isolation and characterization of new cross-linking amino acid “allodesmosine” from hydrolysate...

Vol. 170, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

July 31, 1990 Pages 713-718

ISOLATION AND CHARACTERIZATION OF NEW CROSS-LINKING AMINO

ACID “ALLODESMOSINE” FROM HYDROLYSATE OF ELASTIN

Kyozo Suyama and Fumihiko Nakamura

Molecular Technology of Animal Products, Faculty of

Agriculture, Tohoku University, Sendai, 981 JAPAN

Received June 16, 1990

SUMMARY: A new pentafunctional cross-linking amino acid, termed allo-

desmosine, was isolated from bovine ligamenturn nuchae elastin. This

compound was a very hygroscopic, white amorphous solid with a faint yellow

tinge, soluble in aqueous solvents but not dry methanol: it was characte-

rized by UV, FAB mass and NMR spectroscopy. The compound was shown by UV

and 1 H-NMR to have a pyridinium ring structure similar to desmosine. Mass

spectral analysis indicated a parent compound with a mass of 655. We

postulated that it arose by condensation of a reduced aldol condensation

product of all ysine, all ysine and lysine. s1990 AcademK Press, Inc.

Elastin is a connective tissue protein found in virtually every tissue

and organs. The highest concentration is found in the aorta and pulmonary

vessels(l.2). The degradation of elastin is accelerated in several

diseases such as atherosclerosis and diabetes mellitus(3). Also aberrations

in elastin structure and metabolism have been detected in a number of

heritable and acquired diseases. Elastin is secreted from the cell as a

soluble polypeptide (tropoelastin). As tropoelastin comes in contact with

the extracellular matrix, it becomes insoluble and fibrillar in structure.

This occurs through a series of intra- and intermolecular crosslinks. It is

known that elastin is cross-linked by unique pol yfunctional amino acids

which have a quaternary pyridinium skeleton; desmosine(4), isodesmosine(4),

neodesmosine(5) and pentasine(6). It is demonstrated that a-amino adipic

acid a-semialdehyde(allysine) and lysine are embodied of these cross-

linking amino acids(7,8), however the route of formation is still

uncertain. To gain more understanding about the nature and function of

elastin, we have attempted to isolate hitherto undescribed cross-linking

amino acids. Here, we show that isolation and characterization of such a

new amino acid derived from the cross-linkage in elastin is proposed.

0006-291X/90 $1.50

713 Copyright 0 1990 by Academic Press, Inc.

All rights of reproduction in any form reserved.


EXPERIMENTAL

After acid hydrolysis (GN,HCl; at 110” C for 48h) of purified elastin

from bovine 1 igamentum nuchae, the hydrol ysate was chromatographed on

ion pair HPLC by a SHIMADZU LC-GA pump and SPD-6AV UV detector attached

to a LiChrospher 100 RP-18 125-4tMerck) reverse phase column using a pH

O.lM phosphate buffer/acetonitrile 5:l v/v containing 20mM SDS as a solvent

(final pH 4.0). Desmosine, isodesmosine and other unknown amino acids were

separated and detected at 275nm as shown in Figure 1.

To prepare the unknown amino acid, 50 gram of an acid hydrolysate

of elastin was charged on a large scale activated charcoal column(65 x 180

mm). Major 1 ysine derived cross-links were fractionated from water

followed by elution with 20% v/v aqueous methanol solution. Lysine derived

crosslinking amino acids thus obtained were charged on a preparative HPLC

silica gel column (10 x 240 mm; Lobar LiChroprep Si60, Merck) using ethyl

acetate/acetic acid/water 1.5:1:1 v/v as a solvent. The Fractions were

collected. Confirmation was by HPLC analysis, which indicated the presence

of another amino acid, designated as AD, eluting just after desmosine as

shown in Figure 1. Fractions containing this amino acid were pooled and

evaporated to dryness. Partially purified AD thus obtained was then

charged on the same preparative HPLC column but using a solvent system of

n-propanol/water/25%ammonia 700:330:28, v/v(9). The fractions containing AD

were collected. The UV spectrum of AD dissolved in O.lN HCl was

performed on a JASCO UVIDEC-500 spectrophotometer. Both 1 H- and ! 1 C-NMR

spectra were performed on a JEOL JNM GSX-400 instrument and FAB mass

spectrometry was done by JEOL HX-105 instrument using glycerol as a

matrix.

