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Alv protein plays opposite roles in the transition of amorphous calcium carbonate to calcite and aragonite during shell formation Jingjing Kong, Chuang Liu, Dong Yang, Yi Yan, Yan Chen, Jingliang Huang, Yangjia Liu, Guilan Zheng, Liping Xie, and Rongqing Zhang Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b00025 • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 7, 2018
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Crystal Growth & Design
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Alv protein plays opposite roles in the transition of amorphous calcium carbonate to
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calcite and aragonite during shell formation
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Jingjing Kong1, Chuang Liu1,2, Dong Yang1, Yi Yan1, Yan Chen1, Jingliang Huang1,
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Yangjia Liu1, Guilan Zheng1, Liping Xie1, and Rongqing Zhang1,2*
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1 Protein Science Laboratory of the Ministry of Education, School of Life Sciences,
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Tsinghua University, Beijing 100084 China;
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2 Department of Biotechnology and Biomedicine, Yangtze Delta Region Institute of
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Tsinghua University, Jiaxing, Zhejiang Province, 314006, China;
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*To whom correspondence may be addressed. E-mail:
[email protected].
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Tele: +86-010-62772630.
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For Table of Contents Use Only
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Alv protein plays opposite roles in the transition of amorphous calcium carbonate to
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calcite and aragonite during shell formation
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Jingjing Kong, Chuang Liu, Dong Yang, Yi Yan, Yan Chen, Jingliang Huang,
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Yangjia Liu, Guilan Zheng, Liping Xie, and Rongqing Zhang
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Synopsis
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We found that Alv serving opposite roles in prism and nacre formation. Alv could
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promote nucleation and stimulate calcite crystallization, while inhibit transition of
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aragonite crystallization from ACC by impacting both crystal growth and phase
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transition rate.
22 23
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Crystal Growth & Design
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Abstract
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Amorphous calcium carbonate (ACC) is an important precursor in biominerals such
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as shells, coral, foraminiferal and urchin spine. However, for the mechanism
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underlying the transition from ACC to stable biosynthetic crystals is still poorly
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understood. Herein, we identified a matrix protein referred as Alv in Pinctada fucata,
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which has dramatic opposite functions during the different transition processes from
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ACC to stable crystals-calcite and aragonite in shell formation. The functions of Alv
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were studied by RNA interference, binding of recombinant Alv (rAlv) to chitin,
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calcite and aragonite assay, ACC transition, in vitro crystallization, calcium carbonate
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precipitation, and near-UV CD spectra. We found that rAlv could promote nucleation
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during ACC crystallization, stimulate the transition from ACC to calcite, but suppress
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transition from ACC to aragonite. It is concluded that Alv is involved in the transition
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of ACC, and plays crucial roles in the formation of shells. As far as we know, Alv is
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one of the few reported matrix proteins which plays opposite roles in the transition of
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ACC to calcite and aragonite both in vivo and in vitro. This study could further
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enhance our understanding of the important regulatory role of biomacromolecules in
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biomineralization.
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Introduction
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Molluscan shell is one of the most typical biomineralization models
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oyster Pinctada fucata, the shell is composed of three layers: the outer periostracum,
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middle prismatic and inner nacreous layers 4. The shell is a highly organized
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hierarchical structure with several length scales and possesses excellent mechanical
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properties
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3000-fold greater than that of pure aragonite, which is attributed to the 5% organic
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matrix in shells7. Although organic matrix contains proteins, lipids
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polysaccharides, it is thought that proteins, especially matrix proteins, have been
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proved to be the major components that control the biomineralization process,
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including crystal nucleation, crystal orientation, polymorphism and crystal
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morphology
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calcium carbonate (ACC) precursor
1-3
. In the pearl
2, 5-6
. Constituting of 95% aragonite, the toughness of nacre is roughly
8
and
9-14
. The hierarchical crystalized shells are formed via the amorphous 13, 15
which had been reported to play important
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roles in biomineralization including mollusk shells 16-17, coral18, urchin spine 19-20 et al.
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The transition of ACC to stable crystals is a research emphasis. It has been shown that
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the ACC precursor is formed via aggregation of prenucleated ion clusters which could
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be stabilized by magnesium or macromolecules
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transported to the final mineralization site, where it will be destabilized by Asp-rich
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proteins and transforms into calcite or aragonite 26.
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Currently, dozens of matrix proteins in P. fucata have been identified, including
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Nacrein 4, Pearlin 27, Pif 28-29, KRMP family 30-31, Prisikin-39 32, PfN44 33, Shematrin
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family
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by our group from pearl oyster, P. fucata, which could stabilize ACC
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proteins which could stimulate or suppress the transition were few reported. Until now,
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Pif 97 was reported to stabilize ACC and inhibit calcite growth
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80 could stabilize polymer-induced liquid precursor–like amorphous calcium
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carbonate granules (PILP-like ACGs) by formation of Pif80-CLP and control
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aragonite growth 38.
