Aggrecan Turnover in Human Intervertebral Disc as Determined by the Racemization of Aspartic Acid

We have used the racemization of aspartic acid as a marker for the “molecular age” of aggrecan components of the human intervertebral disc matrix (aggregating and non-aggregating proteoglycans as well as the different buoyant density fractions of aggrecan). By measuring the d/lAsp ratio of the various aggrecan species as a function of age and using the values of the racemization constant, ki, found earlier for aggrecan in articular cartilage, we were able to establish directly the relative residence time of these molecules in human intervertebral disc matrix. For A1 preparations taken from normal tissue, turnover rates of 0.059 ± 0.01 and 0.063 ± 0.01/year correspond to half-life values of 12 ± 2.0 and 11.23 ± 1.9 years for nucleus pulposus and annulus fibrosus, respectively; the turnover rates of 0.084 ± 0.022 and 0.092 ± 0.034/year for degenerate tissue correspond to half-life values of 8.77 ± 2.2 and 8.41 ± 2.8 years, suggesting increased rate of removal of small aggrecan fragments. For the large monomer, fraction A1D1, turnover is 0.13 ± 0.04/year, corresponding to a half-life of 5.56 ± 1.58 years, similar to 3.4 years in human articular cartilage. For the binding region (A1D6), turnover is 0.033 ± 0.0012/year, corresponding to a half-life of 21.53 ± 0.6 years, similar to 23.5 years in articular cartilage. A1 preparations from nucleus pulposus contain a lower proportion of aggregating proteoglycans as compared with annulus fibrosus, suggesting increased proteolytic modification in the nucleus pulposus. d/lAsp values in aggregating and non-aggregating proteoglycans of a 24-year-old individual show similar results, suggesting that the non-aggregating molecules are synthesized initially as aggregating proteoglycans, which thereafter undergo cleavage and detachment from hyaluronan. We have used the racemization of aspartic acid as a marker for the “molecular age” of aggrecan components of the human intervertebral disc matrix (aggregating and non-aggregating proteoglycans as well as the different buoyant density fractions of aggrecan). By measuring the d/lAsp ratio of the various aggrecan species as a function of age and using the values of the racemization constant, ki, found earlier for aggrecan in articular cartilage, we were able to establish directly the relative residence time of these molecules in human intervertebral disc matrix. For A1 preparations taken from normal tissue, turnover rates of 0.059 ± 0.01 and 0.063 ± 0.01/year correspond to half-life values of 12 ± 2.0 and 11.23 ± 1.9 years for nucleus pulposus and annulus fibrosus, respectively; the turnover rates of 0.084 ± 0.022 and 0.092 ± 0.034/year for degenerate tissue correspond to half-life values of 8.77 ± 2.2 and 8.41 ± 2.8 years, suggesting increased rate of removal of small aggrecan fragments. For the large monomer, fraction A1D1, turnover is 0.13 ± 0.04/year, corresponding to a half-life of 5.56 ± 1.58 years, similar to 3.4 years in human articular cartilage. For the binding region (A1D6), turnover is 0.033 ± 0.0012/year, corresponding to a half-life of 21.53 ± 0.6 years, similar to 23.5 years in articular cartilage. A1 preparations from nucleus pulposus contain a lower proportion of aggregating proteoglycans as compared with annulus fibrosus, suggesting increased proteolytic modification in the nucleus pulposus. d/lAsp values in aggregating and non-aggregating proteoglycans of a 24-year-old individual show similar results, suggesting that the non-aggregating molecules are synthesized initially as aggregating proteoglycans, which thereafter undergo cleavage and detachment from hyaluronan. The intervertebral disc (IVD) 2The abbreviations used are: IVD, intervertebral disc; NP, nucleus pulposus; AF, annulus fibrosus; PG, proteoglycan; GAG, glycosaminoglycan; CS, chondroitin sulfate; ANOVA, analysis of variance. is the largest avascular cartilaginous structure. It lies between the vertebral bodies, anchoring them together. IVD plays a primarily mechanical role in transmitting loads through the spine and providing flexibility to the spinal column. The IVD is highly bradytrophic; it is avascular and nourished by diffusion. Although the outer annulus fibrosus (AF) possesses blood vessels in early childhood, the inner nucleus pulposus (NP) remains avascular for the entire life of the organism (1Hirsch S. Schajowicz F. Acta Orthop. Scand. 