Occurrence of lysinoalanine in calcified tissue collagen

Vol. 103, No. 4,198l December

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages 1378-1383

31, 1981

OCCURRENCEOF LYSINOALANINE Daisaburo

Fujimoto,

IN CALCIFIED

Masahiro

Hirama*

TISSUE COLLAGEN

and Takashi

Iwashita*

Department of Chemistry, Hamamatsu University School of Medicine, Hamamatsu 431-31 and *Suntory Institute for Bioorganic Research, Osaka 618, Japan Received November 10, 1981 Summary: Lysinoalanine was identified in hydrolysates of dentine and bone collagen. The compound was isolated and purified by ion exchange chromatography on P-cellulose and QAE-Senhadex columns. Identity with lysinoalanine was demonstrated by IH-nmr spectroand paper chromatography. This is the scopy, amino acid analysis first example of occurrence of lysinoalanine in native proteins. Lysinoalanine linking

amino acid

see ref.1).

This

and cystine treatments formation proteins

(2).

amino acid or lysine

is

proteins

is thought

(for

to be formed

and serine

residues,

a cross-

a review,

from lysine and severe

in 0.2M NaoH at 40°C are needed for

Lysinoalanine

has never

been discovered

the

in native

(1). in calcified

tissues

and swelling, We will

hydrolysates

Materials

in alkali-treated

such as heating

to solubilization crosslinked.

found

residues

Collagen

acid

[NE-(2-amino-2-carboxyethyl)-lysine]

report

of native

is known to be highly

suggesting here

that

the presence

collaqens

it

is

resistant

highly

of

lysinoalanine

from dentine

and bone.

in

and Methods

Human teeth were obtained at oral surgery. Enamel and cementumwere removed from dentine by using a dental bur. Bones were obtained from freshly killed animals. Bone and dentine samples were crushed mechanically, washed with 0.5M NaCl and then decalcified in 0.5M EDTA, pH7.5, at 4Oc for several days. In some experiments, whole teeth were used instead of dentine and decalcified in 0.6M HCl at room temnerature for 48 hr. Essentially the same results were obtained. The demineralized sample (about 500 mq) was hydrolyzed in 6M HCl (30 ml) at llO°C for 24 hr in a sealed tube. The solution was 0006-291X/81/241378-06$01.00/0 Copyrighr 0 1981 by Academic Press, Inc. All righrs of reproduction it1 any/arm reserved.

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evaporated under reduced pressure and the hydrolysate was taken up in water and applied to a P-cellulose (Brown) column (H+form, 1.8 x 20 cm). Elution was performed with a linear gradient formed from 250 ml of water and 250 ml of 0.35M HCl. Fractions (10 ml each) were collected and absorbance at 295 nm was measured to detect pyridinoline (3,4). An aliquot of each fraction was assayed for amino acids by trinitrobenzene sulfonate The peak eluted between bulk amino acids and method (5). pyridinoline (peak A in Fig.1) was collected. The peak fractions obtained in several separate runs were combined, evaporated to dryness and taken up in 10 ml of water. The pH of the solution was adjusted to 9.2 and then the solution was applied to a QAESephadex A25 (Pharmacia) column (1.2 x 15 cm) which had been equilibrated with 5 mM Na2B407. The elution was performed with a linear gradient formed from 150 ml of 5 mM Na2B407 and 150 ml of 100 mM Na2B407. Fractions (7.5 ml each) were collected and aliquots were assayed for amino acids (5). The fractions containing amino acids were pooled, concentrated and desalted on a Biogel P-2 (Bio-Rad) column in 5 mM HCl. Standard lysinoalanine was synthesized by a modification of the method of Bohak (2). N-a-acetyllysine (Sigma, 38 mg), 2acetamidoacrylic acid (Nakarai, 26 mg) and NaOH (16 mg) were mixed in 2 ml water and the solution was kept at 60°C for 48 hr. Then, 2 ml of conc.HCl was added and the mixture was heated at llO°C for 16 hr. Lysinoalanine formed was isolated by P-cellulose chromatography as described above. The 'H-nmr spectra were measured in D20 on a Nicolet NT-360 spectrometer and all chemical shifts were reported in parts per million (6) downfield from TSP [sodium 2,2,3,3-tetradeutero-3(trimethylsilyl)propionatel as external standard. Amino acid analysis was performed on a Hitachi 835-50 amino acid anlyzer using a 2.6 x 250 mm column.