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Herein, we found that Alv protein has opposite functions during transition from ACC
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to calcite and aragonite. Alv, which exists in the prismatic layer of P. margaritifera
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and P. maxima with high homology, was first reported by Marie B et al 39. In 2015,
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our group also found that Alv also exists in the shell of P. fucata using a proteomic
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approach 40. In Liu’s research, Alv was the third most abundant matrix protein in the
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prismatic layer with function poorly understood
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double-stranded RNA (dsRNA) interference in vivo was executed to inhibit the
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function of Alv, what’s more, functional experiments in vitro including binding assay,
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the transition of ACC to stable crystals, in vitro crystallization, circular dichroism
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(CD) spectroscopy, etc. were also executed to study the functions of Alv during ACC
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transition.
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Experimental Section
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Ethics statement
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All methods were executed consistent with approved guidelines. All experimental
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protocols were confirmed by the Animal Experimental Ethics Committee of Tsinghua
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, and ACCBP
21-25
. Then the ACC precursor is
16, 35
. ACCBP is an extrapallial fluid (EPF) protein identified 35-36
, while the
37
. What’s more, Pif
40
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University, Beijing, China.
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Sample preparation
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Two years old adult pearl oyster, P. fucata (with shells 5.5-6.5 cm in length and 30-40
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g of wet weight) were cultured in a pearl farm (Zhanjiang, Guangdong Province,
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China). The oysters were raised in the laboratory at approximately 20°C in a fish tank
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containing aerated artificial seawater of 3% salinity.
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In vivo Alv function interference
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RNAi was employed to suppress the function of Alv in biomineralization. The primers
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designed for the DNA transcription are as follows: Alv-F: 5’-CTAATACGACTCACT
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ATAGGGAGAAGGGAAGGATACCTGAACCTCGAC-3’; and Alv-R: 5’-CTAATA
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CGACTCACTATAGGGAGAAGGACACCCACAGTTTCTACGGAC-3’.
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primers designed for GFP dsRNA are as follows: GFP-F: 5’-CTAATACGACTCACT
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ATAGGGAGAAGGATGGTGAGCAAGGGCGA-3’; and GFP-R: 5’-CTAATACGA
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CTCACTATAGGGAGAAGGACTTGTACAGCTCGTCCATG-3’. A fragment of the
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Alv gene (301 bp) were amplified. dsRNA was synthesized following the Promega
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T7RiboMAXTM protocol (Promega, USA), and DNase was added to digest the DNA
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template. The RNA product was extracted using phenol and chloroform. The RNA
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was diluted in PBS at a dose of 100 µg/200 µl. The negative controls are PBS and
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GFP dsRNA at a dosage of 100 µg/200 µl. PBS or dsRNA of 200 µl were injected
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into the adductor muscle of five uniform size pearl oysters and were injected
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complementarily on the third day. After six days, the mantle tissue of the fifteen
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oysters was excised and qPCR was applied as follows: The pair of primers designed
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for qPCR was as follows: for Alv, F: 5’-GAAGGATACCTGAACCTCGAC-3’; and R:
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5’-ACACCCACAGTTT CTACGGAC-3’. The primers designed for actin 41, which is
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the reference gene, are as follows: F: 5’-CTCCTCACTGAAGCCCCCCTC-3’; and R:
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5’-ATGGCTGGAATAG GGATTCTGG-3’. The qPCR was performed on an ABI
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PCR amplifier (StepOnePlusTM, Life Technologies, USA) following the SYBR®
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Premix Ex Taq™ (TaKaRa, Japan) protocol. The experimental procedure of qPCR is
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as follows: 95°C, 30 s 95°C, 5 s; and 60°C, 30 s for 35 cycles.
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Production and purification of rAlv protein ACS Paragon Plus Environment
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The gene encoding rAlv was designed based on the avoidance of the signal peptide
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and was chemically synthesized with the addition of N-terminal BamHI and
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C-terminal XhoI restriction sites. The target gene was inserted into a pET-28a vector,
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resulting in pET-rAlv. The recombinant plasmid pET-rAlv was transformed into E.
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coli Transetta (DE3) Chemically Competent Cells (TransGen Biotech, China) for the
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expression of the rAlv protein. The amino acids of the recombinant protein included
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Alv protein amino acids, which has hexahistidine (His6) tag in the N-terminus but
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lacking in the signal peptide.
