Pyridinium crosslinks of bone collagen and their location in peptides isolated from rat femur

Biochimica et Biophysica Acta 914 (1987) 233-239

233

Elsevier BBA 32906

Pyridinium crosslinks of bone collagen and their location in peptides isolated from rat femur S i m o n P. R o b i n s a n d A l e x a n d e r D u n c a n Rowett Research Institute, Bucksburn, Aberdeen (U.K.)

(Received 16 February 1987)

Key words: Pyridinoline;Deoxypyridinoline;Collagencrosslink; Collagen maturation; (Rat femur)

The relative proportions of pyridinoline and deoxypyridinoline in bone showed large species variations, although the total number of pyridinium crosslinks in rat, rabbit and bovine bone collagen was only 25-30% of that found in articular cartilage. Three pyridinium-containing peptides were isolated from cyanogen bromide digests of rat femoral bone and were characterized by their M r values and amino-acid compositions. The results showed that pyridinoline and its deoxy analogue were equally distributed at two locations stabilizing the 4D stagger through interactions involving both the N- and C-terminal telopeptide regions. Less than stoichiometric amounts of pyridinium crosslinks were present in the peptides, suggesting that the isolated peptides contained additional (unidentified) maturation products of the bifunctional, reducible crosslinks.

Introduction

The lysine oxidase-mediated crosslinking system for collagen gives rise initially, to intermediate compounds reducible with borohydride [1] that chemically are of two types, depending on whether lysine or hydroxylisine residues are present in the telopeptides [2]. This results in a specificity of crosslinking whereby soft tissues such as skin form aldimine linkages from oxidized lysine residues, whereas cartilage and bone initially contain oxo-imine bonds derived from hydroxylysine aldehydes [2]. Tissue maturation involves the loss of both types of reducible components with concomitant production of apparently more stable bonds [3].

Correspondence: S.P. Robins, Rowett Research Institute, Bucksburn, Aberdeen, AB2 9SB, U.K.

A 3-hydroxypyridinium compound, pyridinoline, identified by Fujimoto and co-workers [4] was shown to constitute a nonreducible crosslink of collagen [5-9]. This compound appeared to be a maturation product of the oxo-imine bifunctional crosslink [10], although the precise mechanism of formation is uncertain [8,9]. Pyridinoline represents the major crosslink of cartilage in which it is located as a trifunctional crosslink at two sites of the molecule [8,9]. A deoxy derivative of pyridinoline involving a lysine rather than hydroxylysine residue in the helix has also been identified [11] and quantified [12]. The presence of this pyridinoline analogue appears to be restricted to bone collagen, although the amounts vary between species [12]. The location of pyridinoline in bone collagen has been investigated by the isolation of crosslinked peptides from enzyme [7] and cyanogen bromide (CNBr) digests [13] with conflicting evidence as to whether the crosslink is present in the C-terminal regions of the al chains. In this paper,

0167-4838/87/$03.50 © 1987 ElsevierSciencePublishers B.V. (BiomedicalDivision)

234

the location of both pyridinium crosslinks in rat bone collagen has been investigated by the isolation and partial characterization of CNBr-peptides. Materials and Methods

Materials Acetonitrile and methanol (HPLC grade) were obtained from Rathburn Chemicals, Walkerburn, U.K. Trifluoroacetic acid ('Spectrosol' grade) was purchased from B.D.H., Poole, U.K. and heptafluorobutyric acid was a product of the Pierce Chemicals, Chester, U.K. NaB3H4 was obtained from Amersham International, Amersham, U.K.

Methods Preparation of tissues. Femurs from 5-month-old rats (Rowett Hooded strain) were cleaned of adhering tissue and the diaphyses were fragmented. The marrow was removed by extensive washing with 0.14 M NaC1/50 mM sodium phosphate (pH 7.4) at 4 ° C and, after rinsing with water, the bones were powdered under liquid N 2 (Spex Mill, Glen Creston, Stanmore, U.K.). An aqueous slurry of bone powder was extracted for 30 min with chloroform/methanol ( 3 : 1 ( v / v ) relative to volume of slurry), washed with acetone and dried under vacuum. Decalcification was effected either by two treatments of 30 min each with 2 M H C O O H (10:1 v / w ) at room temperature or by stirring with several changes of 0.5 M E D T A (pH 7.4) over a period of 5 d at 4 ° C. The decalcified bone was washed with water and freeze-dried. Preparation and chromatography of peptides. Decalcified bone powder was digested with CNBr as described previously [8]. The resultant peptides were fractionated and purified by sequential application of reverse-phase and size-exclusion HPLC methods. For reverse-phase separations, a column (1.0 x 25 cm) packed with Rosil-C18 beads (3 bt; Altech, Carnforth, U.K.) was run in 50 mM N H 4 H C O 3 adjusted to pH 3.2 with either trifluoroacetic acid or heptafluorobutyric acid using a gradient of increasing concentrations of acetonitrile. For size-exclusion chromatography, a TSK G3000SW (600 x 7.5 mm) column or two TSK G2000SW (600 X 7.5 mm) columns in series were