Results Fig.1

shows the chromatographic

of human dentine

0

10

collagen

20

FRACTION

30

pattern

of the

on the P-cellulose

40

50

column.

10

NO.

0

hydrolysate

20

The ultra-

30

FRACTION

40 NO.

Fig.1 P-cellulose chromatography of the hydrolysate of human dentine collagen. Details of the procedure are described in the o-o: Absorbance at 420 nm after the reaction with text. trinitrobenzene sulfonate (5), m-----e: Absorbance at 295 run. Fig,2 QA?+Sephadex chromatography of peak A. procedure are described in the text.

1379

Details

of the

Vol. 103. No. 4,198l

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AND

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ELUTION

3 Amino acid analysis %%O nm is shown.

violet

absorbing

pyridinium peak

peak

crosslink

fractionated

named pyridinoline

components

(Fig.3).

The elution of

identical

mobilities

lH-nmr (quintet, &7.6 Hz,

J=7.6 H-6),

& 5.8 Hz,

H-3'),

Hz, H-2),

4.17

with

of

that

acid-water,

2 x H-5), (dt,

3.56 (dd,

II)

As shown in Fig.2,

were

obtained.

to be exactly

chromatography

J=8.8

lysinoalanine

4:1:2

analyzer

the

same

also

the

have

solvent

(Rf = O.ll),

n-

(Rf = 0.16)

and

(Rf = 0.65). [61.55

2.00

J=13.0

(m, 2 x H-41,

(m, 2 x H-3),

5=13-O & 7.6 Hz,

(dd,

with

15:10:3:12

of compound I

3.23

peak was further

was found

in NH3 vapour

Hz,

and

peak on the amino acid

acid-water,

spectrum

amino acids

chromatography.

on paper

butanol-pyridine-acetic 4:1,

An amino acid

Compound I and lysinoalanine

n-butanol-acetic

phenol-water,

in this

position

lysinoalanine.

systems of

bulk

as a single

as 3-hydroxy-

(3,4).

(compound I and

Compound I eluted

as that

between

by QAE-Sephadex

two major

The absorbance profile

(peak B) has been identified

The amino acids

COMMUNICATIONS

TIME(min)

of compound I.

(peak A) was detected

pyridinoline.

RESEARCH

H-6),

3.20 3.51

& 8.8 Hz, H-3'),

& 5.8 Hz, H-2')] as shown in Fig.

1380

was also 4.

1.82 (dt,

(dd,

4.05

J=13.0 J=13.0

(t,

identical

J=6.3

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Lysinoalanine

HOGi--:“-& ,-rH,-&,-Sn,-

N-&l*-& - &on H

AH,

Fig.4

lli-nmr spectra

From these

data,

it

I;H?

of compound I and lysinoalanine.

can be concluded

that

compound

I is

lysinoalanine. The content

of

lysinoalanine

estimated

by amino

acid

cellulose

chromatography.

in various

analysis

of

The results

Table 1. Content of lysinoalanine Tissue

the

peak

collagens

was

A isolated

by P-

are shown in Table

in collagens

of various

1.

tissues

Residues of leucine equivalents per 1000 total residues*

Ruman(adult) dentine Guinea pigtadult) rib Hen(l.5 year-old) tibia Chick(6 week-old) tibia Bovine(adult) Achilles tendon

0.16 0.021 0.027 0.003 less than 0.001

*Determined by amino acid analysis chromatography.

of peak A fraction

by P-cellulose

1381

isolated

Vol. 103. No. 4,1981

BIOCHEMICAL

Lysinoalanine

was found

in Achilles

tendon

AND BIOPHYSICAL

in dentine

RESEARCH

COMMUNICATIONS

and bone collagen,

but

absent

collagen.

