Biochimica et Biophysica Acta, 31o (I973) 461-468 © Elsevier Scientific Pub l i sh ing C o m p a n y , A m s t e r d a m - P r in t ed in The N e t h e r l a n d s

BBA 36429

INCREASED LYSINE H Y D R O X Y L A T I O N IN RAT BONE AND TENDON COLLAGEN AND LOCALIZATION OF T H E A D D I T I O N A L RESIDUES

M. STOLTZ, H. F U R T H M A Y R " AND I{. T I M P L

Max-Planck-Insti tut fiir Biochemie, Abt. Ki~hn, D-8o33 Martinsried b. Mi~nchen (Germany)

(Received December ISth, 1972)

SUMMARY

The a I and a2 chain obtained from guanidine extracts of rat bone collagen contain IO and 6 additional hydroxylysine residues, in comparison to the respective a chains of rat skin collagen. An increase was also demonstrated for insoluble bone collagen. At least for the a I chains, no evidence was obtained for a corresponding increase in carbohydrate content. Hydroxylysine values intermediate between those of skin and bone are found for the a2 chain of rat tail tendon collagen, yet the a I chain is in this respect indistinguishable from that of skin.

The relationship of the a chains of rat bone to type I collagen was demonstrated by CNBr cleavage and characterization of most of the fragments. This approach also allowed the localization of the additional hydroxylysine residues in three nonhelical cross-linking regions, as well as in various sites of the helical sequence. The findings do not support the idea that glycine in a position adjacent to the carboxyl group of lysine is an absolute requirement for hydroxylation.

INTRODUCTION

Hydroxylat ion of lysine is mediated by specific enzymes1, 2 and takes place during or after the synthesis of collagen a chains. The evidence for two different functions of the hydroxyl group has recently been revieweda,4: firstly, it serves as an exclusive site of carbohydrate a t tachment 5,~ which might control extrusion from the cell and/or morphology of collagen fibrils ; and a second role was indicated by the participation of hydroxylysine components in various cross-linking reactions, in- cluding Schiff base formation or aldol condensation. These hydroxylysine components appear to be more abundant in hard connective tissues (like bone, dentine and car- tilage 7-1°) than in skin. Since lysine can also participate in the same kinds of cross- link, a stabilization by the hydroxyl group (in an as yet unknown manner) was sug- gested for the components derived exclusively from hydroxylysinen, 1~. This sug- gestion was supported by a recent study on an inherited connective tissue disorder

" P r e s e n t address : D e p a r t m e n t of Pa tho logy , Yale Univers i ty , New H a v e n , Conn. , U.S.A.

462 M. STOLTZ e~ al.

of man la, which is characterized by high fragility of collagen and low levels of hydroxy- lysine.

The idea of an unique role of hydroxylysine in modifying structural properties of the connective tissue matr ix is also supported by previous findings of considerable variation in its content depending on its tissue origin. Thus, collagen of cartilage or basement membrane, known to belong to genetically distinct types of collagen, exhibit high hydroxylysine as well as carbohydrate content14,15. The lysine residues of type I collagen, which occur in skin, tendon and bone, are considerably less hydroxylated. However, in the chicken one additional hydroxylysine residue was identified in the nonhelical cross-linking region of bone collagen TM ; similar suggestions have been made recently for rat bone and tendon collagen '7. A thorough investigation of this topic is given in the present report, and demonstrates a large increase in hydroxylysine content for rat bone collagen, which has not yet been observed for any other type I collagen. The sites of additional hydroxylation involve lysine residues in nonhelical as well as in helical sequences. From these findings it is sup- posed that rat bone will be a good model to study cross-linking sites related to hydroxylysine and to approach questions of structural requirements in the hydroxy- lation reaction.

