glycolytic intermediates induce amorphous calcium ......in vitro crystalization of calcium carbonate...

Glycolytic Intermediates Induce Amorphous Calcium

Carbonate Formation in Crustaceans

Ai Sato1§, Seiji Nagasaka1, 2§, Kazuo Furihata1, Shinji Nagata1, Isao Arai3, 4,

Kazuko Saruwatari 3, 5, Toshihiro Kogure3, Shohei Sakuda1 and Hiromichi

Nagasawa1*

1Department of Applied Biological Chemistry, Graduate School of Agricultural and Life

Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan

2Graduate School of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi,

Oura-gun, Gunma 374-0193, Japan

3Department of Earth and Planetary Science, Graduate School of Science, The

University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan

4Central Research Institute, Mitsubishi Materials Corporation, 1002-14 Mukohyama,

Naka-shi, Ibaraki 311-0102, Japan

5National Institute for Materials Science International Center for Materials

Nanoarchitectonics Softchemistry Group, 1-1 Namiki, Tsukuba-shi, Ibaraki 305-0044,

Japan

Nature Chemical Biology: doi:10.1038/nchembio.532

Supplementary Methods

Materials

The freshwater crayfish Procambarus clarkii were purchased from a local dealer. They

were maintained in indoor aquaria at room temperature and provided with artificial

pellets every day. In this study, crayfish at the premolt stage were obtained artificially

by bilateral eyestalk ablation at the intermolt stage, or by injection of

20-hydroxyecdysone (5 μg) once a day for 3 days. Exoskeleton at the intermolt stage

and gastroliths at the premolt stage were dissected out, treated with 2% Triton X-100

(Nacalai tesque, Kyoto, Japan) for 10 min to remove tissues, washed in milli-Q water

several times, and stored at -20 °C until use. Animals were anesthetized on ice prior to

dissection.

Elemental analysis of exoskeleton and gastroliths

About 200 mg of exoskeleton or gastroliths were decomposed in concentrated nitric

acid (150 °C, 4 h, Wako, Tokyo, Japan). The resultant solution was analyzed on an

inductively coupled plasma atomic emission spectrometry (ICP-AES, model

SPS-1200VR, SEIKO, Chiba, Japan) for quantification of calcium and phosphorus.

In vitro Crystalization of calcium carbonate

The pH of the NaHCO3 solution (0.1 M) containing phosphate, PEP, 3PG, citrate or

compounds extracted from gastroliths was adjusted to 8.5 by addition of 1 M NaOH or

1 M HCl solution. Each solution (2.0 ml) was added to 2.0 ml of 0.1 M CaCl2 and the

mixture was stirred for 1 min. The resulting supersaturated solution was left for 1 or 7

days at room temperature. Then the precipitates formed were obtained by filtration with


a glass filter with a pore size of 0.3 μm and washed with deionized water. The glass

filter holding the precipitates was dried in a vacuum desiccator at room temperature.

Extraction and preparation of organic matrices and their separation by

ultrafiltration

Gastroliths were ground to powder in liquid nitrogen. The powder was dissolved in 1 M

acetic acid at a ratio of 1 g to 20 ml at 4°C for 3 days. The solution was centrifuged

(3500 x g, 20 min, 4°C), and the supernatant was collected. It was separated into two

fractions, low- and high-molecular-weight fractions, by ultrafiltration (Amicon Ultra,

MWCO 10 k, Millipore, MA, USA). The solvent of the high-molecular-weight fraction

was substituted by 50 mM Tris-HCl (pH 7.5). Calcium ions were removed from the

low-molecular-weight fraction by passing through a column of Dowex MB 50 (H+)

(Dow Chemical Company, Michigan, USA). The flow-through fraction was lyophilized

and dissolved in milli-Q water.

SEM

The glass filter holding calcium carbonate precipitates was mounted on carbon

double-sided tape attached to aluminum stubs and coated with Pt-Pd. SEM images were

obtained on an S-4000 scanning electron microscope (HITACHI, Tokyo, Japan).

XRD

The X-ray diffraction of calcium carbonate precipitates formed in vitro was measured

on a RINT2100 X-ray diffractometer (Rigaku, Tokyo, Japan) with Cu-Kα radiation at

40 kV and 20 mA. The glass filter holding precipitates was set on a glass holder, and the


diffraction was measured. The crystal polymorph of the precipitates was identified by

comparing its X-ray diffraction pattern with that of each authentic crystal polymorph.