1953; 22: 184-231Crossref Scopus (221) Google Scholar, 2Maroudas A. Stockwell R.A. Nachemson A. Urban J. J. Anat. 1975; 120: 113-130PubMed Google Scholar). The disc has a complex structure and contains very few cells embedded in an extracellular matrix. These cells have the essential function of maintaining and repairing the matrix by synthesizing matrix macromolecules and by producing proteinases for matrix breakdown. Thus, disc function is dependent on a balance between synthesis and matrix breakdown. Normally, when this balance is maintained, damaged tissue can be restored by cellular repair responses. In pathology, when there is imbalance between matrix synthesis and breakdown, the matrix composition and organization are altered, and the cellular repair responses are inadequate. Hence, the degraded matrix can no longer carry loads effectively, which leads to the degeneration of the disc. During aging, many changes involving the proportions and biochemical properties of aggrecan occur. In particular, the structure and composition of aggrecan changes both with aging and with degeneration. These changes involve an increase in the relative contents of keratan sulfate and protein and a decrease in the molecular weight of aggrecan (3Bayliss M.T. Methods in Cartilage Research. Academic Press, London1990: 220-222Google Scholar). The important question of whether these changes in composition of the aggrecan with aging represent changes in biosynthesis or are due to the accumulation of degraded aggrecan fragments can be addressed by measuring the accumulation of the d-aspartic acid isomer and hence the residence time or the “molecular age” of these molecules. In nature, amino acids are synthesized as l-isomers. Spontaneous racemization slowly converts the l-form of amino acids into a racemic mixture of l- and d-forms. Aspartic acid is one of the most rapidly racemizing amino acids (4Helfman P.M. Bada J.L. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2891-2894Crossref PubMed Scopus (270) Google Scholar, 5Bada J.L. Methods Enzymol. 1984; 106: 98-116Crossref PubMed Scopus (133) Google Scholar), allowing the measurement of the concentration of d-isomers in living subjects in proteins that are not renewed or that slowly turn over. It is well known that an age-dependent racemization occurs in various human and animal tissues containing metabolically stable, long-lived proteins, e.g. in enamel and dentine (4Helfman P.M. Bada J.L. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2891-2894Crossref PubMed Scopus (270) Google Scholar), white matter of the brain (6Man E.H. Sandhouse M.E. Burg J. Fisher G.H. Science. 1983; 220: 1407-1408Crossref PubMed Scopus (194) Google Scholar), eye lens (7Masters P.M. Bada J.L. Zigler J.S.J. Nature. 1977; 268: 71-73Crossref PubMed Scopus (291) Google Scholar, 8Helfman P.M. Bada J.L. Nature. 1976; 262: 279-281Crossref PubMed Scopus (303) Google Scholar), aorta (9Powell J.T. Vine N. Grossman M. Atherosclerosis. 1992; 97: 201-208Abstract Full Text PDF PubMed Scopus (206) Google Scholar), cartilage and skin (10Verzijl N. DeGroot J. Thorpe S.R. Bank R.A. Shaw J.N. Lyons T.J. Bijlsma J.W.J. Lafeber F.P.J.G. Baynes J.W. TeKoppele J.M. J. Biol. 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In the present work, turnover of the different populations of proteoglycans (PGs) in human IVD (aggregating and non-aggregating PGs, as well as different buoyant density fractions of the aggrecan) has been determined using racemization of aspartic acid as a tool for assessing the molecular age of long-lived proteins. By using the published racemization constant (ki) earlier found for articular cartilage (15Maroudas A. Bayliss M.T. Uchitel-kaushansky N. Schneiderman R. Gilav E. Arch. Biochem. Biophys. 