M A T E R I A L S A N D M E T H O D S

Male Wistar strain rats (weight lOO-15o g) were made lathyritic by feeding with fl-aminopropionitrile 18. The skin was removed, shaved and immediately homo- genized in deionized 8 M urea (15 ml per g wet weight). The tail tendons were treated similarly with 7 M guanidine hydrochloride of pH 7.5-8 (3oo ml for the tendons of 2o animals). The long bones of the extremities were removed and both ends, corre- sponding totally to 5o% of the entire length of each bone, were discarded to avoid contamination by cartilagineous tissue as much as possible. The middle part was then cut into pieces of about 2-mm size and homogenized with a large volume of o.35 M EDTA (pH 8.1). The insoluble residue was collected by centrifugation (IO min, 25oo × g) and repeatedly washed with the same solvent. This procedure essentially removed the bone marrow and yielded a white product. The inorganic components were subsequently extracted by stirring the residue in the EDTA solution (cf. ref. 19) for 6 days at 4 °C, changing the solvent every day. The insoluble organic matrix was finally homogenized in 7 M guanidine as above.

To achieve sufficient solubilization of the collagen a chains, the homogenates prepared in urea or guanidine were shaken for 2 h at room temperature followed by centrifugation (3o min, 2o ooo × g). The slightly opalescent supernatants were ex- haustively dialyzed for 3-4 days at 4 °C against o.o6 M sodium acetate, pH 4.8, the starting buffer used in CM-cellulose chromatography. The remaining residue was subiected to a second extraction by guanidine for 24 h and was finally washed with distilled water. I t was operationally defined as insoluble collagen.

The extracts including the precipitate formed during dialysis were treated for 3o min at 45 °C to denature reformed triple-helical collagen. After centrifugation (15 min, 25o0 × g) the a chains were separated from appropriate aliquots of the supernatant by chromatography on CM-cellulose 2° at 38 °C. The a I chains thus ob- tained appeared essentially pure. A second chromatography under identical con-

H Y D R O X Y L Y S I N E IN RAT BONE COLLAGEN 463

ditions was required to purify the a2 chains, as judged by polyacrylamide gel electro- phoresis performed in the presence of sodium dodecyl sulfate 21.

Cleavage of the a chains with CNBr was performed as recommended 22. Isolation of individual CNBr peptides was achieved by methods already approved for sepa- ration of analogous peptides of rat, calf and rabbit skin collagen 23 26. The first chro- matography was done on Bio-Gel P-4 which separates a I -CBI from the other pep- tides 25. Final pulification of a I -CBI was accomplished on phosphocellulose 25. The remaining peptides could be distributed into four fractions upon chromatography on Bio-Gel P-I5O (refs 24, 26). Further purification of aI-CB3, aI-CB6, aI-CB 7 and aI-CB8 was achieved by rechromatography on the same column and by CM-cellulose chromatography at pH 3.6 (ref. 23). Separation of the peptides aI-CB2, aI-CB 4 and aI-CB5 was performed by phosphocellulose chromatography 27, followed by runs on Bio-Gel P-Io. From the a2 chain only the peptide a2-CBI was isolated 28. To charac- terize the nonhelical region in aI-CB6, limited cleavage by chymotrypsin and sepa- ration of the fragments on Bio-Gel P-Io was applied TM.

The amino acid composition was determined as described 24. Carbohydrate at tached to hydroxylysine was quanti tated on the amino acid analyzer after alkaline hydrolysis 6, employing a modified technique developed recently in our laboratory ~9.

RES U L T S

Electrophoretically pure a I and a2 chains were obtained in a weight ratio of about 2 :I by CM-cellulose chromatography of extracts from skin, tendon and bone of lathyritic rats. Comparison of their amino acid compositions (Table I) revealed a striking difference in the relative proportion between hydroxylysine and lysine, although the sum of both remained constant for each discrete type of u chain. A further difference was found in the increased content of 3-hydroxyproline in tendon collagen. Other significant differences could not be detected, except that the a I chain of rat skin collagen essentially lacked about 20 amino acids from the carboxy- terminal, nonhelical region is. This is considered to be the reason for the lower content of tyrosine as well as of some other amino acids in that chain.