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Calcite, aragonite and chitin binding assay
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Binding activity of rAlv protein to aragonite, calcite and chitin, was detected by a
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modification of Suzuki’s method 28. A weighed 20 mg substrate (calcite, aragonite and
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chitin) sample was placed in a 1.5 ml Eppendorf tube and incubated with 1 ml of 30
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µg/ml rAlv or BSA at RT for 1 h. The supernatant of the mixture was removed, the
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substrate was washed with 200 µl distilled-water for three times and then washed with
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200 µl 0.2 M NaCl buffer three times. The sample was then boiled with denaturing
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solution (30 mM Tris-HCl, pH 8.0, 1% sodium dodecyl sulfate, 10 mM dithiothreitol)
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for 15 min. The supernatant of each washing step was concentrated and subjected to
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SDS-PAGE.
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Transition from ACC to stable crystals
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According to Pan’s method
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rAlv/BSA or Tris-NaCl buffer (20Mm Tris, 500mM NaCl, pH=7.5) was prepared as
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solution A, and 50 mM NaCO3 was prepared as solution B. Solution A was added
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with 100 mM MgCl2 in the aragonite system. Solution A and solution B were cooled
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on ice for 1 h before being mixed well at 4°C with equal volumes in inclosed 15 ml
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centrifuge tube. Deposited calcite crystals were washed with acetone, dried, and
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analyzed by X-ray diffraction (XRD) after 30 min and 60 min. After 24 h and 48 h,
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deposited aragonite crystals were collected and analyzed similar to the calcite crystals.
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These analyses were repeated three times to obtain a consistent result, and are
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presented as the mean ± standard deviation (SD).
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To detect the dynamic phase change from ACC to more stable phase, we also execute
33
, in the calcite system, 50 mM CaCl2 and 30 µg/ml
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ACC transition investigated by polarized microscopy modified by Su et al.36.The
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ACC was synthesized using a method adapted from the procedure described by Koga
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et al.42. In addition, the ACC used during the transition from ACC to aragonite was
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synthesized with 50mM magnesium ions. We observed the process every 10 seconds
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for 3 minutes by polarized microscope (Leica DMR, Germany).
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In vitro crystallization
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Two types of crystallization solutions were prepared according to the modification of
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Xu’s method 43. A weighed 0.2 g sampled of CaCO3 was added to 20 ml Milli-Q water
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and filled with CO2 for 4 h. Redundant CaCO3 solid was removed by a 0.22 µm filter
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membrane and then filled with CO2 into solution for 1 h. The silicified glass was
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placed on a six-well tissue culture plate. The saturated Ca(HCO3)2 solution was mixed
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with rAlv protein and BSA, and the mixture was dripped onto a silicified glass with a
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final volume of 20 µl. Samples were placed in a stable environment at RT and reacted
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for 24 h. The glass was cleaned with diluted water after the reaction, dried under RT
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and scanned using SEM. In the meantime, according to Liu’s method 44-45, 170 µl cy5
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(LiTTLE-PA Sciences Company, China) dye was mixed with 900 µl of 1 mg/ml rAlv
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protein, which was dissolved in PBS buffer before, for 2 h in the dark at RT. Then, the
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mixture was desalted using a desalting column to remove excess dye. Cy5-rAlv was
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mixed with the crystallization system and cy5-BSA was added as a control. The
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mixtures with final volumes of 20 µl were dripped onto the glass bottoms of cell
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culture dishes (Nest, China) for stochastic optical reconstruction microscopy
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(STORM; Nikon A1, Japan) imaging
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dark. Nano Measurer 1.2 procedure was used to measure the diameters of the crystals.
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Photoshop CC 2015 (Adobe, USA) was used to count the number of crystals. The
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results are presented as the mean ± standard deviation (SD).
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Assessment of activity on calcium carbonate precipitation
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The activities of rAlv on calcite and aragonite calcium carbonate precipitation were
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assessed by a modification of the method of Suzuki 48. In the calcite system, 10 mM
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CaCl2 and 10 mM NaCO3 was prepared to mix in an equal volume with different
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concentrations of rAlv; while 20 mM CaCl2 with 40 mM MgCl2 and 20 mM NaCO3
46-47
. The whole reaction was performed in the
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was prepared to mix in an equal volume with different concentrations of rAlv in the
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aragonite reaction system. As the magnesium could stabilize ACC, we extended
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precipitation time of aragonite system. The data of precipitation were collected every
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30 s for 5 min in the calcite system and 10 min in the aragonite system by measuring
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the absorbance at 570 nm using a Model 550 Microplate Reader (Bio-Rad, USA). The
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results were repeated three times and are presented as the mean ± standard deviation
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(SD).
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CD spectroscopy
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Near-UV CD spectra measurements were obtained with a Chirascan plus
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spectropolarimeter (Applied Photophysics, UK) using cuvettes with a 0.1 mm path
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length. The concentration of rAlv was 200 µg/ml, and the detective wave length was
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between 190 nm and 260 nm. The CD spectroscopy was executed three times to
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obtain a consistent result. CDNN software was used to analyze secondary structure
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variation.
