Alv protein plays opposite roles in the transition of amorphous calcium

3 days ago - Products. Journals A–Z · eBooks · C&EN · C&EN Archives · ACS Legacy Archives · ACS Mobile · Video. User Resources. About Us · ACS ...
0 downloads 0 Views 4MB Size
Subscriber access provided by Kaohsiung Medical University

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 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

Crystal Growth & Design

1

Alv protein plays opposite roles in the transition of amorphous calcium carbonate to

2

calcite and aragonite during shell formation

3

Jingjing Kong1, Chuang Liu1,2, Dong Yang1, Yi Yan1, Yan Chen1, Jingliang Huang1,

4

Yangjia Liu1, Guilan Zheng1, Liping Xie1, and Rongqing Zhang1,2*

5

1 Protein Science Laboratory of the Ministry of Education, School of Life Sciences,

6

Tsinghua University, Beijing 100084 China;

7

2 Department of Biotechnology and Biomedicine, Yangtze Delta Region Institute of

8

Tsinghua University, Jiaxing, Zhejiang Province, 314006, China;

9

*To whom correspondence may be addressed. E-mail: [email protected].

10

Tele: +86-010-62772630.

11

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

12

For Table of Contents Use Only

13

Alv protein plays opposite roles in the transition of amorphous calcium carbonate to

14

calcite and aragonite during shell formation

15

Jingjing Kong, Chuang Liu, Dong Yang, Yi Yan, Yan Chen, Jingliang Huang,

16

Yangjia Liu, Guilan Zheng, Liping Xie, and Rongqing Zhang

17

Synopsis

18

We found that Alv serving opposite roles in prism and nacre formation. Alv could

19

promote nucleation and stimulate calcite crystallization, while inhibit transition of

20

aragonite crystallization from ACC by impacting both crystal growth and phase

21

transition rate.

22 23

ACS Paragon Plus Environment

Page 2 of 33

Page 3 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

Crystal Growth & Design

24

Abstract

25

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,

29

which has dramatic opposite functions during the different transition processes from

30

ACC to stable crystals-calcite and aragonite in shell formation. The functions of Alv

31

were studied by RNA interference, binding of recombinant Alv (rAlv) to chitin,

32

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

34

during ACC crystallization, stimulate the transition from ACC to calcite, but suppress

35

transition from ACC to aragonite. It is concluded that Alv is involved in the transition

36

of ACC, and plays crucial roles in the formation of shells. As far as we know, Alv is

37

one of the few reported matrix proteins which plays opposite roles in the transition of

38

ACC to calcite and aragonite both in vivo and in vitro. This study could further

39

enhance our understanding of the important regulatory role of biomacromolecules in

40

biomineralization.

41

Introduction

42

Molluscan shell is one of the most typical biomineralization models

43

oyster Pinctada fucata, the shell is composed of three layers: the outer periostracum,

44

middle prismatic and inner nacreous layers 4. The shell is a highly organized

45

hierarchical structure with several length scales and possesses excellent mechanical

46

properties

47

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

50

proved to be the major components that control the biomineralization process,

51

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

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

Page 4 of 33

54

roles in biomineralization including mollusk shells 16-17, coral18, urchin spine 19-20 et al.

55

The transition of ACC to stable crystals is a research emphasis. It has been shown that

56

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

59

proteins and transforms into calcite or aragonite 26.

60

Currently, dozens of matrix proteins in P. fucata have been identified, including

61

Nacrein 4, Pearlin 27, Pif 28-29, KRMP family 30-31, Prisikin-39 32, PfN44 33, Shematrin

62

family

63

by our group from pearl oyster, P. fucata, which could stabilize ACC

64

proteins which could stimulate or suppress the transition were few reported. Until now,

65

Pif 97 was reported to stabilize ACC and inhibit calcite growth

66

80 could stabilize polymer-induced liquid precursor–like amorphous calcium

67

carbonate granules (PILP-like ACGs) by formation of Pif80-CLP and control

68

aragonite growth 38.

69

Herein, we found that Alv protein has opposite functions during transition from ACC

70

to calcite and aragonite. Alv, which exists in the prismatic layer of P. margaritifera

71

and P. maxima with high homology, was first reported by Marie B et al 39. In 2015,

72

our group also found that Alv also exists in the shell of P. fucata using a proteomic

73

approach 40. In Liu’s research, Alv was the third most abundant matrix protein in the

74

prismatic layer with function poorly understood

75

double-stranded RNA (dsRNA) interference in vivo was executed to inhibit the

76

function of Alv, what’s more, functional experiments in vitro including binding assay,

77

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

79

transition.

