ARCHIVES OF BIOCHEMISTRYAND BIOPHYSICS Vol. 198, No. 1, November, pp. 205-211, 1979

Biosynthesis of Collagen Crosslinks in Rabbit Articular Cartilage in Vivo FREDERIC Department

of Orthopaedic

SHAPIRO, DIANE BRICKLEY-PARSONS, AND MELVIN J. GLIMCHER Surgery,

Harvard Medical School, Children’s Boston, Massachusetts 0.2115

Hospital

Medical

Center,

Received April 19, 1979; revised July 23, 1979 The biosynthesis in viva of the two reducible aldimine crosslinks of immature rabbit articular collagen, hydroxylysinohydroxynorleucine and hydroxylysinonorleucine, is demonstrated. The peak amount of crosslink was detected l-2 weeks following labeling of the cartilage with [WIlysine. The subsequent diminution which occurred was due primarily to a decrease in the amount of hydroxylysinohydroxynorleucine. Natural reduction of the aldimine crosslinks in wivo did not occur. Glucosylgalactosyl hydroxylysine and galactosylhydroxylysine, in a 1.45/1.00 ratio, were synthesized. Seventy-three percent of the hydroxylysine residues were glycosylated. rH]NaBH, reduction of non-W-labeled cartilage showed diminished amounts of reducible crosshnk with time and the presence of hexosyl lysines and hexosyl hydroxylysines in mature articular cartilage.

Several post-translational changes occur during the biosynthesis of collagen molecules (l), including hydroxylation of the proline and lysine residues to hydroxyproline and hydroxylysine, glycosylation of certain hydroxylysine residues, and formation of intramolecular and intermolecular crosslinks (2). In the post-translational glycosylation of collagen, galactose and glucosylgalactose are attached by an 0-glycosidic linkage to hydroxylysine residues to form galactosylhydroxylysine and glucosylgalactosylhydroxylysine (3). The major recognized intermolecular crosslinks of articular cartilage collagen on chemical reduction are hydroxylysinohydroxynorleucine (HylOHNle)’ and hydroxylysinonorleucine (HylNle) (4,5). Glycosylated reducible crosslinks, glucosylgalactosyl HylOHNle, have also been recognized (6). Considerable uncertainty persists, however, concerning the ultimate fate of the reducible aldimine crosslinks. Previous studies have demonstrated a marked decrease in the amounts of these 1 Abbreviations used: HylOHNle, hydroxylsinohydroxynorleucine; HylNle, hydroxylysinonorleucine; GGH, glucosylgalactosyl hydroxylysine; GH, galactosyl hydroxylysine. 205

intermolecular crosslinks in mature tissues (7). One of the postulated mechanisms for the decrease is that natural reduction occurs in viva such that the number of molecules available for sodium borohydride reduction decreases (8). Others have discussed conversion to related compounds (9). Evidence against natural reduction has been presented (10-12) and no natural reduction of the two aldimine crosslinks was detected in [14C]lysine-labeled embryonic chick bone or sternal cartilage in organ culture (13). Articular cartilage is an avascular tissue, receiving its nutrition primarily via diffusion from synovial fluid. High levels of [14C]lysine were made available for incorporation into chondrocytes by instillation directly onto the surface of the cartilage at open operation. The [14C]lysine was then identifiable following its incorporation into collagen as lysine, hydroxylysine, the glycosylated hydroxylysines, and the two reducible aldimine crosslinks. This paper demonstrates the decreased amounts of the reducible intermolecular crosslinks with maturation in rabbit articular cartilage. The r4C]lysine-labeling studies demonstrate the biosynthesis in vivo of the intermolecular crosslinks and of the glycosylated hydroxy0003-9861/‘79/130205-07$02.00/O Copyright 0 1979by AcademicPress, Inc. All rights of reproduction

in any form reserved.

