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Tsinghua Science and Technology 2008年第4期13卷 439-446页

作者：王雪范为民谢莉萍张荣庆 Institute of Marine Biotechnology Department of Biological Sciences and Biotechnology Tsinghua University Beijing 100084 China Institute of Marine Biotechnology Department of Biological Sciences and Biotechnology Tsinghua University Beijing 100084 China Protein Science Laboratory of the Ministry of Education Tsinghua University Beijing 100084 China

Plasma membrane calcium ATPase （PMCA） plays a critical role in transporting Ca^2＋ out of the cytosol across the plasma membrane which is essential both in keeping intracellular Ca^2＋ homeostasis and in biomineralization. In this paper we cloned and localized a gene encoding PMCA from the pearl oyster Pinctada fucata. This PMCA shares similarity with other published PMCAs within the functional domains. Reverse transcription-polymerase chain reaction analysis shows that it is expressed ubiquitously. Furthermore, in situ hybridization reveals that it is expressed in the inner epithelial cells of the outer fold and in the outer epithelial cells of the middle fold, as well as the edge near the shell, which suggests that PMCA may be involved in calcified layer formation. The identification and characterization of oyster PMCA can help to further understand the structural and functional properties of molluscan PMCA, as well as the mechanism of maintaining Ca^2＋ homeostasis and the mechanism of mineralization in pearl oyster.

关键词： biomineralization Ca^2+ homeostasis pearl oyster plasma membrane calcium ATPase Pinctada fucata

Cloning, Characterization, and Expression Analysis of Calreticulin from Pearl Oyster Pinctada fucata

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Tsinghua Science and Technology 2008年第4期13卷 466-473页

作者：范为民胡轶霖李长忠谢莉萍张荣庆 Institute of Marine Biotechnology Department of Biological Sciences and Biotechnology Tsinghua University Beijing 100084 China Institute of Marine Biotechnology Department of Biological Sciences and Biotechnology Tsinghua University Beijing 100084 China Protein Science Laboratory of the Ministry of Education Tsinghua University Beijing 100084 China

Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sarcoplasmic/endoplasmic reticulum. A large amount of calcium is absorbed from the medium and transported to mineralization sites during biomineralization in pearl oyster. This paper describes the cloning of the full-length cDNA of calreticulin from Pinctada fucata, namely PCRT. PCRT encodes a deduced 414-amino acid protein, which includes a predicted 17- amino acid signal peptide and an endoplasmic reticulum retrieval sequence HDEL. The protein shows 63%-76% sequence identity and shares some common characteristics with calreticulins from other species. Semi-quantitative RT-PCR indicates that PCRT is ubiquitously expressed in all tissues tested with the highest expression in the hemolymph and the mantle. In situ hybridization analysis of PCRT in the mantle showed strong signals in the inner fold, the inner side of middle fold, and the inner side of outer fold of the mantle epithelium, All these results suggest PCRT might be involved in Ca^2＋ transport and storage during oyster biomineralization.

关键词： calreticulin cDNA cloning expression analysis biomineralization Pinctada fucata mollusc

Cloning, Characterization, and Distribution of an mRNA Encoding a H^+-ATPase α Subunit in the Mantle of Pearl Oyster, Pinctada fucata

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Tsinghua Science and Technology 2007年第4期12卷 405-412页

作者：陈蕾孙绪德白宏伟尹善德谢莉萍熊训浩张荣庆 Institute of Marine Biotechnology Department of Biological Sciences and Biotechnology Tsinghua University Department of Anaesthesology Tangdu Hospital the Fourth Military Medical University Department of Hepatobiliary Surgery the Naval General Hospital Department of Gynaecology and Obstetrics the Naval General Hospital

Mitochondrial ATP synthase is responsible for the production of the majority of the cellular ATP, which is composed of two major units： Fo and F1. Although much is known about the active complex （5 subunits （αβγδε））, the role of the α subunit in the catalytic mechanism remains unclear, particularly in bivalve animals. This study first cloned and identified the full-length sequence of the mitochondrial H^＋-ATP synthase α subunit cDNA gene in Pinctada fucata using the reverse transcriptase polymerase chain reaction （RT-PCR） technique. The Pinctada fucata mitochondrial H^＋-ATP synthase α subunit contains 1991 nucleotides, with the translation start site at nt 48 （ATG） and the stop codon at nt 1660 （TAA）, encoding a polypeptide 553 amino acids in length, which shares high similarity to that of other animals （81% identity to fruit fly, 82% to carp, and 83% to humans）. Alignment analysis of the well-conserved amino acid domains in the ATPase α subunit, the call3 signal transduction domain, showed that two residues （Asp^358 and Asn^359） differ from any other ATP synthase α subunit. In situ hybridization analysis was used to reveal the wide-spread distribution of mitochondrial H^＋-ATP synthase in various tissues in Pinctada fucata. This work will help further research on pearl energy metabolism to increase the output and quality of pearls to more efficiently utilize our rich pearl oyster resources.