RESULTS AND DISCUSSION

The purity of the unknown amino acid, AD, was confirmed by HPLC and

TLC analysis . On elution systems of HPLC that will resolve desmosine and

isodesmosine, AD eluted just after desmosine and fractionated as a single

peak (Figure 1). The amino acid AD gave a heavy ninhydrin single spot on

silica gel TLC using a solvent system of ethyl acetate/acetic acid/water

l:l:l, v/v (Rf:0.098; both Rfs of desmosine and isodesmosine were 0.166).

Compound AD was a very hygroscopic, white solid with a faint yellow tinge,

soluble in aqueous solvents but not dry methanol; attempts to induce

crystallization were unsuccessful. It was thought that the hygroscopic

character might be due to the presence of the quaternary pyridinium

skeleton.

The UV spectrum of AD is shown in Figure 2 and is compared with

that of desmosine. Compound AD showed UV absorption maximum at 234

and 268 nm inflections at 274 nm which were completely identical to those

of desmosine. From the results, it is deduced that AD has the same

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Time(min)

234 234

1.5. 1.5.

10 10 AD AD

0.5. 0.5.

a, a,

: : 2 2 0 0

b b In In

2 2 1.5, 1.5,

1.0. 1.0.

0.5. 0.5. Desmosine Desmosine

0 2 0 200 250 300 350

Wavelength (nm)

Figure I. HPLC of acid hydrolysate of bovine ligamentum nuchae elastin and

compound AD.

UK: unknown compounds.

Column: LiChrospher 100 RP-18 125-4.

Solvent: O.lM phosphate buffer/acetonitrile (5:l,v/v) containing

20mM SDS at pH 4.0.

Flow rate: 1.0 ml/min.

Figure 2. Ultra violet absorption spectra of AD and desmosine in O.lN HCI.

400 450 500 550 600 650 700 M/Z

Figure 3. Positive ion FAB mass spectrum of AD (C.3&IN6010 = 655.786 ; Allodesmosine).

715


substitution pattern on the quaternary pyridinium skeleton as desmosine.

The FAB mass spectrum of AD gave a molecular weight of 655, consistent

with an elemental composition of C~eHslN~Oln as shown in Figure 3, whereas

desmosine gave a molecular weight of 526. For comparison and to enable us

to make tentative proton assignments, 1 H-NMR spectra were performed on

standard desmosine as well as AD. Figure 4 shows the 1 H-NMR spectrum of AD

in D20. The compound AD shows a 1 H-NMR spectrum very similar to that of

desmosine especially with two equivalent aromatic protons attached to

pyridinium ring at the 2 and 6 positions at 8.60 ppm. Compound AD was

similar in many respects to the desmosine, yet had a significantly larger

number of protons representing methylene groups. The 13C-NMR spectrum and

proposed chemical structure of AD with assignment is shown in Figure 5.

The in vivo synthesis of this compound probably follows closely to that

proposed for the desmosine, that is, one reduced aldol condensation product

of allysine, two molecules of al 1 ysine condensed to lysine to form AD

as shown in Figure 6. Although the mechanistic details are not clear, one

must invoke a reductive alkylation to obtain the final products, analogous

to lysinonorleucine(9) and hydroxyaldol-histidine (10) formation. An amino

acid, AD, was also detected in bovine lung, aorta and skin by ion pair

HPLC. We propose in future to refer to compounds AD as “allodesmosine”.

CCH(ND2)CooD AD

PPM I ‘, ” I ” I ” ” I ” ” I ” ” I ” ” r

6 7 6 5 4 3 2 1

Figure 4. ' H-NMR spectrum of AD. The spectrum was recorded on JEOL JNM GSX-400.

716


170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20

DEPT b % t C”,CH3 t c -

PPM

Figure 5. 13C-NMR spectra of AD.

The spectra were recorded on JEOL JNM GSX-400 with proton noise

decoupled (A) and DEPT(distortionless enhancement by polarization

transfer) method (B).

-OOCCH(t?H,)CH,CH,CHCH,CH,CH,CH,CH(ljH,)COO- bH0

OOCCH(t?H,)CH$H~H, 1

yH,CH,CH,CH( NH$OO- OHC CHO >

cc? 2

%H,KOO-

Allodesmosi ne

Figure 6. Postulated mechanism of formation of allodesmosine.

717


ACKNOWLEDGMENT

This work was supported in part by Grants-in-Aid for Scientific

Research (No. 01560303) from the Ministry of Education, Science and Culture

of Japan.

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isolation and characterization of new cross-linking amino acid “allodesmosine” from hydrolysate...

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