The guanidine-insoluble residue of rat bone had an amino acid composition characteristic for collagen (Table I). I t also exhibited an increased hydroxylysine content, although not as high as that found for solubilized a chains. The a I chains were also prepared from bones of normal rats and were obtained in a very low yield of less than o . I % of total collagen. Amino acid analysis revealed 12 hydroxylysine and 24 lysine residues per chain.

The content of glycosylated hydroxylysine was not extremely different in the a I chains of skin and bone origin (Table II). No large change in the proportion be- tween glucosyl-galactosyl-hydroxylysine and galactosyl-hydroxylysine was evident, as found, for example, for insoluble collagen of human skin and bone 3°.

The CNBr peptides, except the dipeptide aI-CBo (Glx-Hse), were prepared from the a I chain of rat bone collagen and purified by established methods. The chro- matographic profiles exactly resembled those described for type I a I chains of various skin collagens 23-2e and are therefore not shown. The pat tern also indicated a stoi- chiometric proportion between each of the peptides isolated. Their amino acid compositions (Table I I I ) clearly account for all the amino acids of the original a I

464 M. STOLTZ c t

T A B L E i

A M I N O ACID C O M P O S I T I O N OF C O L L A G E N ( / I C H A I N A N D (12 C H A I N F R O M R A T S K I N , T A I L T E N D O N

A N D B O N E

E x p r e s s e d as res idues per cha in rounded off to the nea res t whole number . Actua l va lues are g iven in p a r e n t h e s e s if less t h a n io res idues are found.

A m i n o acid aI chain a2 chain

Sk in Tai l Bone Sk in Tai l Bone tendon tendon

Insoluble collagen, bone

3-Hydroxypro l i ne 1 ( 0 . 7 ) 2 ( 2 . I ) I ( I .O) - - 2 ( I . 8 ) - -

4 -Hydroxyp ro l i ne 96 98 i o i 90 83 88 i oo Aspar t ic acid 48 49 49 46 47 46 49 Threon ine 2 2 2 2 2 2 2 2 2 I 2 2 2 0

Serine 43 44 43 45 44 43 41 G lu t amic acid 81 84 85 78 8o 78 88 Prol ine 132 128 127 lO9 113 lO9 133 Glycine 337 343 341 349 347 35 ° 333 Alanine t o 7 i I i I 14 1 o5 105 i o9 i 12 Valine 2i 22 21 34 34 33 25 Meth ion ine 8 (7-5) 8 (7.5) 7 (7-3} 4 (4.4) 4 (3 .6) 3 (2.9) 6 (5.9) Isoleucine 7 (6.6) 7 (6.8) 7 (6.8) 21 21 2o 13 Leucine 22 .:3 22 37 38 37 27 r y r o s i n e 3 (3.3) 5 (4.5) 4 (4.4) 4 (3.7) 4 (3.8) 4 (3.9) 5 (4 .8) P h e n y l a l a n i n e J 4 15 15 13 13 13 14 H y d r o x y l y s i n e 5 (4 .8) 5 (5.4) I5 8 (8.2) 12 14 12 Lys ine 33 34 24 27 22 21 20 His t id ine 2 (2.3) 2 (2.2) 2 ( 2 . 2 ) IO l o 1 o 5 (4 .8)

Arginine 53 53 55 53 55 55 52 Total* lO35 lO55 lO55 lO55 lO55 lO55 lO55

* Tota l s were ca lcu la ted for the a i cha in on the basis of recen t sequence s tud ies (Ktihn, K., persona l commun ica t i on ) . The s ame l eng th is a s s u m e d for t he a2 chain. The size of t he sk in col- lagen a2 cha in was corrected according to the in fo rma t ion (ref. i8) t h a t abou t 20 amino acids are lacking in 9 o°//o of the molecules.

T A B L E II

H Y D R O X Y L Y S I N E - B O U N D C A R B O H Y D R A T E IN T H E C O L L A G E N ( / I C H A I N F R O M RAT S K I N A N D BONP;

Expre s sed as res idues per chain.