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Crystal and shell characterization
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The morphologies of the cleaned shells and crystals were captured by SEM after
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being sprayed with gold nanoparticles for 60 s. Raman spectra of the crystals were
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recorded with an excitation wavelength of 514 nm, provided by a Renishaw RM2000
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spectrometer (Renishaw, UK), and the argon laser was limited to a power of 4.6 mW.
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The spectra were scanned for 60 s from 100 to 1500 cm–1. XRD was also used to
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identify the form of calcium carbonate from the ACC phase transformation. XRD
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analysis was executed on a D8 ADVANCE (Bruker, Germany) with an X-ray
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diffractometer over the 2θ range of 10-90°.
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Statistical analysis
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All figures were created using Origin 8 (OriginLab, USA) and Photoshop CC 2015
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(Adobe, USA).
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Results
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The function of Alv during shell formation
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To explore the matrix protein which is responsible for ACC transition, we have
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investigated many new proteins found by shell proteomics of P. fucata ACS Paragon Plus Environment
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. The
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functions of the matrix proteins during shell formation were studied by RNAi, and
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Alv protein (GenBankTM Accession No. KR872410) was very likely to be responsible
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for ACC transition during shell formation.
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Relative mRNA levels were analyzed at six days after injection of dsRNA. The Alv
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expression level in the GFP dsRNA-injected group was similar to that in the
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PBS-injected group, but with a distinct decrease to almost 25% in the Alv
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dsRNA-injected group (Fig. S1). Compared with the control group, the surfaces of the
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prismatic layer in Alv dsRNA-injected group were full of cavities and the margins
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were destroyed (Fig. 1a). In the meantime, nacreous tablets of the Alv
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dsRNA-injected group overgrew where the normal flat nacreous tablets were absent
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(Fig. 1a). Raman spectra were executed to investigate the crystal polymorph of the
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inner surface of the shell from the Alv dsRNA-injected group and GFP
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dsRNA-injected group (Fig. 1b). Compared with the control group, the Raman spectra
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of spots from the Alv dsRNA-injected group had a higher baseline, indicating that
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there was a large amount of organic materials on the surface of the shell (Fig. 1b).
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What’s more, the disappearance of characteristic peaks of calcite (around 158 cm-1,
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284 cm-1 and 717 cm-1) and aragonite (around 148 cm-1, 208 cm-1 and 708 cm-1)
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confirmed the existence of ACC on the surface of shells in RNA interference groups.
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These interesting findings caught our attention and we then performed binding assay
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and ACC transition assay to identify the function of Alv in the in vitro calcium
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carbonate crystallization system.
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Fig. 1. SEM images and Raman analysis of RNAi experiment. a. SEM images of
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shells in the RNAi experiments. b. Raman spectra of the inner shell surface in P.
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fucata. P, prismatic layer; N, nacreous layer. Images of control (P/N): enlargement
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images of the prismatic and nacreous layers of shells from GFP dsRNA-injected P.
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fucata. Images of Alvi (P/N): enlargement images of the prismatic and nacreous layers
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of shells from Alv dsRNA-injected P. fucata. Raman spectra of spots on the inner
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shell surface from different groups.
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Binding ability of rAlv to chitin, calcite and aragonite
235
Previously, framework proteins were suggested interacting with chitin according to
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the models proposed by Weiner et al 2, 49. Binding assay was executed to confirm that
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Alv could serve as a structural role in shell formation, where the remnant of rAlv was
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detected after incubation with CaCO3 crystals or chitin.
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The recombinant protein was expressed with a His6-tag in the N-terminus in E. coli
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(Fig. S2a). Western blot (Fig. S2a) and mass spectrometry (Fig. S2c) confirmed that
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the purified recombinant protein was the recombinant Alv protein of P. fucata.
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As shown in Fig. S2b, lane 1, lane4 and lane 7 were remnants washed after water.
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Lane 2, lane 5 and lane 8 were remnants washed after 0.2 M NaCl. Lane 3, lane 6 and
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lane9 were remnants washed after denaturing solution. BSA was removed by water,
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indicating the weak binding of BSA to chitin, calcite and aragonite. Additionally, the
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lane 3, lane 6 and lane 9 of BSA were less than those of rAlv. The binding ability
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between rAlv and calcite/aragonite indicate that Alv might play important roles in the
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formation of calcite and aragonite 31-32, 50. While the comparison of lane 3, lane 6 and
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lane 9, the binding ability of rAlv to chitin was stronger than binding to calcite and
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aragonite, which was consistent with the location of Alv in the EDTA-insoluble
251
matrix. What’s more, the binding ability of rAlv to aragonite is stronger than that of
252
rAlv to calcite.