80

Experimental Section

81

Ethics statement

82

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

ACS Paragon Plus Environment

. In the present work,

Page 5 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

Crystal Growth & Design

84

University, Beijing, China.

85

Sample preparation

86

Two years old adult pearl oyster, P. fucata (with shells 5.5-6.5 cm in length and 30-40

87

g of wet weight) were cultured in a pearl farm (Zhanjiang, Guangdong Province,

88

China). The oysters were raised in the laboratory at approximately 20°C in a fish tank

89

containing aerated artificial seawater of 3% salinity.

90

In vivo Alv function interference

91

RNAi was employed to suppress the function of Alv in biomineralization. The primers

92

designed for the DNA transcription are as follows: Alv-F: 5’-CTAATACGACTCACT

93

ATAGGGAGAAGGGAAGGATACCTGAACCTCGAC-3’; and Alv-R: 5’-CTAATA

94

CGACTCACTATAGGGAGAAGGACACCCACAGTTTCTACGGAC-3’.

95

primers designed for GFP dsRNA are as follows: GFP-F: 5’-CTAATACGACTCACT

96

ATAGGGAGAAGGATGGTGAGCAAGGGCGA-3’; and GFP-R: 5’-CTAATACGA

97

CTCACTATAGGGAGAAGGACTTGTACAGCTCGTCCATG-3’. A fragment of the

98

Alv gene (301 bp) were amplified. dsRNA was synthesized following the Promega

99

T7RiboMAXTM protocol (Promega, USA), and DNase was added to digest the DNA

100

template. The RNA product was extracted using phenol and chloroform. The RNA

101

was diluted in PBS at a dose of 100 µg/200 µl. The negative controls are PBS and

102

GFP dsRNA at a dosage of 100 µg/200 µl. PBS or dsRNA of 200 µl were injected

103

into the adductor muscle of five uniform size pearl oysters and were injected

104

complementarily on the third day. After six days, the mantle tissue of the fifteen

105

oysters was excised and qPCR was applied as follows: The pair of primers designed

106

for qPCR was as follows: for Alv, F: 5’-GAAGGATACCTGAACCTCGAC-3’; and R:

107

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:

109

5’-ATGGCTGGAATAG GGATTCTGG-3’. The qPCR was performed on an ABI

110

PCR amplifier (StepOnePlusTM, Life Technologies, USA) following the SYBR®

111

Premix Ex Taq™ (TaKaRa, Japan) protocol. The experimental procedure of qPCR is

112

as follows: 95°C, 30 s 95°C, 5 s; and 60°C, 30 s for 35 cycles.

113

Production and purification of rAlv protein ACS Paragon Plus Environment

The

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

114

The gene encoding rAlv was designed based on the avoidance of the signal peptide

115

and was chemically synthesized with the addition of N-terminal BamHI and

116

C-terminal XhoI restriction sites. The target gene was inserted into a pET-28a vector,

117

resulting in pET-rAlv. The recombinant plasmid pET-rAlv was transformed into E.

118

coli Transetta (DE3) Chemically Competent Cells (TransGen Biotech, China) for the

119

expression of the rAlv protein. The amino acids of the recombinant protein included

120

Alv protein amino acids, which has hexahistidine (His6) tag in the N-terminus but

121

lacking in the signal peptide.

122

Calcite, aragonite and chitin binding assay

123

Binding activity of rAlv protein to aragonite, calcite and chitin, was detected by a

124

modification of Suzuki’s method 28. A weighed 20 mg substrate (calcite, aragonite and

125

chitin) sample was placed in a 1.5 ml Eppendorf tube and incubated with 1 ml of 30

126

µg/ml rAlv or BSA at RT for 1 h. The supernatant of the mixture was removed, the

127

substrate was washed with 200 µl distilled-water for three times and then washed with

128

200 µl 0.2 M NaCl buffer three times. The sample was then boiled with denaturing

129

solution (30 mM Tris-HCl, pH 8.0, 1% sodium dodecyl sulfate, 10 mM dithiothreitol)

130

for 15 min. The supernatant of each washing step was concentrated and subjected to

131

SDS-PAGE.

132

Transition from ACC to stable crystals

133

According to Pan’s method

134

rAlv/BSA or Tris-NaCl buffer (20Mm Tris, 500mM NaCl, pH=7.5) was prepared as

135

solution A, and 50 mM NaCO3 was prepared as solution B. Solution A was added

136

with 100 mM MgCl2 in the aragonite system. Solution A and solution B were cooled

137

on ice for 1 h before being mixed well at 4°C with equal volumes in inclosed 15 ml

138

centrifuge tube. Deposited calcite crystals were washed with acetone, dried, and

139

analyzed by X-ray diffraction (XRD) after 30 min and 60 min. After 24 h and 48 h,

140

deposited aragonite crystals were collected and analyzed similar to the calcite crystals.