1998; 350: 61-71Crossref PubMed Scopus (180) Google Scholar), we were able to determine the turnover rate of the different PG populations and a possible relationship with respect to their molecular age and origin. Tissue Sampling—Lumbar and thoracic spines from healthy (ages 0–62, n = 15) and pathological (ages 30–77, n = 14) discs (usually one from each individual) were obtained postmortem or during routine surgical procedures for treatment of herniation or disc degeneration. In this study, healthy discs will be referred to as “normal,” and pathological ones will be referred to as “degenerate.” All discs were divided into NP and AF zones, diced or cryosectioned into 20-μm slices, and stored at –20 °C until analyzed. Extraction of Aggrecan (A1)—For extraction of A1 from NP and AF, 10 volumes of 4 m guanidinium chloride (GuHCl) containing proteinase inhibitors in 50 mm Tris buffer, pH 7.4, at 4 °C for 48 h were used (16Bayliss M.T. Ali S.Y. Biochem. J. (1978). 1978; 176: 683-693Crossref PubMed Scopus (126) Google Scholar). The extracts were clarified by centrifugation at 4 °C, 10,000 rpm for 40 min and exhaustively dialyzed against the same buffer containing no GuHCl. A1 preparations were separated from other tissue proteins by associative cesium chloride (CsCl) equilibrium density gradient centrifugation at a starting density of 1.5 g/ml using 50,000 rpm at 10 °C for 48 h (17Bayliss M.T. Roughley P.J. Biochem. J. 1985; 232: 111-117Crossref PubMed Scopus (30) Google Scholar). A1 was then recovered from the densest fraction of the gradient (density greater that 1.59 g/ml CsCl). All A1 fractions were assayed for density, exhaustively dialyzed against distilled water, freeze-dried, and analyzed for glycosaminoglycan (GAG), (18Farndale R.W. Buttle D.J. Barrett A.J. Biochim. Biophys. Acta. 1986; 883: 173-177Crossref PubMed Scopus (2907) Google Scholar) and protein content. The A1 preparations contain aggrecan and its degradation products but not the small PGs, which are present in the tissue but which at a lower buoyant of Aggrecan fractions were separated by of density gradient centrifugation in the of 4 m mm Tris buffer, pH at a starting density of 1.5 g/ml °C, 50,000 rpm for 48 and into aggrecan of buoyant density and molecular weight (15Maroudas A. Bayliss M.T. Uchitel-kaushansky N. Schneiderman R. Gilav E. Arch. Biochem. Biophys. 1998; 350: 61-71Crossref PubMed Scopus (180) Google Scholar). All fractions were assayed for density, exhaustively dialyzed against distilled water, freeze-dried, and analyzed for (18Farndale R.W. Buttle D.J. Barrett A.J. Biochim. Biophys. Acta. 1986; 883: 173-177Crossref PubMed Scopus (2907) Google Scholar) and protein content. In and aggrecan were recovered from the aggregating of the A1 fraction by the A1 preparations were by through a at a rate of using m pH as the were divided into aggregating at the and non-aggregating in the and The aggregating PG was to the aggrecan by of density gradient centrifugation in the of 4 m at a starting density of 1.5 The fractions were assayed for and protein of of from of the tissue were analyzed using the binding (18Farndale R.W. Buttle D.J. Barrett A.J. Biochim. Biophys. Acta. 1986; 883: 173-177Crossref PubMed Scopus (2907) Google Scholar), with chondroitin sulfate as a acid as a of was determined by the Biochem. PubMed Scopus Google Scholar). acid is present in the human IVD at between and of the on age and Biochem. J. 1976; PubMed Scopus Google Scholar), and will to the of by acid content. its extraction and lower will its in the buoyant density A1 It is that acid to the of by the of of d/lAsp ratio was determined by to and (10Verzijl N. DeGroot J. Thorpe S.R. Bank R.A. Shaw J.N. Lyons T.J. Bijlsma J.W.J. Lafeber F.P.J.G. Baynes J.W. TeKoppele J.M. J. Biol. Chem. 2000; 275: 39027-39031Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar, Biochem. 1984; PubMed Scopus Google Scholar). All d/lAsp were for the of and present in and for racemization during the were by the of the for d/lAsp age or by the d/lAsp ratio for A1 preparations from human or from bone of and rates and of A1 and fractions were on by Maroudas (15Maroudas A. Bayliss M.T. Uchitel-kaushansky N. Schneiderman R. Gilav E. Arch. Biochem. Biophys. 