Extraction and partial purification of phosphorus compounds

Exoskeleton and gastroliths were separately ground to powder in liquid nitrogen. Each

powder was dissolved in 1 M acetic acid at a ratio of 200 mg to 4 ml at room

temperature for 24 h. Calcium ions were removed from the soluble fractions by passing

through a column of Dowex MB 50 (H+) (Dow Chemical Company, Michigan, USA).

The flow-through fraction was lyophilized and used as partially purified preparations.

NMR measurement

The partially purified preparations were dissolved in D2O (Merck, New Jersey, USA).

One- and two-dimensional NMR spectra were measured at 20°C on a JMN-A500

spectrometer (500 MHz for 1H, 125.65 MHz for 13C, 202 MHz for 31P, 500 MHz for

HSQC, 500 MHz for HMBC, JEOL, Tokyo, Japan). The 31P, 1H-HMBC spectra were

measured with 512 points in f2, 256 points in f1, a 3000.30 Hz spectral width in f2, a

5000.00 Hz spectral width in f1. The 13C, 1H-HSQC spectra were measured with 512

points in f2, 256 points in f1, a 2980.63 Hz spectral width in f2, a 25062.66 Hz spectral

width in f1. The 13C, 1H-HMBC spectra were measured with 512 points in f2, 512 points

in f1, a 3038.59 Hz spectral width in f2, a 22644.93 Hz spectral width in f1. As an internal

standard compound, 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid (TSP) (0 ppm, Aldrich,

Missouri, USA) was used for 1H-NMR. As external standard compounds, 1,4-dioxane

(67.4 ppm) for 13C-NMR and 70 % phosphoric acid (0 ppm) for 31P-NMR were used.


Mass spectral analysis

Mass spectra were measured on a time-of-flight mass spectrometer JMS-T100LC

AccuTOF (JEOL, Tokyo, Japan) equipped with an electrospray ionization source in the

negative ion mode. The partially purified preparations were dissolved in Milli-Q water

and applied to the mass spectrometer. The ion-source temperature was 250°C. The mass

analyzer was scanned from m/z 110 to 195 for the full scan analysis.

Quantitative analysis of organic compounds by NMR

The partially purified preparations were dissolved in D2O containing 1.67 mM TSP.

Quantitative analysis was performed by calculating the relative integration ratio of the

characteristic signal of the target organic compounds to that of a known amount of the

internal standard in a 1H-NMR spectrum. Signal integrations obtained from the methyl

proton signal (0 ppm) of TSP, the methylene proton signal (5.5 ppm) of PEP (Wako,

Tokyo, Japan), the methyne proton signal (4.5 ppm) of 3PG (Sigma, Missouri, USA)

and the methylene proton signal (3.0 ppm) of citrate were measured. The amount of

phosphate was calculated by subtracting the amounts of PEP and 3PG from that of total

amount of phosphorus compounds.

The ratio of phosphorus to calcium in the precipitates formed in vitro

About 1 mg of the precipitates obtained from the in vitro crystallization experiment was

dissolved in 1 M acetic acid. Then elemental analyses of the solution were carried out

on ICP-AES. The ratio of phosphorus to calcium was calculated from the data obtained.

RNA isolation and RT-qPCR


Total RNA was isolated from each of the gastrolith disk epithelia and the other part of

the stomach by using ISOGEN (NIPPON GENE, Tokyo, Japan) according to the

manufacture’s instruction, and digested with DNase I (Takara Bio, Shiga, Japan). cDNA

was prepared from 3 μg of RNA using Rever Tra Ace (TOYOBO, Osaka, Japan) and

oligo-(dT) according to the manufacture’s instruction. cDNA (12.5 ng) in 5 μl were

used in a 25 μl PCR reaction (FastStart Universal SYBR Green Master: Roche

Molecular Biochemicals, Indianapolis, IN, USA). The PCR reaction products were

quantified in a 7300 Real-Time PCR System (Applied Biosystems). The sequences of

primers used in this study are as follows: forward,

5’-AACCAACCCTGTGGCTATGAA-3’; reverse,

5’-CTTGGCAACGTTGGTGAAGA-3’ for crayfish phosphoenolpyruvate carboxy

kinase (PEPCK); forward, 5’-GTTGGAGATGAGGGAGGTTTTG-3’; reverse,

5’-GGATAAGGTTGAGGGCATCCT-3’ for crayfish enolase; forward,

5’-CATGGCCTTCCGTGTTCCT-3’; reverse, 5’-CCAAGGCGGACAGTCAAATC-3’

for crayfish glyceraldehydes-3-phosphate dehydrogenase (GAPDH); forward,

5’-GGACAGAAAGTGAAGGCAGAT-3’; reverse,

5’-ATCCTGAGCAGCCAACATAGC-3’ for crayfish ribosomal protein.