206

SHAPIRO, BRICKLEY-PARSONS,

lysines and assess the changes in their quality and quantity with tissue maturation. MATERIALS

AND METHODS

Animals and Surgical

Procedures

Articular cartilage samples. Articular cartilage was retrieved following sacrifice. A No. 15 scalpel blade was used to shave the cartilage from the articular surface. Care was taken not to include the peripheral cartilage adjacent to the synovial-perichondrial reflection. In those animals less than 6 months of age, care was taken not to remove cartilage down to bone as some of the cartilage participating in the endochondral mechanisms of epiphyseal osteogenesis would have been included. In this study knee joint cartilage refers to distal femoral, proximal tibial, and patellar cartilage; shoulder to proximal humeral and glenoidal cartilage; and hip to proximal femoral and acetabular cartilage. ~F]L@w labeling of a&c&r cartilage. Immature 3-month-old white New Zealand rabbits were used for biosynthesis studies in vivo. The animals were anesthetized with intravenous sodium pentothal (Diabutal), supplemented by ether inhalation and 1% Xylocaine locally. The knee, hip, or shoulder joints were opened to display the articular cartilage. No joints which became infected were used for biochemical analysis. L-[14C]Lysine, 25 to 50 &i, in 0.5 cc sterile water was placed directly onto the articular cartilage using a 25-gauge needle. Excellent labeling of the articular chondrocytes was obtained invariably with this technique (vide infra). The same amount of [WIlysine was not taken up. by each articular cartilage mass but as the calculations of activity for each particular joint sample were expressed as a percentage of 14Ccounts in each peak this fact was not of practical significance. No postoperative immobilization was used. Sacrifice occurred at 0.5, 1, 4, 24, and 72 h; 1 and 2 weeks; 1,2,3,6, and 18 months. In the 6- and Wmonth samples and in certain of the earlier time periods, several joint cartilages were pooled for analysis.

Histologic

and Autoradiographic

Studies

Autoradiographs were done on humeral head sections of 3-month-old rabbits sacrificed at varying periods from 20 min to 2 months after instillation of 10 FCi rH]lysine. The cartilage was left intact on the bone to allow for easy assessment of the extent and depth of radioisotope penetration. The tissues were fixed in 10% neutral buffered formalin, decalcified in 25% formic acid, and embedded in paraffin. Sections 6 pM thick were cut, dipped in NTBr emulsion (Kodak),

AND GLIMCHER

and exposed for 3 weeks prior to developing and staining with hematoxylin and eosin.

Reduction of Cartilage IjH]NaBH,

Collagen with

The reducible aldimine crosslink profile was determined from rabbits 1 week, l-6 months, and 1, 2, 3 years old. Following retrieval the cartilage was cut into small pieces, approximately 0.5 x 4 x 2 mm. The tissue was not homogenized. The cartilage was washed three times with distilled water and reacted with rH]NaBH, (New England Nuclear Corp., Boston, Mass) (10 Wmol, 0.01 mmoVl0 mg tissue) in 0.1 M phosphate buffer, pH 7.4, for 1 h at 25°C. Excess borohydride was destroyed by adding glacial acetic acid to bring the pH below 4 for 10 min. The samples were then washed with water, dried in a vacuum desiccator of phosphorous pentoxide for 12+ h, and weighed. Both the W-labeled cartilage and unlabeled cartilage were treated this way.

Hydrolysis

Conditions

Acid hydrolysis was performed in 3 N HCl sealed in vacua under nitrogen at 105°C for 48 h. The samples were then washed with water and dried on a Buchler Evapomix. Acid hydrolysis was done both on [WIlysine samples which had been reduced with rH]NaBH, and which had not been reduced, and on cartilage not labeled with r4C]lysine. Base hydrolysis was performed in 2 ml of 2 N KOH sealed in vacua under nitrogen in alkali-resistant tubes. The samples were hydrolyzed at 105°C for 24 h. The hydrolysate was neutralized with cold 3 M HClO,. The precipitate KClO, was separated by centrifugation and washed three times with distilled water. Supernatants and washings were pooled and dried on a Buchler evaporator.

Column

Chromatography

Acid. The dried hydrolysates were initially drawn up in 0.2 M sodium citrate, pH 2.2, prior to being eluted on an automatic amino acid analyzer with a buffer of pH 5.28; 2.7-min fractions were collected. Base. The dried hydrolysates were initially drawn up in 0.2 M sodium citrate, pH 2.8, and eluted on an automatic amino acid analyzer with the buffer at pH 5.23.

Scintillation

Counting

Two milliliters of sample, 10 ml of Instagel, and 0.5 ml water were placed in a plastic vial, shaken, and allowed to set at 4°C for 3 h. A Packard Tri-Carb scintillation counter was used. As the samples were double-labeled with 3H and W, the W channel was narrowed to entirely exclude any SH spillover, and all calculations are based on the “C counts.