Galactosvl- Glucosyl- Percent of total hydroxylysine galactosyl- hydroxylysine

hydroxylysine glycosylated

Skin 0. 7 2.1 58 Bone 1. 3 2. 7 27

c h a i n . T h e a d d i t i o n a l h y d r o x y l y s i n e r e s i d u e s , w h e n c o m p a r e d w i t h h o m o l o g o u s

p e p t i d e s f r o m r a t s k i n c o l l a g e n g i c h a i n 23, a r e d i s t r i b u t e d a m o n g s e v e r a l p e p t i d e s ,

a s i n d i c a t e d i n T a b l e I I I .

D i f f e r e n c e s i n h y d r o x y l y s i n e c o n t e n t a r e f o u n d i n t h e t w o n o n h e l i c a l r e g i o n s

o f t h e c o l l a g e n a I c h a i n . O n e is c o n f i n e d t o t h e p e p t i d e a I - C B I l o c a t e d n e a r t h e a m i n o -

t e r m i n a l e n d , w h i c h c o n t a i n e d h y d r o x y l y s i n e w h e n d e r i v e d f r o m b o n e ( T a b l e I I I ) ,

b u t l y s i n e w h e n d e r i v e d f r o m s k i n a I - C B I ( r e f s 23, 31) . A s e c o n d n o n h e l i c a l s e q u e n c e

H Y D R O X Y L Y S I N E I N R A T B O N E C O L L A G E N 465

T A B L E I I I

AMINO ACID COMPOSITION OF C~N~Br PEPTIDES FROM THE RAT BONE COLLAGEN GI CHAIN

E x p r e s s e d a s r e s i d u e s p e r p e p t i d e r o u n d e d o f f t o t h e n e a r e s t w h o l e n u m b e r . A c t u a l v a l u e s a r e g i v e n i n p a r e n t h e s e s i f l e s s t h a n i o r e s i d u e s a r e f o u n d . A d a s h d e n o t e s l e s s t h a n o . I r e s i d u e .

a I - C B I a I -CB2 a I - C B 3 a I -C B 4 a I - C B 5 a I -CB6 a I - C B 7 a I -CB8 S u m ai-CI36- C3"

3 - H y d r o x y p r o l i n e . . . . . I (0 .8) - - i (o .8) 2 - - 4 - H y d r o x y p r o l i n e - - 5 (5 .1) 15 6 (5 .9) 3 (2 .7) 18 25 2 9 i o i - - A s p a r t i c a c i d i ( i . o ) - - 7 (7 ,4) 3 (3 .1) 3 (3 .O) i i 12 12 4 9 2 (2 .2) T h r e o n i n e - - - - 2 (2 .1) I ( i . o ) i ( I .O) 4 (4 -1 ) 7 (6 .7) 5 (5 .4) 2 0 - - S e r i n e 3 (2 .6) 2 (1 .9) 3 (3 .1) - - 2 (1 .9) 12 9 (9 .3) i i 42 z (2 .0) H o m o s e r i n e I ( i . i ) i (I .O) I ( i . o ) I ( I . I ) i ( i . i ) - - i (1 .2) i ( I . 3 ) 7 - - G l u t a m i c a c i d I ( 1 . i ) 4 (4 .4) 16 3 (3 .3) 3 (3 .3) 16 18 22 83 5 (4 .7) P r o l i n e 2 (1 .9 ) 7 (6-7) 14 6 (5 .7) 2 (2 .4) 3 ° 35 32 1 2 8 3 (3 .1) G l y c i n e 3 (3 .1) 12 5 ° 16 12 6 9 8 9 91 3 4 2 2 (2 .3) A l a n i n e i ( I . I ) 2 (2 .3) 2 0 3 (3 .4) 3 (3 .1 ) 21 31 3 9 1 2 o V a l i n e 2 (2 .0) - - 4 (4 .0 ) - - - - 2 (1 .9) 7 (7 .2 ) 6 (5 .7) 21 - - I s o l e u c i n e . . . . . 3 (2 .6) 3 (2 .9) I (1 .4) 7 - - L e u c i n e - - i (0 .9) 3 (3 .° ) 2 (2 .0) i ( I . I ) 4 (4 .4) 4 (4 .2) 4 (4-4) 19 i (1 .2) T y r o s i n e 2 ( I . 7 ) - - - - - - 2 ( I . 7 ) - - 4 2 (1 .8) P h e n y l a l a n i n e - - I (I .O) 3 ( 2 . 9 ) - - I (0 .9) 3 (2 .9) 3 (3 ,3) 3 (3 .1) 14 2 (2 .1) H y d r o x y l y s i n e i (0 .9 ) - - I ( 1 . 2 ) - 2 (1 .9) 3 (2 .6) 5 (4 .8) 3 (2 .9) 15 1 (0 .9) L y s i n e - - 4 (3 .8 ) 2 (2 .0) I (0 .9) 4 (4-3) 6 (6 .4) 7 (6 .8) 2 4 - - H i s t i d i n e . . . . i ( I .O) I (0 .8) - - 2 - - A r g i n i n e - - i (I .O) 6 (6 .1) 4 (3 .9) i ( i . o ) 12 13 15 52 1 (0 .8) T o t a l 17 3 6 1 4 9 4 7 3 7 2 1 6 2 6 8 2 8 2 l O 5 2 21 A d d i t i o n a l H y l t o