Discussion Lysinoalanine but has never dentine This

been found

therefore,

that

in native

remains

collagen

serves

fibers

linked.

However, this

tendon

collagen,

tissue

with

phosphoproteins and werenot

lysinoalanine

is

removed during

in

Thus,

as the

collagen

but

cross-

known crosslinks

For example,

at least

that It

is

the

rather

lysinoalanine

absent

in Achilles the

content

lower

Since

in part,

lysinoalanine is

of the

than is

stability

tissues.

decalcification

an enzyme may be involved

1382

noncollagen-

noncollagenous

tissues

to be covalently

the proteins.

unites

known that

in calcified

calcification

were found

In any case,

are known

collagen.

are present

in the

in

seems to be highly

(6,7).

may explain,

tissues

tissues.

stability.

collagen

may be

to swelling

of previously

collagen.

phosphoproteins

in calcified

crosslink

resistant

collagen

possibility

ous proteins

of lysino-

function

in calcified

tissues

tissue it

amino acid

in soft

unusual

in calcified

a role

fibers

the content

in Achilles

Another

play

fibers

in bone and dentine

of calcified

study,

to alkali.

as an intermolecular

in calcified

of pyridinoline

tendon

In this

One possible

and highly

collagen

explain

of this

Collagen

collagen

present

(1).

example of occurrence

to be elucidated.

insoluble

compared with

first

function

fibers.

to be highly

that

proteins

proteins

proteins.

lysinoalanine

cannot

in native

the

The biological tissues

in alkali-treated

and bone samples had not been exposed

is,

alanine

has been found

(8,9)

and may

Some of the

bound to collagen in EDTA (8). in the formation

of

BIOCHEMICAL

Vol. 103, No. 4,198l

Compound II

in Fig.2

8 min before

lysinoalanine

structure

this

of

AND

BIOPHYSICAL

seems also

RESEARCH

a new amino acid.

on the amino acid

compound is

COMMUNICATIONS

now being

analyzer.

It

eluted

The

studied.

Acknowledgements We thank Prof. K. Motegi and Dr. K. Sakamoto, Department of Oral Surgery, Hamamatsu University School of Medicine, for supplying tooth samples and for help in the isolation of dentine. This work was supported in part by a Grant-in-Aid for Scientific Research (547126) and a Grant-in-Aid for Special Project Research (56109002) from the Ministry of Education, Science and Culture, Japan. References 1. 2. 3. 4. 5. 6. 7. 8. 9.

Sternberg, M. and Kim., C. Y. (1977) in Protein Crosslinking (Friedman, M. ed.) ~~-73-84, Plenum Press, New York Bohak, 2. (1964) J. Biol. Chem. 239, 2878-2887 Fujimoto, D., Akiba, K. and Nakamura, N. (1977) Biochem. Biophys. Res. Commun. 76, 1124-1129 Fujimoto, D., Moriguchi, T., Ishida, T. and Hayashi, H. (1978) Biochem. Biophys. Res. Commun. 84, 52-57 Goldfarb, A. R. (1966) Biochemistry 5, 2570-2574 Fuj imoto, D. and Moriguchi, T. (1978) J. Biochem. 83, 863867 H. (1980) Biochem. Biophys. Res. Eyre, D.R. and Oguchi, Commun. 92, 403-410 Veis, A. and Curley-Joseph, J. (1979) J. Dent. Res. 58, 16251633 Lee, S. L. and Glimcher, M. L. (1981) Calcif. Tissue Res. 33, 385-394

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