141

These analyses were repeated three times to obtain a consistent result, and are

142

presented as the mean ± standard deviation (SD).

143

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

ACS Paragon Plus Environment

Page 6 of 33

Page 7 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

Crystal Growth & Design

144

ACC transition investigated by polarized microscopy modified by Su et al.36.The

145

ACC was synthesized using a method adapted from the procedure described by Koga

146

et al.42. In addition, the ACC used during the transition from ACC to aragonite was

147

synthesized with 50mM magnesium ions. We observed the process every 10 seconds

148

for 3 minutes by polarized microscope (Leica DMR, Germany).

149

In vitro crystallization

150

Two types of crystallization solutions were prepared according to the modification of

151

Xu’s method 43. A weighed 0.2 g sampled of CaCO3 was added to 20 ml Milli-Q water

152

and filled with CO2 for 4 h. Redundant CaCO3 solid was removed by a 0.22 µm filter

153

membrane and then filled with CO2 into solution for 1 h. The silicified glass was

154

placed on a six-well tissue culture plate. The saturated Ca(HCO3)2 solution was mixed

155

with rAlv protein and BSA, and the mixture was dripped onto a silicified glass with a

156

final volume of 20 µl. Samples were placed in a stable environment at RT and reacted

157

for 24 h. The glass was cleaned with diluted water after the reaction, dried under RT

158

and scanned using SEM. In the meantime, according to Liu’s method 44-45, 170 µl cy5

159

(LiTTLE-PA Sciences Company, China) dye was mixed with 900 µl of 1 mg/ml rAlv

160

protein, which was dissolved in PBS buffer before, for 2 h in the dark at RT. Then, the

161

mixture was desalted using a desalting column to remove excess dye. Cy5-rAlv was

162

mixed with the crystallization system and cy5-BSA was added as a control. The

163

mixtures with final volumes of 20 µl were dripped onto the glass bottoms of cell

164

culture dishes (Nest, China) for stochastic optical reconstruction microscopy

165

(STORM; Nikon A1, Japan) imaging

166

dark. Nano Measurer 1.2 procedure was used to measure the diameters of the crystals.

167

Photoshop CC 2015 (Adobe, USA) was used to count the number of crystals. The

168

results are presented as the mean ± standard deviation (SD).

169

Assessment of activity on calcium carbonate precipitation

170

The activities of rAlv on calcite and aragonite calcium carbonate precipitation were

171

assessed by a modification of the method of Suzuki 48. In the calcite system, 10 mM

172

CaCl2 and 10 mM NaCO3 was prepared to mix in an equal volume with different

173

concentrations of rAlv; while 20 mM CaCl2 with 40 mM MgCl2 and 20 mM NaCO3

46-47

. The whole reaction was performed in the

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

Page 8 of 33

174

was prepared to mix in an equal volume with different concentrations of rAlv in the

175

aragonite reaction system. As the magnesium could stabilize ACC, we extended

176

precipitation time of aragonite system. The data of precipitation were collected every

177

30 s for 5 min in the calcite system and 10 min in the aragonite system by measuring

178

the absorbance at 570 nm using a Model 550 Microplate Reader (Bio-Rad, USA). The

179

results were repeated three times and are presented as the mean ± standard deviation

180

(SD).

181

CD spectroscopy

182

Near-UV CD spectra measurements were obtained with a Chirascan plus

183

spectropolarimeter (Applied Photophysics, UK) using cuvettes with a 0.1 mm path

184

length. The concentration of rAlv was 200 µg/ml, and the detective wave length was

185

between 190 nm and 260 nm. The CD spectroscopy was executed three times to

186

obtain a consistent result. CDNN software was used to analyze secondary structure

187

variation.

188

Crystal and shell characterization

189

The morphologies of the cleaned shells and crystals were captured by SEM after

190

being sprayed with gold nanoparticles for 60 s. Raman spectra of the crystals were

191

recorded with an excitation wavelength of 514 nm, provided by a Renishaw RM2000

192

spectrometer (Renishaw, UK), and the argon laser was limited to a power of 4.6 mW.

193

The spectra were scanned for 60 s from 100 to 1500 cm–1. XRD was also used to

194

identify the form of calcium carbonate from the ACC phase transformation. XRD

195

analysis was executed on a D8 ADVANCE (Bruker, Germany) with an X-ray

196

diffractometer over the 2θ range of 10-90°.

197

Statistical analysis

198

All figures were created using Origin 8 (OriginLab, USA) and Photoshop CC 2015

199

(Adobe, USA).