s k i n ' * I (0 .9 ) - - i ( 1 . o ) - i (0 .8) I (0 .8) 4 (4 .1) 2 (1 .8) i o I (0 .9)

" C a r b o x y - t e r m i n a l c h y m o t r y p t i c p e p t i d e o f a I - C B 6 . ** N u m b e r o f a d d i t i o n a l h y d r o x y l y s i n e r e s i d u e s i n b o n e c o l l a g e n p e p t i d e s i f c o m p a r e d w i t h t h e c o r r e -

s p o n d i n g C N B r p e p t i d e s o f r a t s k i n c o l l a g e n ( re f . 23 ) . T h e r e q u i r e d d a t a f o r a I - C B 6 a n d a I - C B 6 - C 3 a r e t a k e n f r o m r a t t e n d o n c o l l a g e n ( re f . 18) .

of 25 amino acids ls,3~ constitutes the carboxy-terminal part of the peptide aI-CB6. For localization of the additional hydroxylysine residue in bone collagen, aI-CB6 was cleaved by chymotrypsin and the fragments CI, C2 and C3 were prepared from that peptide and characterized by amino acid analysis. The single lysine residue of peptide C3, known to be entirely nonhelicaP ~, was completely replaced by hydroxylysine (Table III).

A further nonhelical sequence occurs at the amino-terminal region of the col- lagen a2 chain and is represented by the CNBr peptide a2-CBI (refs 28, 31). Isolation of a2-CBI from tendon and bone collagen revealed a composition exactly resembling the known composition of the homologous peptide obtained from skin collagen3k However, the single lysine residue was entirely hydroxylated in the bone collagen peptide.

D I S C U S S I O N

Beside some small changes, which are obviously artefacts appearing during extraction TM, the only differences observed between the a chains of rat skin, tendon and bone collagen are restricted to hydroxylysine and to a lesser degree to hydroxy- proline. The increase in hydroxylysine content of bone collagen c~I and a2 chain (IO

466 M. STOL'rZ e~ a/.

and 6 residues, respectively) approaches values characteristic for the type I I collagen of cartilage 14,33. However, the occurrence of two types of a chains in the proportion 2 :I, as well as the pat tern of CNBr peptides which is characteristic of type I collagen, did not lend support for the possibility that the increase is caused by large contami- nation with cartilage-type collagen. This assumption is also supported by a relatively low carbohydrate content in bone collagen a I chain, as opposed to high values ob- served for cartilage collagen14m. Previous comparative studies on chick skin and bone collagen revealed less of an increase in lysine hydroxylation, which probably involved only one site 16. Preliminary studies on the a I chain of calf bone supported tile same conclusion (unpublished). No such differences were observed for insoluble human collagen of bone and skin 3°. The present finding therefore indicated a rather unique feature of rat bone collagen. For rat tail tendon collagen a still different pattern was observed, since only the a2 chain is involved in increased lysine hydroxylation.