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Effects of rAlv on the transition of ACC to stable crystals
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ACC is a precursor of stable calcium carbonate
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function of rAlv in the transition rate of ACC to stable calcium carbonate crystals to
256
investigate whether it could affect calcite and aragonite deposition (Fig. 2). To
257
characterize the polymorphs of deposited crystals, we tested the structure and
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proportion of polymorphs by XRD. During the process of ACC transitioning to calcite,
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there is an intermediate phase as vaterite when crystals are deposited
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38.1 ± 0.7% vaterite and 61.9 ± 0.7% calcite in deposited crystals 30 minutes after
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mixture of 50 mM CaCl2, 50 mM NaCO3 and 30 µg/ml rAlv; while there were 64.7 ±
262
0.4% vaterite/35.3 ± 0.4% calcite with 30 µg/ml BSA and 52.1 ± 0.5% vaterite/47.9 ±
263
0.5% calcite in buffer group. One hour after reaction, almost all the crystals in rAlv
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group (95.8 ± 0.5%) transformed to calcite; however, there was still 16.4 ± 0.3%
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vaterite in the BSA group and 34.4 ± 0.3% vaterite in buffer group (Fig. 2a). In brief,
51
. Therefore, we examined the
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. There was
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rAlv could stimulate transition from ACC to calcite. In contrast, rAlv inhibited the
267
transition from ACC to aragonite crystals. ACC would transform to magnesium
268
calcium carbonate before the formation of aragonite in the solution of crystallization
269
system with magnesium
270
proved that matrix proteins could control calcium carbonate crystallization in pearl
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oyster shell formation 53. With addition of 50mM magnesium in reaction system, the
272
BSA group and buffer group had 71.8% ± 0.8% and 55.5 ± 0.6% aragonite 24 h after
273
reaction respectively, while there was 10.2 ± 0.3% aragonite and 89.8 ± 0.3%
274
magnesium calcium carbonate in deposited crystals in the rAlv group. After 48 h, the
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aragonite content in the rAlv group was 13.9 ± 0.2%, while the ratio in BSA group
276
and buffer group were 78.6% ± 0.6% and 94.9 ± 0.8% (Fig. 2b). To explore the effect
277
of concentration of magnesium during ACC transition, we also executed the assay
278
with 25mM magnesium (Fig. S3a). After 24 hours, there is 6.99 ± 0.3% aragonite in
279
rAlv group and no aragonite formation in BSA group. More significantly, there are
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76.75 ± 0.9% aragonite in rAlv group but 23.26 ± 0.8% in BSA group. That is, low
281
dosage of magnesium (25mM) could still stimulate transition from ACC to aragonite,
282
while high dosage magnesium (50mM) surprisingly inhibit transition of ACC to
283
aragonite compared with BSA. Magnesium might be an important element that
284
stabilizes ACC to form stable crystals which need further study 33, 35.
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To exclude the effect of pH during ACC transition, we detected the pH level of
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aragonite and calcite reaction systems in different time (Fig. S3b/c). And there is no
287
significant different between the values of rAlv and BSA group which indicates pH is
288
not the key factor influence ACC transition in this assay.
289
The ACC transformation investigated by polarized microscopy experiment showed
290
that the droplet of rAlv dissolved in Tris-buffer (20mM Tris, 500mM NaCl, pH=7.5)
291
could stimulate transition from ACC to crystals in calcite system compared with the
292
control (Fig. S4a), while inhibit transition from ACC to crystals in aragonite system
293
(Fig. S4b).
294
In conclusion, rAlv could stimulate the transition from ACC to calcite but inhibited
295
the transition from ACC to aragonite, which is consistent with the consequence after
33, 52
. The existence of magnesium calcite in the shell also
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interference of Alv dsRNA.
297 298
Fig. 2. Transition of ACC to stable crystals. a. Transition of ACC to calcite. C,
299
calcite; and V, vaterite. b. Transition of ACC to aragonite. M, magnesium calcium
300
carbonate; A, aragonite.