200

Results

201

The function of Alv during shell formation

202

To explore the matrix protein which is responsible for ACC transition, we have

203

investigated many new proteins found by shell proteomics of P. fucata ACS Paragon Plus Environment

40

. The

Page 9 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

Crystal Growth & Design

204

functions of the matrix proteins during shell formation were studied by RNAi, and

205

Alv protein (GenBankTM Accession No. KR872410) was very likely to be responsible

206

for ACC transition during shell formation.

207

Relative mRNA levels were analyzed at six days after injection of dsRNA. The Alv

208

expression level in the GFP dsRNA-injected group was similar to that in the

209

PBS-injected group, but with a distinct decrease to almost 25% in the Alv

210

dsRNA-injected group (Fig. S1). Compared with the control group, the surfaces of the

211

prismatic layer in Alv dsRNA-injected group were full of cavities and the margins

212

were destroyed (Fig. 1a). In the meantime, nacreous tablets of the Alv

213

dsRNA-injected group overgrew where the normal flat nacreous tablets were absent

214

(Fig. 1a). Raman spectra were executed to investigate the crystal polymorph of the

215

inner surface of the shell from the Alv dsRNA-injected group and GFP

216

dsRNA-injected group (Fig. 1b). Compared with the control group, the Raman spectra

217

of spots from the Alv dsRNA-injected group had a higher baseline, indicating that

218

there was a large amount of organic materials on the surface of the shell (Fig. 1b).

219

What’s more, the disappearance of characteristic peaks of calcite (around 158 cm-1,

220

284 cm-1 and 717 cm-1) and aragonite (around 148 cm-1, 208 cm-1 and 708 cm-1)

221

confirmed the existence of ACC on the surface of shells in RNA interference groups.

222

These interesting findings caught our attention and we then performed binding assay

223

and ACC transition assay to identify the function of Alv in the in vitro calcium

224

carbonate crystallization system.

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

225 226

Fig. 1. SEM images and Raman analysis of RNAi experiment. a. SEM images of

227

shells in the RNAi experiments. b. Raman spectra of the inner shell surface in P.

228

fucata. P, prismatic layer; N, nacreous layer. Images of control (P/N): enlargement

229

images of the prismatic and nacreous layers of shells from GFP dsRNA-injected P.

230

fucata. Images of Alvi (P/N): enlargement images of the prismatic and nacreous layers

231

of shells from Alv dsRNA-injected P. fucata. Raman spectra of spots on the inner

232

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

ACS Paragon Plus Environment

Page 10 of 33

Page 11 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

Crystal Growth & Design

236

the models proposed by Weiner et al 2, 49. Binding assay was executed to confirm that

237

Alv could serve as a structural role in shell formation, where the remnant of rAlv was

238

detected after incubation with CaCO3 crystals or chitin.

239

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.

242

As shown in Fig. S2b, lane 1, lane4 and lane 7 were remnants washed after water.

243

Lane 2, lane 5 and lane 8 were remnants washed after 0.2 M NaCl. Lane 3, lane 6 and

244

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

247

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

249

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

258

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

260

38.1 ± 0.7% vaterite and 61.9 ± 0.7% calcite in deposited crystals 30 minutes after

261

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

ACS Paragon Plus Environment

33

. There was

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

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

ACS Paragon Plus Environment

Page 12 of 33

Page 13 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

Crystal Growth & Design

296

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

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

Page 14 of 33

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

ACS Paragon Plus Environment

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

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

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

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).

ACS Paragon Plus Environment

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

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

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

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

ACS Paragon Plus Environment

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

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.

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

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

ACS Paragon Plus Environment

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

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

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

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

ACS Paragon Plus Environment

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

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

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

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

.

ACS Paragon Plus Environment

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

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

(1) Boskey, A. L., Biomineralization: Conflicts, challenges, and opportunities. J. Cell.

509

Biochem. 1998, 1998 83-91.

510

(2) Addadi, L.; Joester, D.; Nudelman, F.; Weiner, S., Mollusk shell formation: A source of

511

new concepts for understanding biomineralization processes. Chem.-Eur. J. 2006, 12,

512

981-987.

513

(3) Suzuki, M.; Nagasawa, H., Mollusk shell structures and their formation mechanism. Can.

514

J. Zool. 2013, 91, 349-366.

515

(4) Miyamoto, H.; Miyashita, T.; Okushima, M.; Nakano, S.; Morita, T.; Matsushiro, A., A

516

carbonic anhydrase from the nacreous layer in oyster pearls. Proc. Natl. Acad. Sci. U. S. A.

517

1996, 93, 9657-9660.