1998; 350: 61-71Crossref PubMed Scopus (180) Google Scholar, A. Gilav E. Tissue 1992; PubMed Scopus Google Scholar) on turnover with for aggrecan and are in in the of the in accumulation between NP and AF and aggregating and non-aggregating was determined using The of the in accumulation between normal and degenerate tissue and between age and from A1 or fractions was analyzed using the and was to represent analysis of the increase in protein to for normal and degenerate tissue NP and was The of of the in between NP and AF was at the = of in Aggrecan from and the values of d/lAsp for the A1 fractions obtained from NP and AF of normal and degenerate human IVD as a function of to the d/lAsp values were as of aspartic acid as the ratio in the A1 preparations with age in a = = and n = = for normal NP and AF, and n = = and n = = for degenerate NP and AF, was in the rate of accumulation of the with age in NP as compared with AF for both normal and degenerate For d/lAsp for of obtained from normal tissue values of normal NP and are in the same In to the entire aggrecan no is for aggrecan is known to be with respect to molecular and composition J. Biol. Chem. 1977; Full Text PDF PubMed Google Scholar, J. Matrix Biol. PubMed Scopus Google Scholar), the increase of d/lAsp for A1 taken with the of the values that there is a turnover of the various aggrecan the accumulation of in A1 obtained from NP of normal and degenerate human IVD as a function of ANOVA, a was between the values of d/lAsp for normal and degenerate tissues age The values of d/lAsp are lower in degenerate aggrecan as compared with For in the age accumulation of the was as in the normal discs as compared with the degenerate in accumulation between age normal and degenerate was between and and between and in accumulation are between normal and degenerate tissue and as a function of we that the during aging are and during degeneration. were obtained for AF not the of the of A1D1, the d/lAsp ratio was determined for each of the different buoyant density fractions from the gradient of It is well known that cartilage the density gradient to their molecular the the the greater the density at which the J. Biol. Chem. 1977; Full Text PDF PubMed Google Scholar). the of the ratio of taken from normal NP of and in a density gradient at a starting density of 1.5 Thus, the fraction contains the of weight of the fractions represent of aggrecan of content. The fraction contains and the decrease in weight of protein with age is The composition of to from a NP of a individual is in of aggrecan of different buoyant density obtained from nucleus pulposus of a weight recovered of of weight of density in a 4 a between d/lAsp of normal and degenerate for different age and are the between NP and The was due to the small of and was due to the that no was found between the values of d/lAsp for NP as compared with AF ANOVA, a of between the different in accumulation between and was found between and normal as well as between degenerate and normal of in the degenerate is similar to that in the normal age due to increased protein turnover in the degenerate tissue values of obtained from NP and AF of normal and degenerate human intervertebral ± ± ± ± ± ± ± ± 2.8 in a was found for the A1 an increase in accumulation of the is in It was found to be the in from both normal and degenerate as in the of articular cartilage N. DeGroot J. Bank R. Bayliss M.T. Johannes W.J.B. Lafeber F.P.J.G. Maroudas A. TeKoppele J.M. Matrix Biol. 2001; 20: 409-417Crossref PubMed Scopus (97) Google this is the for from the aggregating PG fraction by of the A1 preparations on not The of in with age as in obtained from and cartilage (15Maroudas A. Bayliss M.T. Uchitel-kaushansky N. Schneiderman R. Gilav E. Arch. Biochem. Biophys. 1998; 350: 61-71Crossref PubMed Scopus (180) Google Scholar). between and the of racemization is and that the racemization rate is a constant for each these that the large in and composition these lower density fractions is by a The and turnover rate is for the large the values of d/lAsp were for the and normal discs (ages and In the of the lower accumulation was for the degenerate tissue as compared with the normal tissue the of modification of the aggrecan structure and accumulation of was to the A1 a from a healthy 24-year-old of fractions aggregating fractions large non-aggregating fractions and small non-aggregating fractions For the AF A1 preparations from