Alv protein plays opposite roles in the transition of amorphous calcium

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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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ACS Paragon Plus Environment

Crystal Growth & Design 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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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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Abstract

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Amorphous calcium carbonate (ACC) is an important precursor in biominerals such

26

as shells, coral, foraminiferal and urchin spine. However, for the mechanism

27

underlying the transition from ACC to stable biosynthetic crystals is still poorly

28

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

33

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.

41

Introduction

42

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

46

properties

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3000-fold greater than that of pure aragonite, which is attributed to the 5% organic

48

matrix in shells7. Although organic matrix contains proteins, lipids

49

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

52

morphology

53

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

57

be stabilized by magnesium or macromolecules

58

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

75

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

78

(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

81

Ethics statement

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All methods were executed consistent with approved guidelines. All experimental

83

protocols were confirmed by the Animal Experimental Ethics Committee of Tsinghua

34

, 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


. In the present work,

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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

89

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

108

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

The


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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



Page 8 of 33

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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

180

(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

199

(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

40

. 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.



225 226

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.

233 234

Binding ability of rAlv to chitin, calcite and aragonite

235

Previously, framework proteins were suggested interacting with chitin according to


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236

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

240

(Fig. S2a). Western blot (Fig. S2a) and mass spectrometry (Fig. S2c) confirmed that

241

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,

245

indicating the weak binding of BSA to chitin, calcite and aragonite. Additionally, the

246

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

248

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

250

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.

253

Effects of rAlv on the transition of ACC to stable crystals

254

ACC is a precursor of stable calcium carbonate

255

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,

259

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

264

group (95.8 ± 0.5%) transformed to calcite; however, there was still 16.4 ± 0.3%

265

vaterite in the BSA group and 34.4 ± 0.3% vaterite in buffer group (Fig. 2a). In brief,

51

. Therefore, we examined the


33

. There was


266

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

271

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

275

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

280

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.

285

To exclude the effect of pH during ACC transition, we detected the pH level of

286

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


Page 14 of 33

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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


α-glycine

which

by

were

Page 15 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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


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).


Page 16 of 33

Page 17 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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


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


Page 18 of 33

Page 19 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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.



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


Page 20 of 33

Page 21 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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



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


Page 22 of 33

Page 23 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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



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

.


Page 24 of 33

Page 25 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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

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Alv protein plays opposite roles in the transition of amorphous calcium

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