run in 50 mM N H 4 H C O 3 trifluoroacetic acid buffer (pH 3.2). Peptide samples were denatured by heating at 60 ° C for 10 min, immediately prior to chromatography. Isolated peptides were desalted on a column (30 × 1 cm) of Bio-Gel P2 with 0.1 M acetic acid as eluent. Analysis of crosslinks. Reducible crosslinks were quantitatively determined in hydrolysates of NaB3H4-treated peptide samples, by ion-exchange chromatography, as described previously [14]. Pyridinium crosslinks were assayed by reversephase HPLC [12] using a Vydac TP218 column (250 × 5 mm) run in 0.1% heptafluorobutyric acid with an acetonitrile gradient. Pyridinium crosslinks were monitored by their fluorescence emission at 400 nm using a Shimadzu RF-530 fluorimeter (excitation at 295 nm). As recommended by Eyre and co-workers [12], it was necessary to make up the samples in 1% heptafluorobutyric acid. Sample volumes were, however, kept low (50-100/~1) to minimize interference from a fluorescent artefact, eluting close to the position of the crosslinks, that was detected in heptafluorobutyric acid purchased from several different suppliers. Other procedures. Acid hydrolysis was carried out in sealed, evacuated tubes with constant-boiling HC1 at 110 ° C for 22 h, after which time the HC1 was removed in vacuo. Complete amino-acid analyses were performed using a Locarte analyser. Hydroxyproline measurements for quantification of crosslinks in hydrolysates of bone collagen were performed by a colorimetric method [15]. Results

The distribution of pyridinoline and its deoxy analogue in the rat differed from that in rabbit and bovine tissues (Table I). Thus, although pyridinoline was the major crosslink in all cartilage samples, the proportion of deoxypyridinoline in bone varied from about 10% of the total pyridinium residues in rabbit femur to around 50% of these residues in the rat (Table I). One of the aims of this work was to determine whether the two pyridinium crosslinks in rat bone were present in similar locations within the molecular framework. Chromatography of CNBr-peptides of rat diaphyseal bone by reverse-phase HPLC (Fig. 1)

235 al

0.6

-CB8

E

c O O

"1-CB72-CB4_ I X

A

~q~ _~ -

\

I

\

.-

.\

I /

h

\'.

/ ~ ~2cB5 o

u. I

i

30

40

I

I

I

50 60 Elution time (mins)

70

Fig. 1. Separation of CNBr peptides from rat bone collagen by reverse-phase HPLC. The peptides (5 mg), dissolved in starting buffer (50 mM NH4HCO3/trifluoroacetic acid (pH 3.2), with 14% acetonitrile), were denatured and chromatographed at 1.0 ml/min with the column maintained at 50 o C, using a linear gradient of 14 to 38% acetonitrile over 90 rnin. Fractions (1 min) were collected and those containing fluorescent material denoted by the bar were pooled for further analysis.

showed a single b r o a d b a n d , c o n t a i n i n g virtually all of the fluorescent material, that eluted close to the position of the a 2 - C B 4 peptide. This pooled fraction exhibited a shift in excitation m a x i m u m to 325 n m at n e u t r a l pH, characteristic of this type of c o m p o u n d [4,16]. Very similar peptide p a t t e r n s were o b t a i n e d for both formic acid- a n d EDTA-decalcified material (data n o t shown). Gel filtration o n a T S K G3000 c o l u m n of the p y r i d i n i u m - c o n t a i n i n g fraction from the reverse-

phase c o l u m n separated the fluorescent material into two m a i n fractions (Fig. 2). A fluorescent peak (denoted H1) which eluted just after the excluded material was r e c h r o m a t o g r a p h e d b y reverse-phase H P L C using h e p t a f l u o r o b u t y r i c acid rather than trifluoroacetic acid to give a single peak for which the A230 a n d fluorescence traces were again c o i n c i d e n t (data n o t shown). This peptide gave a single b a n d o n SDS-polyacryl-

0 7

TABLE I QUANTITATIVE ANALYSIS OF PYRIDINIUM CROSSLINKS Analyses by HPLC of pyridinoline and its deoxy analogue were performed directly on hydrolysates of femoral cartilage or bone. Values are expressed as residues/molecule with reference to the hydroxyproline content.