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

Page 26 of 33

518

(5) Li, X. D.; Chang, W. C.; Chao, Y. J.; Wang, R. Z.; Chang, M., Nanoscale structural and

519

mechanical characterization of a natural nanocomposite material: The shell of red abalone.

520

Nano. Lett. 2004, 4, 613-617.

521

(6) Rousseau, M.; Lopez, E.; Stempfle, P.; Brendle, M.; Franke, L.; Guette, A.; Naslain, R.;

522

Bourrat, X., Multiscale structure of sheet nacre. Biomaterials 2005, 26, 6254-6262.

523

(7) Jackson, A. P.; Vincent, J. F. V.; Turner, R. M., The Mechanical design of nacre. P. Roy.

524

Soc. B.-Biol. Sci. 1988, 234, 415-+.

525

(8) Farre, B.; Dauphin, Y., Lipids from the nacreous and prismatic layers of two Pteriomorpha

526

Mollusc shells. Comp. Biochem. Phys. B. 2009, 152, 103-109.

527

(9) Falini, G.; Albeck, S.; Weiner, S.; Addadi, L., Control of aragonite or calcite polymorphism

528

by mollusk shell macromolecules. Science 1996, 271, 67-69.

529

(10) Levi, Y.; Albeck, S.; Brack, A.; Weiner, S.; Addadi, L., Control over aragonite crystal

530

nucleation and growth: An in vitro study of biomineralization. Chem.-Eur. J. 1998, 4, 389-396.

531

(11) Shimomura, N.; Ohkubo, N.; Ichikawa, K., Control of the production amount and

532

polymorphism of calcium carbonate by biomimetic mineralization. Chem. Lett. 2002, 2002 902-903.

533

(12) Ichikawa, K.; Shimomura, N.; Yamada, M.; Ohkubo, N., Control of calcium carbonate

534

polymorphism

535

nanotechnology. Chem.-Eur. J. 2003, 9, 3235-3241.

536

(13) Marin, F.; Luquet, G.; Marie, B.; Medakovic, D., Molluscan shell proteins: Primary

537

structure, origin, and evolution. In Current Topics in Developmental Biology, Schatten, G. P.,

538

Ed. Elsevier Academic Press Inc: San Diego, 2008; Vol. 80, pp 209-276.

539

(14) Takeuchi, T.; Sarashina, I.; Iijima, M.; Endo, K., In vitro regulation of CaCO3 crystal

and

morphology

through

biomimetic

mineralization

ACS Paragon Plus Environment

by

means

of

Page 27 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

Crystal Growth & Design

540

polymorphism by the highly acidic molluscan shell protein Aspein. FEBS Lett. 2008, 582,

541

591-596.

542

(15) Marin, F.; Luquet, G., Molluscan shell proteins. CR. Palevol. 2004, 3, 469-492.

543

(16) Xiang, L.; Kong, W.; Su, J. T.; Liang, J.; Zhang, G. Y.; Xie, L. P.; Zhang, R. Q.,

544

Amorphous Calcium Carbonate Precipitation by Cellular Biomineralization in Mantle Cell

545

Cultures of Pinctada fucata. Plos One 2014, 9, 8.

546

(17) Weiss, I. M.; Tuross, N.; Addadi, L.; Weiner, S., Mollusc larval shell formation: Amorphous

547

calcium carbonate is a precursor phase for aragonite. J. Exp. Zool. 2002, 293, 478-491.

548

(18) Mass, T.; Giuffre, A. J.; Sun, C.-Y.; Stifler, C. A.; Frazier, M. J.; Neder, M.; Tamura, N.;

549

Stan, C. V.; Marcus, M. A.; Gilbert, P. U. P. A., Amorphous calcium carbonate particles form

550

coral skeletons. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, E7670-E7678.

551

(19) Politi, Y.; Arad, T.; Klein, E.; Weiner, S.; Addadi, L., Sea urchin spine calcite forms via a

552

transient amorphous calcium carbonate phase. Science 2004, 306, 1161-1164.

553

(20) Gong, Y. U. T.; Killian, C. E.; Olson, I. C.; Appathurai, N. P.; Amasino, A. L.; Martin, M. C.;

554

Holt, L. J.; Wilt, F. H.; Gilbert, P. U. P. A., Phase transitions in biogenic amorphous calcium

555

carbonate. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 6088-6093.

556

(21) Aizenberg, J.; Lambert, G.; Weiner, S.; Addadi, L., Factors involved in the formation of

557

amorphous and crystalline calcium carbonate: A study of an ascidian skeleton. J. Am. Chem.

558

Soc. 2002, 124, 32-39.