PEPCK, enolase and GAPDH signals were normalized to ribosomal protein signals

determined in parallel for each sample.

Preparation of intracellular metabolite

Intracellular metabolites were prepared from the gastrolith disk epithelia and the other

part of the stomach by treatment with a chloroform/methanol solution using a vortex,

followed by ultrafiltration of the aqueous layer with a 5-kD cut off filter (Millipore, MA,


USA).

CE/MS analysis

DB-WAX capillary (50 μm i.d. x 80 cm, Agilent, CA, USA) was used for separation of

organic acids. Ammonium acetate (50 mM) was used as a running buffer. Each sample

(30 nl) was injected to the capillary (50 mbar, 30 seconds). The electrophoresis was

operated under the -30 kV voltage and 50 mbar pressure. ESI-MS was operated in the

negative ion mode. The concentration of each organic acid in samples was quantified by

comparing the peak areas of the organic acids in the standard solution with those in

samples. Piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) was used as an internal

standard.

In vitro incubation of gastrolith disk epithelia

A pair of gastrolith disk epithelia were rinsed with crayfish phosphate buffered saline

which was prepared according to Lin et al. (2008). One of the paired gastrolith disk

epithelia was preincubated in 0.7 ml of the L-15 medium (Sigma-Aldrich, Missouri,

USA) at room temperature for 30 minutes. Then it was incubated in a new 0.7 ml L-15

medium (5 μM mercaptoethanol, 1 μM phenylthiourea, 60 μg/ml penicillin, 50 μg/ml

streptomycin, 50 μg/ml gentamicin) in a 24-well plate at room temperature for 30 h.

After incubation, PIPES was added to the medium as an internal standard. This medium

was ultrafiltered (MWCO 5 k, Millipore, MA, USA) and lyophilized.

Statistical analysis

One-way ANOVA tests were used for analysis of differences between various samples.


P values <0.05 are considered significant.

Supplementary References

Lin, X., Söderhäll, K. & Söderhäll, I. Transglutaminase activity in the hematopoietic

tissue of a crustacean, Pacifastacus leniusculus, importance in hemocyte homeostasis.

BMC Immunology 9, 58 (2008)


Calcium (mg/g) Phosphorus (mg/g)

192.75

301.75

Mean S.E.M.

2.71

5.83

Mean

6.15

9.04

S.E.M.

0.35

1.07

Supplementary Table 1. The concentrations of phosphorus and calcium in the exoskeleton and gastroliths

Exoskeleton(N = 10)

Gastrolith(N = 12)

Mean S.E.M.

P/Ca (mol/mol)

0.041

0.041

0.002

0.017

Eggshell(Calcite, N = 5)

371.60 9.54 0.57 0.02 0.002 0.000

Supplementary Results


PEP

3PG

Citrate

δP

-4.5

0.1

δH

4.45

4.15

2.85

δC

167.18

145.15

109.92

175.88

71.08

67.77

174.85

44.39

74.45

178.50

Supplementary Table 2. Chemical shifts of 13C, 1H, and 31P of PEP, 3PG and citrate.

C-No.

1

2

3

1'

2'

3'

1''

2''

3''

4''

5''

6''

5.86b

3.03

5.50a

44.39

174.85

2.85 3.03


Stomach

Gastroliths

Exoskeleton

Supplementary figure 1. The exoskeleton and gastroliths of P. clarkii.Prior to molting, a pair of gastroliths is formed at the anterior part of the stomach. Scale bar in the image corresponds to 1 cm.


20 25 30 35 40 45 50

a b

Supplementary figure 2. In vitro precipitation of calcium carbonate in the absence of high- or low-molecular-weight compounds.a, An SEM image of the calcium carbonate precipitated in the absence of high- or low-molecular-weight compounds. The typical rhombohedral shapes of calcite were observed. Scale bar corresponds to 10 µm. b, An XRD pattern of the precipitates in the absence of high- or low-molecular-weight compounds. Calcite-specific signals were detected.

Inte

nsity

2θ (deg.) CuKα


20 25 30 35 40 45 502θ (deg.) CuKα

C

C

C C C CC

Inte

nsity

20 25 30 35 40 45 502θ (deg.) CuKα

Inte

nsity

Low-molecular-weight compounds

High-molecular-weight compounds

Supplementary figure 3. ACC spherules formed in the presence of low-molecular-weight compoundsextracted from gastroliths.SEM images and XRD patterns of the calcium carbonate precipitated in the solution containing low- (50 mg) or high-molecular-weight compounds extracted from gastroliths (500 mg). Calcite-specific peaks are represented by "C". Scale bars in the images correspond to 1 µm.


