of a signalpeptide, which was consistent with previous reports. Thequestion about how the 67 kDa-LR located finally on thecell membrane is still to be answered. The absence of anN-terminal leader sequence was observed also in someother membrane-associated proteins previously. Notableexamples are the transferrin receptor [33] and the asialogly-coprotein receptor [34]. The mechanisms responsible forthe integration, insertion, or association of these unusualreceptors to the cell membrane are not yet elucidated.One possible membrane association mechanism is thatanother predicted polypeptide, either alone or in conjunc-tion with the 37 kDa LRP, may possess a signal peptideor a signal patch formed by the juxtaposition of aminoacids that are physically separated before protein foldingor before association of the two subunits.

Thirty-seven kilo Dalton LRP is considered to beinvolved in several physiological processes such as implan-tation, invasive phenotype of trophoblastic tissue, angio-genesis, T-cell biology, mechanical sensing, as well as cellattachment and migration. The tissue expression analysisbased on RT-PCR showed that 37 kDa LRP transcript iswidely expressed in mantle, foot, gill, gonad, digestivegland, and adductor muscle tissues of the adult individualof P. fucata as a household gene (Fig. 4). In the followingin situ hybridization, strong hybridization signals for37 kDa LRP mRNA were detected in the epithelial cellsalong the mantle edge. The mantle, that is an importantpart of the molluscan body, is responsible for the forma-tion of the shell. The outer epithelial cells of mantle areactive in proliferation and tissue remodeling. Also secre-tory cells are found in the epithelium layer in this area.As shown in Fig. 5, hybridization signals of 37 kDa LRPmRNA are arranged along the mantle edge in a very regu-lated way, like an outline sketching of the mantle, suggest-ing its involvement in the function of these epithelial cells.According to previous reports, 67 kDa LR, the mature

form of 37 kDa LRP, could be an upstream functionalreceptor of the MAPK pathway. Some researches revealedthat the basal phosphorylation extent of ERK, JNK andp38 was significantly higher in cell lines expressing reduced67 kDa LR [35]. MAPK pathway is involved in a variety ofprocesses in cell cycle, such as proliferation and differenti-ation. The role of 67 kDa LR in this pathway, however,still needs to be further examined.

Pinctada fucata is predominantly used for pearl produc-tion in marine water in China as it is abundantly distrib-uted throughout the saltwater habitats of the country.During pearl cultivation, the quality of pearl is directlyrelated to the function of the epithelial cells. Detailed phys-iological studies on function of epithelial cells, however,have not been conducted adequately, mainly due tounavailability of constantly cultured cell in vitro. In thisstudy, 37 kDa LRP of the pearl oyster P. fucata was firstcloned and its tissue expression was examined to deducethe function of it in mantle epithelial cells. The resultscan be helpful to understand the signal transduction inthe mantle epithelial cells, and can provide primary supportfor the cultivation of mantle epithelial cell and tissue regen-eration in vitro.

Acknowledgements

This work was supported by National Natural ScienceFoundation of China (Grant No. 30530600) and NationalHigh Technology Reserch and Development Program ofChina (Grant Nos. 2006AA09Z413, 2006AA09Z441).

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