0.5

c u

0.3

Pyridinoline Deoxypyridinoline Total Bovine (2-3 years) articular cartilage 1.15 diaphyseal bone 0.34 Rabbit (12-18 months) articular cartilage 0.82 diaphyseal bone 0.18 Rat (15-20 weeks) sternal cartilage 0.65 articular cartilage 0.59 diaphyseal bone 0.11 metaphyseal bone 0.03 a n.d., not detected

O.

0.01 0.06

1.16 0.40

30

40

50

60

E l u t i o n t i m e (rains)

n.d. a 0.02

0.82 0.20

n.d. 0.03 0.09 0.04

0.65 0.62 0.20 0.07

Fig. 2. Size-exclusionHPLC of crosslinked peptides from the reverse-phase separations. The pyridinium-containing pept~des were heat denatured and chromatographed at room temperature on a TSK G3000SW column (600 × 7.5 mm) at 0.4 ml/min with 50 mM NH4HCO3/trifluoroacetic acid (pH 3.2). Fractions (1 min) were collected and the fluorescence-containing material denoted by the bars were separately pooled for further purification. LMW, low molecular weight.

236

amide gel electrophoresis with an M r of 42 500, using known collagen CNBr-peptides as standards. The lower-molecular-weight fraction was rechromatographed on TSK G2000 to give two fluorescent peptides (Fig. 3), denoted L1 and L2, which on SDS-polyacrylamide gel electrophoresis had M r values of 23500 and 19000, respectively. The amino-acid compositions of the crosslinked peptides H1, L1 and L2 are shown in Table II in comparison with the compositions of their most likely constituent peptides, based on known amino-acid sequence and composition data [17,18]. There was good agreement between the proposed and observed compositions, particularly for 3-hydroxyproline, which is present only in a single location of the al chain in the CB6 peptide, and for the C-terminal homoserine residues. The possible involvement of N-termimil telopeptides of the

a2 chain cannot be excluded, and this may account for some discrepancies in the contents of valine, leucine and tyrosine (Table II). The combined hydroxylysine and lysine values were lower in the isolated peptides, presumably reflecting the formation of crosslinks. Also shown in Table II are the relative amounts of pyridinium crosslinks in the peptide hydrolysates, together with analyses of the reducible, bifunctional cross-links obtained by reduction of a portion of the peptides with KBH 4 before hydrolysis. Analysis of the hydrolysates by HPLC showed that the ratio pyridinoline/deoxypyridinoline in peptides H1, L1 and L2 was 1.0, 1.1 and 1.3, respectively, thus exhibiting similar proportions to that in the original bone sample (Table I). Analysis of the initial bone powder after borohydride reduction revealed a bifunctional crosslinks content of 0.25 residues per collagen

TABLE II A M I N O - A C I D COMPOSITIONS OF CROSSLINKED PEPTIDES ISOLATED FROM RAT BONE COLLAGEN Values for peptides H1, L1 and L2 are the means of duplicate analyses of the hydrolysates and are expressed as residues per peptide assuming the likely compositions given for comparison in the adjacent columns. H1

3-Hyp 4-Hyp Asp Thr a Scar a Hse b GIu Pro Gly Ala. Val lie Leu Tyr Phe Hyl Lys His Arg Pyridinium Di-HLNL) + HLNL

1.6 36 25 8.4 23 0.6 27 56 144 37 4.2 5.4 10 1.6 3.1 3.0 6.8 2.7 23 0.1

2x al -CB6 + al-CB5 2 33 23 9 24 1 27 60 140 39 4 6 9 2 3 5 8 3 21

a Corrected for hydrolytic loss. b Includes lactone.