559

(22) Raz, S.; Hamilton, P. C.; Wilt, F. H.; Weiner, S.; Addadi, L., The transient phase of

560

amorphous calcium carbonate in sea urchin larval spicules: The involvement of proteins and

561

magnesium ions in its formation and stabilization. Adv. Funct. Mater. 2003, 13, 480-486.

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

562

(23) Ren, D. N.; Feng, Q. L.; Bourrat, X., Effects of additives and templates on calcium

563

carbonate mineralization in vitro. Micron 2011, 42, 228-245.

564

(24) Gebauer, D.; Volkel, A.; Colfen, H., Stable Prenucleation Calcium Carbonate Clusters.

565

Science 2008, 322, 1819-1822.

566

(25) Pouget, E. M.; Bomans, P. H. H.; Goos, J. A. C. M.; Frederik, P. M.; de With, G.;

567

Sommerdijk, N. A. J. M., The Initial Stages of Template-Controlled CaCO3 Formation

568

Revealed by Cryo-TEM. Science 2009, 323, 1455-1458.

569

(26) Tao, J.; Zhou, D.; Zhang, Z.; Xu, X.; Tang, R., Magnesium-aspartate-based crystallization

570

switch inspired from shell molt of crustacean. Proc. Natl. Acad. Sci. U. S. A. 2009, 106,

571

22096-22101.

572

(27) Miyashita, T.; Takagi, R.; Okushima, M.; Nakano, S.; Miyamoto, H.; Nishikawa, E.;

573

Matsushiro, A., Complementary DNA cloning and characterization of pearlin, a new class of

574

matrix protein in the nacreous layer of oyster pearls. Mar. Biotechnol. 2000, 2, 409-418.

575

(28) Suzuki, M.; Saruwatari, K.; Kogure, T.; Yamamoto, Y.; Nishimura, T.; Kato, T.; Nagasawa,

576

H., An Acidic Matrix Protein, Pif, Is a Key Macromolecule for Nacre Formation. Science 2009,

577

325, 1388-1390.

578

(29) Suzuki, M.; Iwashima, A.; Kimura, M.; Kogure, T.; Nagasawa, H., The Molecular Evolution

579

of the Pif Family Proteins in Various Species of Mollusks. Mar. Biotechnol. 2013, 15, 145-158.

580

(30) Zhang, C.; Xie, L. P.; Huang, J.; Liu, X. L.; Zhang, R. Q., A novel matrix protein family

581

participating in the prismatic layer framework formation of pearl oyster, Pinctada fucata.

582

Biochem. Bioph. Res. Co. 2006, 344, 735-740.

583

(31) Liang, J.; Xu, G.; Xie, J.; Lee, I.; Xiang, L.; Wang, H.; Zhang, G.; Xie, L.; Zhang, R., Dual

ACS Paragon Plus Environment

Page 28 of 33

Page 29 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

Crystal Growth & Design

584

Roles of the Lysine-Rich Matrix Protein (KRMP)-3 in Shell Formation of Pearl Oyster, Pinctada

585

fucata. Plos One 2015, 10.

586

(32) Kong, Y.; Jing, G.; Yan, Z.; Li, C.; Gong, N.; Zhu, F.; Li, D.; Zhang, Y.; Zheng, G.; Wang,

587

H.; Xie, L.; Zhang, R., Cloning and Characterization of Prisilkin-39, a Novel Matrix Protein

588

Serving a Dual Role in the Prismatic Layer Formation from the Oyster Pinctada fucata. J. Biol.

589

Chem. 2009, 284, 10841-10854.

590

(33) Pan, C.; Fang, D.; Xu, G.; Liang, J.; Zhang, G.; Wang, H.; Xie, L.; Zhang, R., A Novel

591

Acidic Matrix Protein, PfN44, Stabilizes Magnesium Calcite to Inhibit the Crystallization of

592

Aragonite. J. Biol. Chem. 2014, 289, 2776-2787.

593

(34) Yano, M.; Nagai, K.; Morimoto, K.; Miyamoto, H., Shematrin: A family of glycine-rich

594

structural proteins in the shell of the pearl oyster Pinctada fucata. Comp. Biochem. Phys. B.

595

2006, 144, 254-262.

596

(35) Su, J. T.; Zhu, F. J.; Zhang, G. Y.; Wang, H. Z.; Xie, L. P.; Zhang, R. Q., Transformation of

597

amorphous calcium

598

Crystengcomm 2016, 18, 2125-2134.

599

(36) Su, J.; Liang, X.; Zhou, Q.; Zhang, G.; Wang, H.; Xie, L.; Zhang, R., Structural

600

characterization of amorphous calcium carbonate-binding protein: an insight into the

601

mechanism of amorphous calcium carbonate formation (vol 453, pg 179, 2013). Biochem. J.