CROSSLINKS OF CARTILAGE

TABLE I

RESULTS

Reducible Collagen Crosslink as a Function of Age

Composition

High levels of reducible aldimine crosslinks were found in the cartilage from immature rabbits. After reduction the two intermolecular crosslinks identified were HylOHNle and HylNle. No histidinyl hydroxymerodesmosine was found (2). As the animals approached maturity the amount of crosslinks present decreased steadily. At six months of age the levels were about 1/rd and at 1 year only l/lath the level at 1 week (Table I). Appreciable amounts of the reducible aldimine crosslinks persisted in mature articular cartilage. The total counts in the reduced aldimine fractions at 1, 2, and 3 years were much lower than those seen in immature cartilage but they remained level. Two new peaks of 3H activity appeared, however, in the mature cartilage. They were identified by their elution positions as the hexosyl hydroxylysines and hexosyl lysines previously identified by others (14, 15). Uptake of Radioactive Cartilage

207

COLLAGEN

Lysine by Articular

Following placement of lysine directly onto the cartilage surface, autoradiography demonstrated radioisotope incorporation into the chondrocytes throughout the entire articular cartilage, and this became evenly distributed throughout the extracellular matrix. At 20 min almost all the label was intracellular. It was seen in increasing amounts in the matrix at progressively longer time intervals.

DECREASE WITH AGE INTHE CONTENTOF REDUCIBLE ALDIMINE CROSSLINKS IN RABBIT ARTICULAR CARTILAGE"

Age 1 week l-2 months 3-4 months 6 months 1 year 2 years 3 years

“H cpm in HylOHNle and HylNleimg dry tissue (*SD) 796 451 + 119 358 + 160 284 c 103 79 k 46 78 k 36 71 _t 8

Number of specimens 2 9 15 5 9 14 6

u All tissues were reacted in [“H]NaBH, under standard conditions. These cartilage samples were not labeled with [‘4C]lysine. Values are expressed as averages ?SD of 5-15 specimens except for l-week time period.

0.5 to ‘72 h the decrease was rapid. The eventual level attained, which persisted unchanged for several months, showed 52% of the counts in lysine and 48% in hydroxylysine (Fig. 1). The profiles obtained in the borohydride reduced cartilage and in the non-borohydride-reduced cartiIage were the same and were assessed together. Biosynthesis Crosslinks

of Reducible Aldimine in Vivo

In the 14C-labeled cartilage reduced with sodium [3H]borohydride, no crosslink was seen at 30 min. At 1 h, 14Ccounts were seen in the two reducible crosslink peaks. The amount of crosslink present, expressed as a ratio of the 14Ccounts in the two reducible aldimine peaks against the total 14Ccounts Hydroxytation of Lysine in Articular recovered in the hydroxylysine peak conCartilage tinued to increase until 2 weeks. SubAnalysis was made of the relative amount sequently, it fell off and stabilized around and temporal sequence of hydroxylysine 3 months (Table II). The proportion of formation by adding all counts in the recoverable counts in the aldimine fractions lysine and hydroxylysine peaks and deter- remained the same from 3 months following mining the proportion in each peak. One labeling (i.e., rabbit 6 months of age) until half hour following [14CJlysine labeling, 1.5 years. The ratio of the two crosslinks 85% of the counts were in Iysine and 15% changed with the marked early prewere in hydroxylysine. The lysinelhydroxydominance of HylOHNle diminishing with lysine ratio diminished with time. From time (Table III). The shift appeared to be

208

SHAPIRO, BRICKLEY-PARSONS,

AND GLIMCHER

per collagen molecule. For purposes of this assessment it was assumed that there were 60 residues of hydroxylysine (20 per (Ychain) per molecule of collagen. The half-residues per molecule were calculated as (counts per minute in aldimine fractions/counts per minute in hydroxylysine) x60. The highest number of reducible aldimine residues per molecule detected was at 1 week postlabeling (Table V). Biosynthesis of Glycosylated Hydroxylysine Compounds FIG. 1. The progressive hydroxylation of [WIlysine with time is seen. This figure represents 54 separate analyses from 0.5 h to 18 months following labeling. The total numbers of counts in the lysine and hydroxylysine fractions have been added and their ratio calculated.

due to an absolute decrease in HylOHNle. Table IV indicates that the proportion of HylNle relative to hydroxylysine remained steady, except for a slight increase from 24 h to 2 weeks, while the amount of HylOHNle relative to hydroxylysine continued to decline. Crosslink Residues per Collagen