0.7 22 14 5.5 18 2.6 20 32 78 23 4.8 3.0 6.3 3.6 2.8 0.6 4.9 1.9 11 0.1

al -CB6 + 2x al-CB1 + al-CB5 1 18 15 5 19 3 17 33 80 23 6 3 5 5 2 3 5 2 11

L2

0.7 18 11 4.4 16 1.5 15 31 68 20 4.2 2.6 5.1 3.9 2.1 0.2 2.3 1.2 11 0.1

al -CB6 + 2x al-CB1 1 15 12 4 17 2 14 31 68 20 6 3 4 5 1 2 3 1 10

0.1 419

v

L1

256

219

237 0.8

,~2 CB6

00

] ,L

0.4

I

CB5

Peptide H 1

~

c~

]'-

cB6

0.2

I

Pephde L 1

75 100 Elution time (mins)

CB1

125

Fig. 3. Size-exclusion HPLC of the low-molecular-weight material from the G3000SW chromatogram. The peptides were fractionated under conditions similar to those given in the legend to Fig. 2, using two TSK G2000SW columns (600 × 7.5 mm) in series at a pumping rate of 0.3 ml/min. Peptide material designated (L)I and (L)2 indicated by the bars were separately pooled and desalted for analysis.

molecule, determined relative to hydroxyproline content. Discussion The three crosslinked peptides isolated in this study were identified on the basis of their relative molecular weights and amino-acid compositions. There exists some doubt about which of the N-terminal telopeptides was involved, but few other ambiguities were encountered and the presence of 3-hydroxyproline confirmed the involvement of the al-CB6 peptide in each of the crosslinked peptides. To account for the compositions of the isolated peptides, pyridinium crosslinks must be situated at two locations involving both the N- and C-terminal telopeptide regions (Fig. 4), each of which stabilizes the 4D stagger [19] of adjacent collagen molecules. The detection of reducible, bifunctional crosslinks in peptide L1

CB6

-r

I

I

Peptide L2

Fig. 4. The location of pyridinium-containing peptides H1, L1 and L2 isolated from CNBr digests of rat bone collagen, shown in each case by the bold lines. Each of the peptides involved the trifunctional linkage between the C-terminal peptide, alCB6, and either and N-terminal telopeptides (CB1) or al-CB5 at the 'overlap' region. Peptide L1 also contained a bifunctional crosslink stabilizing the 4D stagger of adjacent molecules.

was attributed to the presence in the peptide of an additional bond linking the C-terminal of an od chain to peptide td-CB5 (Fig. 4). These results are therefore in agreement with those of Kuboki et al. [7] in which a pyridinolinecontaining peptide isolated from a tryptic digest of bovine dentine collagen was shown to be derived from two M-CB6 peptides linked to a sequence at the junction of al-CB4 and ~-CB5. A similar location for pyridinoline in type I collagen was inferred by Fujimoto [5] based on the analysis of a crosslinked peptide isolated from a thermolysin digest of bovine achilles tendon. These findings

238

are consistent with a number of earlier reports citing similar locations in bone collagen for the precursor, bifunctional crosslinks [20-22]. By contrast, a recent report [13] has suggested that in bovine bone, dentine and achilles tendon, pyridinoline crosslinks were associated only with the C-terminal peptide of the ct2 chain; these authors contended that the al-CB6 peptide is involved in high-molecular-weight polymeric material [23] that does not contain pyridinoline [13]. The reason for the discrepancies between these findings is unclear; in the present study, no evidence was found for the association of pyridinoline with c~2-CB3,5 (Fig. 1), nor with peptides having similar electrophoretic mobility. Analysis of CNBr digests of bone achilles tendon by similar techniques also showed no pyridinium crosslinks associated with the a2-CB3,5 peptide (data not shown). Nevertheless, the involvement of the c~2-CB3,5 peptide of bovine dentine collagen in crosslinking by reducible, bifunctional components has been reported [24]. The fact that the proportions of pyridinoline and deoxypyridinoline were virtually the same in each of the isolated peptides indicated that the two crosslink analogues occupy the same loci in the molecule. Deoxypyridinoline appears to arise, therefore, through incomplete hydroxylation of the appropriate lysine residues in the helix. The interspecies differences in the relative amounts of the deoxy derivative ([12], Table I) probably reflect differences in lysine hydroxylase activities, which may occur indirectly through altered rates of helix formation. Less than stoichiometric amounts of pyridinium crosslinks were detected in each of the isolated peptides. Although pyridinoline is known to be susceptible to photochemical decomposition [25,26], the peptide-bound form is more stable than the free crosslink [25] and it is unlikely that this type of degradation is wholly responsible for the low yields of crosslink. In previous studies, pyridinoline recoveries in peptides isolated from cartilage [8,9] and dentine [7] were 40-70% of the stoichiometric amount. The low amounts of crosslink in the apparently homogeneous peptides isolated from bone collagen in the present study suggests that some form of crosslink other than pyridinium compounds was present, such that