Supplementary figure 4. The effects of high- or low-molecular-weight compounds extracted from gastroliths on the formation of calcium carbonate in vitro.SEM images of the calcium carbonate precipitated in the solution containing low-molecular-weight compounds extracted from 5 mg (a) or 50 mg (b) of gastroliths, high-molecular-weight compounds extracted from 5 mg (c), 50 mg (d) or 500 mg (e) of gastroliths, and 50 mM Tris-HCl (pH 7.5, control) (f). ACC spherules were observed in the presence of low-molecular-weight compounds, whereas rhombohedral crystals were observed in the presence of high-molecular-weight compounds and in control experiment. The right panels are magnified images of the white flames in the corresponding left panels. Scale bars correspond to 6 µm (left panels) or 1 µm (right panels).

d

f

e

c

a

b


5.0 0.0 -5.0Chemical shift (ppm)

Gastroliths

Exoskeleton


-10.0

-10.0

Supplementary figure 5. 31P-NMR (D2O, 202 MHz) spectra of the crude extracts from gastroliths and the exoskeleton. Chemical shifts of the 31P-NMR spectra are dependent on pH of the solution.




Gastroliths

Exoskeleton

-10.0

-10.0

31P-NMR

Supplementary figure 6. 31

P-NMR (D2O, 202 MHz) spectra of the partially purified fractions from gastroliths and the exoskeleton.


6.0

5.9

5.8

5.7

5.6

5 5

5.4

5.3

140.0150.0160.0170.06.0

5.9

5.8

5.7

5.6

5.4

5.3

5 5

115.0 110.0

5.3

5.4

5.5

5.6

5.7

5.8

5.9

6.0-4.0 -5.0

3.8

3.9

4.0

4.1

4.2

4.3

4.4

4.5050.090.0130.0170.0

3.8

3.9

4.0

4.1

4.2

4.3

4.4

3.8

3.9

4.0

4.1

4.2

4.3

4.4

4.570.0 65.075.0

2.6

2.7

2.8

2.9

3 0

3.1

3.2

3.330.070.0110.0150.0190.0

2.6

2.7

2.8

2.9

3.0

3.1

3.2

3.340.045.050.0

13C (ppm)

1H (ppm

)

31P (ppm)

1H (ppm

)

1H (ppm

)

31P (ppm)

13C (ppm)

13C (ppm)13C (ppm)

13C (ppm)13C (ppm)

4.5-1.01.0

13C, 1H-HMBC 13C, 1H-HSQC 31P, 1H-HMBC

H-3a

H-3b

C2C1C3

H-3'

H-2'

C2'C1'

C2' C3'

P

P

H-2''H-4''

C1''C5''

C6''C3''

C2''C4''

C2''C4''

a b3

2

1

1' 2'

3'

1'

2'' 3'' 4

5''

6'

105.0

HOOCC

OPO3H2

CHH

HOOCH2C C

OH

COOHCH2

COOH

Supplementary figure 7. Two-dimensional NMR spectra (HMBC, HSQC) of partially purified samples from the exoskeleton and gastroliths.1H spectra are displayed along the horizontal axes. 13C spectra or 31P spectra are displayed along the vertical axes. PEP, 3PG and citrate were identified as major components of the partially purified samples by a combination of one-dimensional NMR and two-dimensional NMR spectral analyses.

HOOC COH

H2C OPO3H2H


120 140 160 180m/z

167.0

185.0

191.0

120 140 160 180m/z

167.0

185.0

191.0

50

40

30

20

10

0

Inte

nsity

x 1

03

50

40

30

20

10

0

Inte

nsity

x 1

03

Gastroliths

Exoskeleton

Supplementary figure 8. Mass spectra of partially purified samples from the exoskeleton and gastroliths. (M-H)- ion peaks for PEP, 3PG and citrate were observed at m/z 167.0, 185.0 and 191.0, respectively.The existence of PEP, 3PG and citrate were confirmed.


20 25 30 35 40 45 50

5 mM

2.5 mM

1 mMPhosphate

20 25 30 35 40 45 50

5 mM

2.5 mM

1 mM

Citrate

20 25 30 35 40 45 50

5 mM

2.5 mM

1 mM

0.5 mM 3PG

20 25 30 35 40 45 50

5 mM

2.5 mM

1 mM

0.5 mM PEP2θ (deg.) CuKα

2θ (deg.) CuKα

2θ (deg.) CuKα

2θ (deg.) CuKα

Supplementary figure 9. XRD spectra of the calcium carbonate precipitates.A CaCl2 solution (0.1 M, 2.0 ml) was added to a NaHCO3 solution (0.1 M, 2.0 ml) containing phosphate, PEP, 3PG or citrate, and the mixture was left for 1 day at room temperature. Calcium carbonate precipitated in the solution was obtained by filtration.