The a chains used in the present study were obtained from lathyritic animals, taking advantage of the tremendously increased collagen solubility under this experimental condition. Tile possibility of an influence on the extent of lysine hydroxylation by the lathyrogen restricted to bone can be excluded for two reasons. Small amounts of ctI chains could be obtained from bones of normal rats and revealed 7 additional hydroxylysine residues instead of the IO derived from lathyritic rats. As discussed below, at least two hydroxylysine residues are located in nonhelical sequences and may be oxidized to aldehydes in normal bone collagen 22, thus ac- counting for most of the difference. Furthermore, the insoluble collagen from tile bones of lathyritic rats should be least affected by the lathyrogen, but this collagen also exhibited a high content of hydroxylysine. I t still remains to be clarified whether tile insoluble residue contains substantial amounts of a second type of collagen (e.g. type I I I as in skin 34) which might have a lower hydroxylysine content.

Characterization of CNBr peptides facilitated the localization of the additional sites of lysine hydroxylation in bone collagen. Each of the single lysine residues located in the three known nonhelical regions is almost completely hydroxylated. This confirms recent findings from labeling the amino-terminal peptides a I -CBI and ~,2-CBI, with [14Cllysine, although only partial hydroxylation was demonstrated by this approachlL Since tile amino-terminal nonhelical regions are well-characterized sites of cross-linking 4 and similar evidence has been obtained for the carboxy- terminal counterpart in aI-CB6 (ref. 35), an influence on the cross-linking reaction by the introduced hydroxyl group seems possible. The way in which this difference may exert its effect is still unknown, though some suggestions for increased stabil- ization have been made1', 12.

The remaining sites of additional lysine hydroxylation could be assigned to CNBr peptides from the helical region. Recent studies emphasized the potential occurrence of cross-links between nonhelical and helical sequences 3~. I t is, in this context, of interest that those helical CNBr peptides considered to be especially involved (aI-CB5, aI-CB 7 and aI-CBS) are enriched in hydroxylysine in rat bone collagen. From such information, a predominance of cross-links derived from two hydroxylysine residues should be expected, a prediction consistent with results derived from estimation of reducible cross-link components of bone connective tissueT, s. Minor sites of hydroxylation have been demonstrated for discrete lysine residues in aI-CB 5 and aI-CB 3 of skin collagena6,aL Preliminary characterization of

HYDROXYLYSINE IN RAT BONE COLLAGEN 467

tryptic peptides from corresponding CNBr peptides of rat bone collagen detected increases in hydroxylation in exactly those particular sites (unpublished). Butler et aL as reported on an increased hydroxylysine content of insoluble rat dentine collagen. Recent characterization 39 of aI-CB3 from that source demonstrated two additional sites of lysine hydroxylation and suggested identity to type I collagen.

Previous information on the localization of hydroxylysine in helical sequences suggested the requirement of glycine adjacent to lysine in a carboxy-terminal position for effective hydroxylation 3. The demonstration of partially hydroxylated lysine in chick bone aI-CBI (ref. 16) has already denied that such a requirement is absolute. This is supported by the present study since rat bone a I - C B I , as well as aI-CB6-C3, obviously does not support that prediction:

a I-CB I : - A s p - G l u - L y s - S e r - A l a (ref. 3 I ) aI-CB6-C3: - G l n - G l x - L y s , Ser, G lx - (ref. 18, 32, tenta t ive)

The question remains still to be answered if different forms of lysine hydroxylase (c]. ref. I) are adapted to different sequence environments. To approach this problem, rat bone should be considered a good source for isolating these enzymes.

ACKNOWLEDGMENT

We wish to thank Miss V. Tezak and Miss M. Furthmayr for expert technical assistance and Mr W. Strasshofer for help in amino acid analysis. This work was supported by the Deutsche Forschungsgemeinschaff, SFB 37.

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