peptides containing the proposed novel compound(s) copurified with the pyridinium-containing material. Recoveries of 10-20% were reported for bifunctional crosslinks in peptides isolated from bovine dentine collagen [24], a fact that also attributed to the presence of additional (unidentified) crosslinks. Only one of the purified peptides in the present study contained significant quantities of reducible, keto-amine crosslinks, suggesting that the isolated material must contain maturational products of the reducible precursors. The relatively low amounts of reducible crosslinks present in the original bone powder (0.25 residue, molecule) confirmed that maturation of these compounds had occurred. Bone collagen, however, contains much fewer pyridinium crosslinks than cartilage (Table I) indicating that, in the former tissue, pyridinium crosslinks may not be the sole maturation product. Evidence that a number of different pathways for the transformation of keto-amine crosslinks into non-reducible forms has been obtained from several sources [27-29] but none has yet been fully substantiated. The possibility that the pyridinoline crosslinks undergo further modification should also be considered. Further studies of the peptides isolated from bone are in progress in an attempt to establish the nature of the crosslinking moieties. References 1 Bailey, A.J., Robins, S.P. and Balian, G. (1974) Nature 251, 105-109 2 Robins, S.P. (1982) in Collagen in Health and Disease (Weiss, J.B. and Jayson, M.I.V., eds.), pp. 160-178, Churchill Livingstone, Edinburgh 3 Robins, S.P. and Bailey, A.J. (1973) Biochem. J. 135, 657-665 4 Fujimoto, D., Moriguchi, T., Ishida, T. and Hayashi, J. (1978) Biochem. Biophys. Res. Commun. 84, 52-57 5 Fujimoto, D. (1980) Biochem. Biophys. Res. Commun. 93, 948-953 6 Eyre, D.R. and Oguchi, H. (1980) Biochem. Biophys. Res. Commun. 92, 403-410 7 Kuboki, Y., Tsuzaki, M., Sasaki, S., Lin, C.F. and Mechanic, G. (1981) Biochem. Biophys. Res. Commun. 102, 119-126 8 Robins, S.P. and Duncan, A. (1983) Biochem. J. 215, 175-182 9 Wu, J-J. and Eyre, D.R. (1984) Biochemistry 23, 1850-1857 10 Siegel, R.C., Fu, J.C.C., Uto, N., Horiuchi, K. and Fujimoto, D. (1982) Biochem. Biophys. Res. Commun. 108, 1546-1550

239 11 Ogawa, T., Ono, T., Tsuda, M. and Kawanishi, Y. (1982) Biochem. Biophys. Res. Commun. 107, 1251-1257 12 Eyre, D.R., Koob, T.J. and Van Ness, K.P. (1984) Anal. Biochem. 137, 380-388 13 Light, N.D. and Bailey, A.J. (1985) FEBS Lett. 182, 503-508 14 Robins, S.P. (1982) Methods. Biochem. Anal. 28, 329-379 15 Firschein, H.E. and Shill, J.P. (1966) Anal. Biochem. 14, 296-304 16 Robins, S.P. (1983) Biochem. J. 215, 167-173 17 Kang, A.H., Bornstein, P. and Piez, K.A. (1967) Biochemistry 6, 788-795 18 Butler, W.T., Piez, K.A. and Bornstein, P. (1967) Biochemistry 6, 3771-3780 19 Hodge, A.J. and Petruska, J.A. (1963) in Aspects of Protein Structure (Ramachandran, G.N., ed.), pp. 289-306, Academic Press, New York 20 Eyre, D.R. and Glimscher, M.J. (1973) Biochem. J. 135, 393-403

21 Stimler, N.P. and Tanzer, M.L. (1979) J. Biol. Chem. 254, 666-671 22 Kuboki, Y., Takagi, T., Shimokawa, H., Oguchi, J., Sasaki, S. and Mechanic, G.L. (1981) Conn. Tissue Res. 9, 107-114 23 Light, N.D. and Bailey, A.J. (1980) Biochem. J. 185, 373-381 24 Scott, P.G. (1980) Biochemistry 19, 6118-6124 25 Sakura, S. and Fujimoto, D. (1981) J. Biochem. 89, 1541-1546 26 Fujimori, E. (1985) Biochim. Biophys. Acta 828, 104-106 27 Scott, J.E. Hughes, E.W. and Shuttleworth, A. (1981) Biosci. Rep. 1,611-618 28 Housley, T., Tanzer, M.L. Henson, E. and Gallop, P.M. (1975) Biochem. Biophys. Res. Commun. 67, 824-830 29 Yamauchi, M., Noyes, C., Kuboki, Y. and Mechanic, G.L. (1982) Proc. Natl. Acad. Sci. USA 79, 7684-7688