602

2013, 454, 167-167.

603

(37) Bahn, S. Y.; Jo, B. H.; Hwang, B. H.; Choi, Y. S.; Cha, H. J., Role of Pif97 in Nacre

604

Biomineralization: In Vitro Characterization of Recombinant Pif97 as a Framework Protein for

605

the Association of Organic-Inorganic Layers in Nacre. Cryst. Growth Des. 2015, 15,

carbonate nanoparticles into aragonite

ACS Paragon Plus Environment

controlled by

ACCBP.

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

606

3666-3673.

607

(38) Bahn, S. Y.; Jo, B. H.; Choi, Y. S.; Cha, H. J., Control of nacre biomineralization by Pif80

608

in pearl oyster. Sci. Adv. 2017, 3.

609

(39) Marie, B.; Joubert, C.; Tayale, A.; Zanella-Cleon, I.; Belliard, C.; Piquemal, D.;

610

Cochennec-Laureau, N.; Marin, F.; Gueguen, Y.; Montagnani, C., Different secretory

611

repertoires control the biomineralization processes of prism and nacre deposition of the pearl

612

oyster shell. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 20986-20991.

613

(40) Liu, C.; Li, S.; Kong, J.; Liu, Y.; Wang, T.; Xie, L.; Zhang, R., In-depth proteomic analysis

614

of shell matrix proteins of Pinctada fucata. Sci. Rep. 2015, 5.

615

(41) Radonic, A.; Thulke, S.; Mackay, I. M.; Landt, O.; Siegert, W.; Nitsche, A., Guideline to

616

reference gene selection for quantitative real-time PCR. Biochem. Bioph. Res. Co. 2004, 313,

617

856-862.

618

(42) Koga, N.; Nakagoe, Y. Z.; Tanaka, H., Crystallization of amorphous calcium carbonate.

619

Thermochim. Acta 1998, 318, 239-244.

620

(43) Xu, G. F.; Yao, N.; Aksay, I. A.; Groves, J. T., Biomimetic synthesis of macroscopic-scale

621

calcium carbonate thin films. Evidence for a multistep assembly process. J. Am. Chem. Soc.

622

1998, 120, 11977-11985.

623

(44) Liu, C.; Du, J.; Xie, L.; Zhang, R., Direct Observation of Nacre Proteins in the Whole

624

Calcite by Super resolution Microscopy Reveals Diverse Occlusion Patterns. Cryst. Growth

625

Des. 2017, 17, 1966-1976.

626

(45) Liu, C.; Xie, L.; Zhang, R., Heterogeneous distribution of dye-labelled biomineralizaiton

627

proteins in calcite crystals. Sci. Rep. 2015, 5.

ACS Paragon Plus Environment

Page 30 of 33

Page 31 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

Crystal Growth & Design

628

(46) Rust, M. J.; Bates, M.; Zhuang, X., Sub-diffraction-limit imaging by stochastic optical

629

reconstruction microscopy (STORM). Nat. Methods 2006, 3, 793-795.

630

(47) Huang, B.; Jones, S. A.; Brandenburg, B.; Zhuang, X., Whole-cell 3D STORM reveals

631

interactions between cellular structures with nanometer-scale resolution. Nat. Methods 2008, 5,

632

1047-1052.

633

(48) Suzuki, M.; Murayama, E.; Inoue, H.; Ozaki, N.; Tohse, H.; Kogure, T.; Nagasawa, H.,

634

Characterization of Prismalin-14, a novel matrix protein from the prismatic layer of the

635

Japanese pearl oyster (Pinctada fucata). Biochem. J. 2004, 382, 205-213.

636

(49) Weiner, S.; Traub, W., Macromolecules in mollusk shells and their functions in

637

biomineralization. Philos. T. Roy. Soc. B. 1984, 304, 425-&.

638

(50) Yan, Y.; Yang, D.; Yang, X.; Liu, C.; Xie, J.; Zheng, G.; Xie, L.; Zhang, R., A Novel Matrix

639

Protein, PfY2, Functions as a Crucial Macromolecule during Shell Formation. Sci. Rep. 2017,

640

7.

641

(51) Weiner, S.; Mahamid, J.; Politi, Y.; Yurong, M.; Addadi, L., Overview of the amorphous

642

precursor phase strategy in biomineralization. Front. Mater. Sci. 2009, 3, 104-8.

643

(52) Wang, D.; Wallace, A. F.; De Yoreo, J. J.; Dove, P. M., Carboxylated molecules regulate

644

magnesium content of amorphous calcium carbonates during calcification. Proc. Natl. Acad.

645

Sci. U. S. A. 2009, 106, 21511-21516.