关键词： mitochondrial H^+-ATP synthase Pinctada fucata gene cloning

Characteristics of Two Intermediates Trapped in the Unfolding Pathway of Arginine Kinase Induced by Guanidinium Chloride

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Tsinghua Science and Technology 2005年第4期10卷 461-468页

作者：郭晓罡谢莉萍潘继承张荣庆 Department of Biological Sciences and Biotechnology Tsinghua University Beijing 100084 China Protein Science Laboratory of the Ministry of Education Tsinghua University Beijing 100084 China

Equilibrium guanidinium chloride （GdmCl）-induced unfolding of arginine kinase （AK） was investigated by enzymatic activity, intrinsic fluorescence, 8-anilinonaphthalene-1-sulfonic acid （ANS） fluorescence, circular dichroism （CD） spectrum, and size-exclusion chromatography. The measurements showed that AK unfolded through two equilibrium intermediates： the molten globule state and the partly folded state. Both intermediates have no enzyme activity. The molten globule state exists at 0.4-0.8 mol/L GdmCi, perhaps after the N-terminal domain has unfolded but the C-terminal domain is still intact. The partly folded state occurs at 1.1-1.5 mol/L GdmCI with a hydrodynamic volume no more than 1.6-fold larger than the native state and a pronounced far UV-CD signal. Its ANS fluorescence intensity is about 50% of the molten globule state. This partly folded state shares similarities with the “burst” kinetic intermediate of protein folding.

关键词： arginine kinase equilibrium intermediate molten globule state partly folded state

Cloning and Characterization of a Homologous Ca^(2+)/Calmodulin-Dependent Protein Kinase PSKH1 from Pearl Oyster Pinctada fucata

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Tsinghua Science and Technology 2005年第4期10卷 504-511页

作者：戴益平谢莉萍熊训浩陈蕾范为民张荣庆 Institute of Marine Biotechnology Department of Biological Sciences and Biotechnology Tsinghua University Beijing 100084 China Protein Science Laboratory of the Ministry of Education Tsinghua University Beijing 100084 China

Many of the effects of Ca^2＋ signaling are mediated through the Ca^2＋/calmodulin complex and its acceptors, the Ca^2＋/calmodulin-dependent protein kinases, including PSKHI. Studies of the proteins involved in the calcium metabolism in oysters will help elucidate the pearl formation mechanism. This paper describes a full-length PSKH1 cDNA isolated from pearl oyster Pinctada fucata. Oyster PSKH1 shares 65% homology with human PSKH1 and 48% similarity with rat CaM kinase I in the amino acid sequence, and contains a calmodulin-binding domain. The results of semi-quantitative reverse transcription-polymerase chain reaction and in situ hybridization revealed that oyster PSKH1 mRNA is highly expressed in the outer epithelial cells of the mantle pallial and in the gill epithelial cells. These studies provide important information describing the complex Ca^2＋ signaling mechanism in oyster calcium metabolism.

关键词： PSKH1 calcium metabolism biomineralization pearl oyster Pinctada fucata Ca^2+/calmodulin-dependent protein kinases (CaMKs)

Cloning and Characterization of an mRNA Encoding F_1-ATPase Beta-Subunit Abundant in Epithelial Cells of Mantle and Gill of Pearl Oyster, Pinctada fucata

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Tsinghua Science and Technology 2007年第4期12卷 381-388页

作者：刘亮谢莉萍熊训浩范为民陈蕾张荣庆 Institute of Marine Biotechnology Department of Biological Sciences and Biotechnology Tsinghua University

In oyster biomineralization, large amounts of calcium are absorbed from external media, transported to the mineralization site, and finally deposited via a matrix-mediated process, All these activities are very energy intensive; therefore, investigations of the energy metabolism pathways of different oyster tissues will facilitate understanding of oyster biomineralization physiology. A full-length cDNA encoding the F1- ATPase beta-subunit （the F1-β-subunit, a major calalytic subunit of F-ATPase） from the pearl oyster （Pinctada fucata） was cloned using the homology strategy with a pair of degenerated primers based on the conserved regions of other animals＇ F1-β-subunit genes. Sequencing and structural analyses showed that the obtained sequence shared high identity with other animals＇ F1-β-subunits, and had a unique phosphorylation site of PKC and CK II on the external surface of the putative protein. Results from semi-quantitative reverse transcription-polymerase chain reaction and in situ hybridization demonstrated this oyster F1-β-subunit mRNA is abundant in the gill and mantle, and distributed widely in the periostracal groove, the outer folder. and the dorsal region of the mantle and in the gill epithelial cells. These tissues were the main regions that participate in biomineralization processes such as calcium uptake, transport, and matrix secretion. The results indicate that tissues involved in biomineralization have stronger energy metabolic processes and that F1-ATPase might play an important role in oyster biomineralization by providing energy transport.

关键词： biomineralization F1-β-subunit mantle gill Pinctada fucata