301 302
The effect of rAlv in in vitro calcium carbonate crystallization
303
To elucidate the mechanism of rAlv during the formation of stable CaCO3 crystals, in
304
vitro calcite and aragonite crystallization assays were performed. In the calcite
305
crystallization system, saturated Ca(HCO3)2 was introduced to imitate the composition
306
of the biomineralization in P. fucata. The Raman analyses of BSA group confirmed ACS Paragon Plus Environment
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307
the chemical nature of the formed crystal was calcite, with the spectrum of
308
characteristic peaks of 152, 280, 711 and 1085 cm-1. When the concentration of rAlv
309
was 10 µg/ml, the formed calcite crystals (Fig. 3b/f) were similar to those of the
310
control (Fig. 3a/e). However, When increasing rAlv concentration to 20 µg/ml rAlv,
311
the size of calcite became smaller, and the morphology tended to have more
312
crystallographic
313
Berkovitch-Yellin 54(Fig. 3c/g). With the increased concentration of rAlv (Fig. 3d/h),
314
the size became much smaller. The crystals form was still calcite, the Raman
315
spectrum of which showed characteristic calcite peaks (Fig. 3i). The crystal faces of
316
calcite in Figure 3g/h were marked and the overgrown calcite crystals were consisting
317
of fine prisms and truncated corners ((11.0) and (00.1)). While the (10.4) planes were
318
stacked
319
inorganic−organic mixture55-56. On the other side, the morphology could also occur
320
when impurity including protein hinder crystal growth, the binding of protein to
321
crystal face might hinder crystal growth while the other crystal faces would not be
322
hindered57-58. To quantify the changing features with the addition of rAlv, we
323
analyzed the diameters and numbers of formed crystals in both control group and
324
experimental group. According to Fig. 3j/k, the diameters of crystals decreased with
325
the addition of rAlv, while the number of crystals increased intensely (The diameter
326
of crystals from the 30 µg/ml BSA group is 19.404 ± 3.484 µm, while the diameter of
327
crystals from the 30 µg/ml rAlv group is 13.964 ± 1.962 µm. The number of crystals
328
from the 30 µg/ml BSA group is 106 ± 19, and the number of crystals from the 30
329
µg/ml rAlv group is 447 ± 14.).
with
planes
which
micrometer-size
is
similar
crystallites
to
as
prediction
of
mesocrystals
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which
by
were
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Crystal Growth & Design
330 331
Fig. 3. In vitro calcite crystallization experiments in the presence of rAlv or BSA.
332
a. SEM image of calcite crystals grown in the presence of 30 µg/ml BSA. b, c, d.
333
SEM images of calcite crystals grown in the presence of 10/20/30 µg/ml rAlv. e, f, g,
334
h. The enlarged images of the crystals in the box of a/b/c/d respectively. (11.0), (00.1),
335
(10.4) are the crystal faces of calcite. i. The Raman spectra of crystals with 30 µg/ml
336
BSA or 30 µg/ml rAlv. j, k. The diameter and number of crystals from different
337
groups. Values are means ± SD of three independent experiments. Asterisks indicate
338
statistically significant differences (P < 0.05, Student’s test). ACS Paragon Plus Environment
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339 340
The effect of rAlv on the formation of aragonite, generated from the saturated
341
Ca(HCO3)2 with 50 mM Mg2+, was similar to calcite (Fig. 4). The morphology of the
342
aragonites in the control group and experimental group had no significant difference.
343
However, the diameter of crystals became smaller with the addition of rAlv, and the
344
number of crystals increased intensely according to analyses of diameter and number
345
of formed crystals (Fig. 4j/k) (The diameter of crystals from the 30 µg/ml BSA group
346
and the diameter of crystals from the 30 µg/ml rAlv group are 63.522 ± 24.310 µm
347
and 37.857 ± 4.792 µm, respectively. The number of crystals from the 30 µg/ml BSA
348
group and the number of crystals from the 30 µg/ml rAlv group is 9 ± 3 and 58 ± 7,
349
respectively.). Raman analyses of obtained crystals from the 30 µg/ml BSA group and
350
the 30 µg/ml Alv group suggested that all of them were aragonites with absorbance
351
peaks at 153, 205, 706 and 1085 cm-1 (Fig. 4i).
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Crystal Growth & Design
352 353
Fig. 4. In vitro aragonite crystallization experiments in the presence of rAlv or
354
BSA. a. SEM image of aragonite crystals grown with the presence of 30 µg/ml BSA.
355
b, c, d. SEM image of aragonite crystals grown with the presence of 10/20/30 µg/ml
356
rAlv. e, f, g, h. The enlarged images of the crystals, indicated by black box in a/b/c/d
357
respectively. i. The Raman spectra of crystals with 30 µg/ml BSA or 30 µg/ml rAlv.
358
j, k. The diameter and number of crystals from different groups. Values are means ±
359
SD of three independent experiments. Asterisks indicate statistically significant
360
differences (P < 0.05, Student’s test). ACS Paragon Plus Environment
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361 362
To explore how rAlv influence the formation of stable crystals from ACC, the
363
distributions of rAlv proteins during calcite and aragonite crystallization in the
364
presence of cy5-rAlv were observed by stochastic optical reconstruction microscopy
365
(STROM) imaging (Fig. 5). Three forms of fluorescent clusters, which reflect the
366
distribution of cy5-rAlv, were observed 44. The distribution of chain-like clusters was
367
around the edges of crystals. Islet-like and haze-like clusters spread over the inner
368
crystals in both the experimental group and control group. Calcite in the presence of
369
cy5-rAlv has a core of fluorescence composed of islet-like clusters compared with the
370
control group. Aragonite in the experimental group has a core of fluorescence
371
composed of islet-like clusters and an edge composed of much stronger chain-like
372
clusters. In summary, rAlv could provide crystallization sites during crystallization.