646

(53) Bischoff, W. D.; Mackenzie, F. T.; Bishop, F. C., Stabilities of synthetic magnesian

647

calcites in aqueous-solution-comparison with biogenic materials. Geochim. Cosmochim. Ac.

648

1987, 51, 1413-1423.

649

(54) Bisker-Leib, V.; Doherty, M. F., Modeling crystal shape of polar organic materials:

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

650

Applications to amino acids. Cryst. Growth Des. 2003, 3, 221-237.

651

(55) Wada, N.; Horiuchi, N.; Nakamura, M.; Nozaki, K.; Nagai, A.; Yamashita, K., Calcite

652

Crystallization on Polarized Single Calcite Crystal Substrates in the Presence of Poly-Lysine.

653

Cryst. Growth Des. 2018, 18, 872-878.

654

(56) Colfen, H.; Antonietti, M., Mesocrystals: Inorganic superstructures made by highly parallel

655

crystallization and controlled alignment. Angew. Chem.-Int. Edit. 2005, 44, 5576-5591.

656

(57) Ristic, R. I.; DeYoreo, J. J.; Chew, C. M., Does impurity-induced step-bunching invalidate

657

key assumptions of the Cabrera - Vermilyea model? Cryst. Growth Des. 2008, 8, 1119-1122.

658

(58) Weaver, M. L.; Qiu, S. R.; Hoyer, J. R.; Casey, W. H.; Nancollas, G. H.; De Yoreo, J. J.,

659

Inhibition of calcium oxalate monohydrate growth by citrate and the effect of the background

660

electrolyte. J. Cryst. Growth 2007, 306, 135-145.

661

(59) Xie, J.; Liang, J.; Sun, J.; Gao, J.; Zhang, S.; Liu, Y.; Xie, L.; Zhang, R., Influence of the

662

Extrapallial Fluid of Pinctada fucata on the Crystallization of Calcium Carbonate and Shell

663

Biomineralization. Cryst. Growth Des. 2016, 16, 672-680.

664

(60) Jiang, Z.; Li, Y.; Wang, M.; Song, B.; Wang, K.; Sun, M.; Liu, D.; Li, X.; Yuan, J.; Chen, M.;

665

Guo, Y.; Yang, X.; Zhang, T.; Moorefield, C. N.; Newkome, G. R.; Xu, B.; Li, X.; Wang, P.,

666

Self-assembly of a supramolecular hexagram and a supramolecular pentagram. Nat. Commun.

667

2017, 8.

668

(61) Voorhees, P. W., The theory of Ostwald Ripening. J. Stat. Phys. 1985, 38, 231-252.

669

(62) De Yoreo, J. J.; Gilbert, P.; Sommerdijk, N.; Penn, R. L.; Whitelam, S.; Joester, D.; Zhang,

670

H. Z.; Rimer, J. D.; Navrotsky, A.; Banfield, J. F.; Wallace, A. F.; Michel, F. M.; Meldrum, F. C.;

671

Colfen, H.; Dove, P. M., Crystallization by particle attachment in synthetic, biogenic, and

ACS Paragon Plus Environment

Page 32 of 33

Page 33 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

Crystal Growth & Design

672

geologic environments. Science 2015, 349.

673

(63) Gotliv, B. A.; Kessler, N.; Sumerel, J. L.; Morse, D. E.; Tuross, N.; Addadi, L.; Weiner, S.,

674

Asprich: A novel aspartic acid-rich protein family from the prismatic shell matrix of the bivalve

675

Atrina rigida. Chembiochem 2005, 6, 304-314.

676

(64) Politi, Y.; Mahamid, J.; Goldberg, H.; Weiner, S.; Addadi, L., Asprich mollusk shell protein:

677

in vitro experiments aimed at elucidating function in CaCO3 crystallization. Crystengcomm

678

2007, 9, 1171-1177.

679

(65) Han, G.; Chow, P. S.; Tan, R. B. H., Direct Comparison of alpha- and gamma-Glycine

680

Growth Rates in Acidic and Basic Solutions: New Insights into Glycine Polymorphism. Cryst.

681

Growth Des. 2012, 12, 2213-2220.

682

(66) Zhong, C.; Chu, C. C., On the Origin of Amorphous Cores in Biomimetic CaCO3

683

Spherulites New Insights into Spherulitic Crystallization. Cryst. Growth Des. 2010, 10,

684

5043-5049.

685

(67) Krebs, M. R. H.; Bromley, E. H. C.; Rogers, S. S.; Donald, A. M., The mechanism of

686

amyloid spherulite formation by bovine insulin. Biophys. J. 2005, 88, 2013-2021.

687

ACS Paragon Plus Environment