Molecule

The [14C]lysine-labeling technique allowed for an estimation of the crosslink residues

Following reduction with rH]NaBH, and base hydrolysis, synthesis of glucosylgalactosyl hydroxylysine and galactosyl hydroxylysine was detected in immature articular cartilage. The GlcGalHyUGalHyl ratio was 1.4U1.00 at 1 week and 1.47Y1.00at 2 months. Seventy-three percent of the hydroxylysine residues were glycosylated at both time periods. The number of glycosylated hydroxyline residues per collagen molecule was calculated as (cpm GGH + cpm GH/ cpm GGH + cpm GH + hyly) ~60. At 1 week there were 42.7 and at 2 months 44.6 residues per molecule. The Question of in Viva Natural Reduction of the Crosslinks When the 14C-labeled cartilage was not reduced with sodium rH]borohydride prior

FIG. 2. Elution profile of rabbit articular cartilage labeled with [WIlysine retrieved at sacrifice 1 week later. The cartilage was reduced with rH]NaBH, Hydrolysis conditions: 3 N HCl, 105”C, 48 h. Elution: 0.35 M sodium citrate, double peak in fractions 5 to 12 was present in all chromatograms and was lysine metabolites. A, B, C, and D represent unidentified peaks which were the experiment.

at age 3 months and prior to hydrolysis. pH 5.28. The early composed of several constant throughout

CROSSLINKS TABLE

OF CARTILAGE

209

COLLAGEN

II

TABLE

PERCENTAGEOF 14CCOUNTSIN THE REDUCIBLE ALDIMINE FRACTIONSRELATEDTO~OUNTS IN HYDROXYLYSINEFRACTIONS Time following [‘T]lysine labeling

(HylOHNle + HylNle (cpm)/ Hydroxylysine (cpm)) x 100

Number of specimens”

0.5 h lto4h 24to 72 h 1 to 2 weeks 1 month 3 months 6 months 18 months

0 3.56 2 1.05 5.13 k 1.66 5.30 2 2.44 2.44 k 0.83 1.27 1.79 1.04

2 5 6 10 6 2 1 1

” Each sample was from a different animal.

to acid hydrolysis peaks in the aldimine region were not detected. It does not appear that natural reduction of the aldimine crosslinks occurred in viva. It is calculated, considering the limits of resolution with this system, that natural reduction of an amount greater than 15% in the reducible aldimine crosslinks synthesized would have been detected. Nonreduced hydrolysates were done on samples from 1 h to 18 months following labeling. DISCUSSION

The previously described decrease in reducible aldimine crosslinks with age is demonstrated for rabbit articular cartilage. A steady decrease occurs from 1 week of age

III

PROPORTIONSOF% COUNTSINHYDROXYLYSINOHYDROXYNORLEUCINEANDHYDROXYLYSINONORLEUCINEPEAKS Time following 14Clabeling

HylOHNleiHylNle

Number of specimens

12.311.0 5.6/1.0 4.311.0 * 0.94 3.7U.O ? 1.60 2.4/1.0

lh 2-4 h 1.2 weeks 1 month 6 months

2 3 8 6 1

to 1 year of age although reducible aldimines remain detectable and level in mature cartilage. Two additional peaks appear adjacent to the reduced aldimine region in mature articular cartilage and are interpreted to be hexosyl hydroxylysines and hexosyl lysines. These have previously been found in mature tissues, especially cartilage (14, 15). Autoradiographic evidence of the rapid uptake of rH]lysine by chondrocytes throughout all levels of the articular cartilage following direct instillation onto the cartilage surface is demonstrated. This method of labeling provides high concentrations of radioisotope in cartilage without necessitating large systemic injections. Sequential autoradiographs show the appropriate passage of lysine from an early intracellular position to an extracellular, matrix position with time as participation in the biosynthetic pathways occurs.

TABLE IV PROPORTIONALAMOUNTSOFHYDROXYLYSINOHYDROXYNORLEUCINE TOHYDROXYLYSINE ANDOF HYDROXYLYSINONORLEUCINE TOHYDROXYLYSINE~ Time following [%]lysine labeling lto4h 24 to 72 h 1 to 2 weeks 1 month 3 months 6 months 18 months

(cpm HylOHNlei cpm Hydroxylysine) x 100

(cpm HylNlei cpm Hydroxylysine) x 100

4.33 4.30 4.23 1.89 1.89 1.26 0.79

n Determined from 14Ccounts following [3H]NaBH, reduction.