373
The difference of edge distribution of rAlv in calcite experimental group and
374
aragonite experimental group is consistent with binding ability of rAlv with calcite
375
and aragonite, and might be relevant to crystal growth.
376 377
Fig. 5. STORM images of CaCO3 crystals. rAlv was labeled with cy5 and added to
378
the in vitro crystallization assay. Cy5-BSA was used as a negative control. The white
379
arrows pointed to the core of cy5-rAlv, and the blue arrow indicated the edge of
380
chain-like clusters. Scale bars, 10 µm.
381
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Crystal Growth & Design
382
In vitro activity of rAlv on calcium carbonate precipitation
383
The effect of rAlv on calcite calcium carbonate precipitation was examined to verify
384
whether rAlv could affect the crystallization rate of calcite and aragonite (Fig. 6). As a
385
control, the Tris-NaCl buffer and 30 µg/ml BSA were added in separating groups. The
386
calcium carbonate precipitation rate was detected by a microplate reader at an
387
absorbance of 570 nm for 5 min; the highest absorbance was around 0.19. When rAlv
388
was added, the precipitation rate changed intensely. The highest absorbance reached
389
about 0.260 with the addition of 5 µg/ml rAlv. As the absorbance increased with the
390
amount of rAlv increased, the highest absorbance reached around 0.340 with the
391
addition of 30 µg/ml rAlv. In conclusion, the addition of rAlv in the calcite
392
crystallization system increased the crystallization rate in a dose-dependent manner
393
(Fig. 6a). However, in the aragonite CaCO3 precipitation experiment, rAlv decreased
394
the rate of calcium carbonate precipitation. The absorbance value at 570 nm after 10
395
min in the presence of the Tris-NaCl buffer and 30 µg/ml BSA groups was
396
approximate 0.275 and 0.270, respectively; while the absorbance in 5 µg/ml rAlv
397
group was around 0.241 and the absorbance in 30 µg/ml rAlv group reached about
398
0.206 as rAlv increased (Fig. 6b). Overall, rAlv increased the precipitation rate in the
399
calcite crystallization system but decreased the precipitation rate in the aragonite
400
crystallization system.
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401 402
Fig. 6. In vitro activity of rAlv on calcium carbonate precipitation. a. Calcite
403
CaCO3 precipitation rates of rAlv and BSA during calcium carbonate precipitation
404
were indicated by the absorbance of crystals at 570 nm. b. Aragonite CaCO3
405
precipitation rate of rAlv and BSA during calcium carbonate precipitation.
406 407
Interactions between rAlv and ions
408
The opposite effect of rAlv on CaCO3 crystallization may be concerned with the
409
interaction between rAlv and ions
410
and ions, we analyzed near-UV CD spectra of Alv in the presence or absence of Ca2+,
411
Mg2+ and CO32- (Fig. 7). As a control, the addition of NaCl or NaOH which has the
412
same pH as NaCO3 solution, was executed, and had no significant influence on the
413
near-UV CD spectra of the rAlv protein (Fig. S5). Additionally, compared to the
414
natural secondary structure of the rAlv protein, adding CaCl2 could lead to a blue shift
33, 59
. To investigate the interaction between rAlv
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Crystal Growth & Design
415
(Fig. 7a), MgCl2 and Na2CO3 causing red shift (Fig. 7b/c) at the 204 nm peak, which
416
indicated the secondary structure variation in the rAlv protein while the addition of
417
NaCl or NaOH could not. The near-UV CD spectra results imply the interaction
418
between rAlv and crystallization ions. The opposite function of rAlv on transition of
419
ACC to calcite and aragonite (Fig. 2a/b) might due to the interaction of rAlv and
420
magnesium 52-53. It is possible there is supramolecular assembly when adding ions into
421
protein which need to be further studied
422
changes between natural rAlv and rAlv with ions, we used CDNN software to study
423
the different secondary structure domain proportions of CD spectra. As the Table S1
424
shows, secondary structures of the rAlv protein in the presence of Ca2+, Mg2+ and
425
CO32- have less helix, more parallel and more random coil compared with the natural
426
secondary structure of the rAlv protein.
36, 60
. To analyze the secondary structure
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427 428
Fig. 7. Interactions between rAlv and crystallization ions. a. Near-UV CD spectra
429
of rAlv in the presence of different doses of Ca2+. b. Near-UV CD spectra of rAlv
430
with different doses of Mg2+. c. Near-UV CD spectra of rAlv with different doses of
431
CO32-. Black arrows, native protein; green arrows, proteins with 25mM Ca2+, Mg2+ or
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Crystal Growth & Design
432
CO32-.