Supplementary figure 10. The stability of the ACC formed in the presence of phosphate, PEP or 3PG.A CaCl2 solution (0.1 M, 2.0 ml) was added to a NaHCO3 solution (0.1 M, 2.0 ml) containing phosphate (5.0 mM), PEP (2.0 mM) or 3PG (2.0 mM) and the mixture was left for 5 days at room temperature. Calcium carbonate precipitated in the solution was obtained by filtration.

20 25 30 35 40 45 50

Phosphate

3PG

PEP

2θ (deg.) CuKα


b

7.0 6.0 5.0 4.0 3.0 2.0 1.0

190 160 130 100 70 40 10

1H-NMR

13C-NMR

Exoskeleton(M. japonicus)

PEP

3PG

Citrate

PEP

3PG

Citrate


Exoskeleton(E. japonica)

Exoskeleton(E. japonica)


-10.010.0


-10.010.0

aExoskeleton(E. japonica)


H-3b H-3a

H-2'H-3'

H-2''H-4''

C1 C2C3

C1' C2'C3'

C6''

C1''C5'' C3''

C2''C4''

PEP

3PG

Citrate

1 2

3a b

HOOCC

OPO3H2

CHH

Supplementary Figure 11. The existence of 3PG and/or PEP in the exoskeleton of other crustacean species.a, 31P-NMR (D2O, 202 MHz) spectra of partially purified exoskeleton fractions.b, 1H-NMR (D2O, 500 MHz) and 13C-NMR (D2O, 125.65 MHz) spectra of partially purified exoskeleton fractions and those of authentic PEP, 3PG and citrate. These results showed that 3PG and/or PEP are contained in the exoskeleton of the kuruma prawn Marsupenaeus japonicus and the mitten crab Eriocheir japonica.

1'

2'

3'

1''

2'' 3'' 4''

5''

6''

HOOCH2C C

OH

COOHCH2

COOH

Chemical shift (ppm)

Chemical shift (ppm) HOOC C

OH

H2C OPO3H2H


3PG

n = 4n = 4 n = 7 n = 4 0.00

0.10

0.20

0.30

0.40

0.50

0.60

n = 4n = 4 n = 7 n = 4 0.00

0.05

0.10

0.15

0.20

0.25

n = 4n = 4 n = 7 n = 4 0.0000

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

n = 4n = 4 n = 7 n = 4 0.00

0.10

0.20

0.30

0.40

n = 4n = 4 n = 7 n = 4 0.00

0.05

0.10

0.15

0.20

0.25

Intr

acel

lula

r co

ncen

trat

ion

(nmol/mg) Pyruvate Citrate

Intermolt stage Premolt stage

Intr

acel

lula

r co

ncen

trat

ion

Intr

acel

lula

r co

ncen

trat

ion

Intr

acel

lula

r co

ncen

trat

ion

Intr

acel

lula

r co

ncen

trat

ion

(nmol/mg) (nmol/mg)

(nmol/mg)(nmol/mg)

Intermolt stage Intermolt stage

Intermolt stage Intermolt stage

Premolt stage Premolt stage

Premolt stage Premolt stage

2-oxoglutarate Malate

Supplementary figure 12. Quantification of some metabolite in the gastrolith disk epithelia and the other part of the stomach.Some metabolites in the gastrolith disk epithelia or the other part of the stomach were quantified by CE-MS. These results indicate that carbohydrate metabolism in the epithelial cells changes at the premolt and postmolt stages. Data represent the amounts of metabolites in the glycolytic pathway and the TCA cycle normalized to fresh weight and internal standard level Error bars represent S.E.M.


Gastrolithepithelial disks

Stomach

Intermolt stage

Premolt stage

5

10

15

20

25

30

Rel

ativ

e tr

ansc

riptio

n le

vel

0

0

10

20

30

40

50

60

Rel

ativ

e tr

ansc

riptio

n le

vel

0.00.20.40.60.81.01.21.41.6

Rel

ativ

e tr

ansc

riptio

n le

vel

∗∗

∗∗

∗∗

PEPCK

GAPDH

enolase

Supplementary figure 13. Expression levels of each of phosphoenolpyruvate carboxykinase(PEPCK), enolase and glyceraldehyde-3-phosphte dehydrogenase (GAPDH) relative to that of ribosomal protein.Data represent the mean + s.d. (N = 7).


glycolytic intermediates induce amorphous calcium ......in vitro crystalization of calcium carbonate...

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