0.61 0.98 1.07 0.55 0.55 0.53 0.24

Number of specimens 6 6 10 6 2 1 1

210

SHAPIRO, BRICKLEY-PARSONS,

AND GLIMCHER

of the two reducible aldimine crosslinks are seen at 2 weeks whereupon they rapidly ESTIMATION OF CROSSLINKHALF-RESIDUES diminish. The decrease is far too rapid to be PER COLLAGENMOLECULE~*~ accounted for by cohagen turnover and Time following Number of synthesis of newer, nonlabeled crosslinks 14Clabeling Residues/molecule specimens and indicates that the molecules themselves are transformed to as yet unidentified lto4h 2.14 ? 0.56 5 nonreducible compounds. No evidence for 24 to 72 h 3.09 5 0.99 6 natural reduction in tivo of the two aldimine 1 week 3.10 2 1.08 6 crosslinks is seen. When the ‘*C-labeled 2 weeks 2.29 3 cartilage is hydrolyzed without prior sodium 1.47 * 0.50 6 1 month borohydride reduction, labeled peaks do not 3 months 0.78 2 appear in the aldimine region. Had reduction 1 6 months 1.08 occurred in viva the aldimines would have 18 months 0.63 1 been stable to hydrolysis and identifiable. (1Calculated as (cpm in aldiminesicpm in hydroxyIt remains most likely that the reducible lysine) x 60 residues (hydroxylysine). aldimines are incorporated into other crossb Note: these numbers are residues per molecule of link compounds but although new peaks hydroxylysine that are involved in aldimine crosslinks. were carefully searched for none were The numbers of moles of aldimine crosslinks per collagen identified with this system. It is important molecule are half these values. to note, however, that some of the reducible aldimines persist well into mature One-half hour following instillation the life presumably failing to progress to nonmajority of counts are in lysine but the reducible forms. Alternatively, this finding 15% of radioactivity in hydroxylysine might mean that new aldimines and perhaps indicates that hydroxylation and collagen new aldehydes continue to be produced at synthesis are occurring. At this time no low levels in existing collagen long after it crosslinks are detectable. The lysine/hyhas been deposited in the matrix. droxylysine ratio decreases with time as Initially HylOHNle is the major crossprogressively more lysine is hydroxylated link synthesized in terms of amount. The during collagen syntheses. Lysine is present ratio of the two crosslinks decreases with in articular cartilage collagen but also in time and evidence is presented that this is noncollagenous cell and matrix proteins due primarily to an absolute decrease in such as the proteoglyean link proteins HylOHNle. The overall diminution in (16) which have a much faster synthesis reducible crosslink levels therefore is mainly and turnover rate. All the [14C]lysine accounted for by modification of deHylOHNle therefore is not incorporated initially into to mature, nonreducible structures that collagen which accounts for the period of remain to be identified. time required for hydroxylysine and lysine Synthesis of glucosylgalactosyl hydroxyto establish the approximate 1:l ratio that lysine and galactosyl hydroxylysine is also exists in type II collagen of cartilage. A demonstrated for rabbit articular cartilage similar temporal sequence has been seen using this system. following a single intraarticular injection of rH]proline in the rabbit knee (17). ACKNOWLEDGMENTS The biosynthesis of the two reducible This work was supported in part by grants from aldimine crosslinks of rabbit articular the National Institutes of Health (AM 156’71), The cartilage is seen initiahy 1 h following injection. The levels detected increase and John A. Hartford Foundation, Inc., and the New peak at 2 weeks. Their diminution cor- England Peabody Home for Crippled Children, Inc. responds to the diminished content demonREFERENCES strated with increasing age, however the 14Clabel provides an excellent indication of 1. GALLOP, P. M., AND PAZ, M. A. (1976) Physiol. the temporal aspect of change for molecules Rev. 55,418-487. labeled at the same time. The peak levels 2. TANZER, M. L. (1976)in Biochemistry of Collagen TABLE V

CROSSLINKS

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9. 10.

OF CARTILAGE

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17. REPO, R. U., AND MITCHELL, N. (1971) J. Bone Joint Surg. 53B, 541-548.