433
Discussion
434
It is well known that multifunctionality is a common characteristic of many shell
435
matrix proteins; however, it was the first time to find a matrix protein with opposite
436
functions in ACC transition to calcite and aragonite during shell formation. According
437
to Marin’s model, the control of shell synthesis includes two antagonistic mechanisms:
438
crystal nucleation and growth inhibition which are important steps during ACC
439
transition to stable crystals 13. However, in our study, the functions of Alv during shell
440
formation include both crystal nucleation and growth regulation.
441
To determine how Alv works in ACC transition during shell formation, in vitro
442
crystallization assays were achieved
443
crystals decreased with the addition of rAlv; the number of crystals greatly increased
444
in both the calcite and aragonite groups. Although the increase of supersaturation
445
would diminish free energy and make more particles according to Voorhees’s article
446
61
447
control group and the particles are generated in greater numbers. The distribution of
448
rAlv proteins in the whole calcite and aragonite crystallized in the presence of rAlv
449
was detected by STROM imaging, in which a core of fluorescence was found in
450
calcite and aragonite crystals. According to in vitro crystallization results and the rAlv
451
location in the core of crystals, we infer that rAlv participate in nucleation, which is a
452
supplement of the theory that acidic proteins nucleate
453
patterns of the edge of calcite and aragonite are consistent with the binding assay
454
results. The aragonite has much stronger binding ability with rAlv than calcite.
455
According to Cabrera and Vermilyea (C-V) model, the existence of impurity including
456
protein could hinder crystal growth
457
ease desolvation to promote crystal growth65. Alv is a basic protein with PI as 11.34
458
which could stimulate crystal growth, in the same time, Alv could bind crystals which
459
would inhibit crystal growth. Taken together, the binding ability of rAlv to aragonite
460
is too strong so that the suppression is predominant in aragonite system. While the
461
binding of rAlv to calcite is weak and the calcite growth was stimulated. The
and Yoreo’s article
33
. The results showed that the diameter of
62
. rAlv might diminish free energy further compared with
63-64
. The different distribution
57-58
. Han et al indicated that base additive could
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462
precipitation rate depends on number of particles with different phases and the
463
diameter of particles, which reflect nucleation and crystal growth. In a comparable
464
way, the rAlv protein could accelerate calcite CaCO3 precipitation and suppress
465
aragonite precipitation rate according to calcium carbonate precipitation experiment
466
which is accordant to consideration of both nucleation and crystal growth.
467
Additionally, ACC transition experiment showed that rAlv stimulated calcite
468
transition but inhibited aragonite transition by stabilizing magnesium calcium
469
carbonate. To study the molecular mechanism of Alv in ACC transition process,
470
near-UV CD spectra of Alv in the presence or absence of Ca2+, Mg2+ and CO32- was
471
performed. It is found that there were interactions between rAlv and mineralization
472
ions, in which magnesium might be the reason why Alv has function of stabilizing
473
magnesium calcium carbonate
474
assay with different dosage of magnesium, we can infer that the concentration of
475
magnesium is an important element that affect ACC transition. What’s more, the
476
increase of random coil according to CD spectra suggests that a protein complex or
477
supramolecular aggregate is forming as amyloid-like phase which might contribute to
478
form the nucleus for spherulitic crystallization like spherulitic aragonite in this paper
479
66-67
480
Alv could promote calcite nucleation and stimulate calcite growth. At the same time,
481
Alv could promote aragonite nucleation but inhibit aragonite growth. What’s more,
482
Alv could stabilize magnesium calcium carbonate to inhibit transition from ACC to
483
aragonite, but stimulate transition from ACC to calcite. The above-mentioned
484
functions lead to opposite results of a destroyed prismatic layer and an overgrown
485
nacreous layer after the inhibition of Alv function in P. fucata.
486
Conclusion
487
Alv, as a matrix protein existing in both prism and nacre of P. fucata, plays important
488
roles in shell formation. In this study, we figure out Alv has opposite functions in
489
transition from ACC to calcite and aragonite by promoting nucleation and impacting
490
both crystal growth and phase transition rate, which is rare function of matrix protein
491
and could expand understanding of biomineralization especially the role of
26, 33, 35, 52
. Combining the result of ACC transition
.
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Crystal Growth & Design
492
biomacromolecules in the transition of ACC.
493
Acknowledgments
494
This study was funded by the National Natural Science Foundation of China
495
(31572594, 31372502, and 31502139) and National Found for Fostering Talents of
496
Basic Science (J1310020).
497
Conflict of interest
498
The authors declare that they have no conflicts of interest with the contents of this
499
article.
500
Author Contributions
501
Jingjing Kong conceived the project. Jingjing Kong and Chuang Liu performed the
502
experiment and analyzed the data. Jingjing Kong and Rongqing Zhang wrote the
503
manuscript. All authors analyzed the results and approved the final version of the
504
manuscript.
505
Data and materials availability
506
GenBankTM Accession No. KR872410
507
References
508
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by
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