and the aggregating for of the For the NP A1 preparations from the same the aggregating for and of the PGs, Thus, the A1 preparations from the NP contain a lower proportion of aggregating from the AF, suggesting a greater of proteolytic modification in the The fractions were with respect to their d/lAsp content. The that no was between d/lAsp of aggregating and non-aggregating fractions and from the 24-year-old individual that the non-aggregating were synthesized at the same time as the aggregating and that both PG similar in the The that the d/lAsp ratio of the non-aggregating is lower that of aggregating in the individual that of the non-aggregating are and it is most that these are the with of there is degradation of the non-aggregating PGs, and a small will be from the disc by diffusion. that the non-aggregating molecules will be from the aggrecan molecules and hence will be of the content. The that similar d/lAsp values were obtained for the non-aggregating of the individual that a balance between the of small fragments from and cleavage of the fragments from the to fragments present in of Aggrecan and the that the A1 preparations are very in of aggregating and non-aggregating we it to the half-life values of the protein in these preparations obtained from normal and degenerate the racemization rate constant of l- to of aspartic acid (ki) of found for articular cartilage (15Maroudas A. Bayliss M.T. Uchitel-kaushansky N. Schneiderman R. Gilav E. Arch. Biochem. Biophys. 1998; 350: 61-71Crossref PubMed Scopus (180) Google Scholar), and the obtained from the racemization we were able to the rate constant for protein turnover and the corresponding half-life for A1 preparations as well as for the aggrecan as in the for which to the increase in the of protein with are from For A1 preparations taken from normal tissue, turnover rates of 0.059 ± 0.01 and 0.063 ± 0.01/year correspond to half-life values of 12 ± 2.0 and 11.23 ± 1.9 years for NP and AF, respectively; the turnover rates of 0.084 ± 0.022 and 0.092 ± 0.034/year for degenerate tissue correspond to half-life values of 8.77 ± 2.2 and 8.41 ± 2.8 years suggesting an increased rate of removal of small aggrecan fragments. The large is due to the of the A1 The protein turnover rate for aggrecan from normal tissue was NP and AF The of the turnover rate constant for the large in fraction is 0.13 ± which to a half-life of 5.56 ± 1.58 years as compared with 3.4 years in human articular cartilage (15Maroudas A. Bayliss M.T. Uchitel-kaushansky N. Schneiderman R. Gilav E. Arch. Biochem. Biophys. 1998; 350: 61-71Crossref PubMed Scopus (180) Google Scholar), the turnover for the binding region is 21.53 ± 0.6 years as compared with 23.5 years found for articular cartilage (15Maroudas A. Bayliss M.T. Uchitel-kaushansky N. Schneiderman R. Gilav E. Arch. Biochem. Biophys. 1998; 350: 61-71Crossref PubMed Scopus (180) Google values of aggrecan taken from normal human ± ± ± ± ± ± ± ± ± ± ± ± 0.6 in a In this an was to determine the turnover rates and half-life values of different PG populations in human IVD by using the accumulation of the isomer as a marker for residence time in the turnover of a species in the tissue a residence time and hence a of and repair of matrix on the other that it is for cells to repair which with of the residence time of a molecular species is one to changes in composition to of synthesis or Aggrecan from human IVD in that with respect to the and composition of the protein as well as the of the is very similar to that of aggrecan in articular cartilage (15Maroudas A. Bayliss M.T. Uchitel-kaushansky N. Schneiderman R. Gilav E. Arch. Biochem. Biophys. 1998; 350: 61-71Crossref PubMed Scopus (180) Google Scholar). of these during and degeneration of the tissue and to a of molecular M.T. Biochem. PubMed Scopus Google Scholar). A1 by of gradient centrifugation show changes from the fraction at the buoyant density to that recovered from the buoyant density These changes an increase in the relative of a decrease in the relative of acid and hence of a decrease in the relative of and hence in the of keratan sulfate and a decrease in the ratio of to A1 as a an increase in the of protein with is due to a very turnover of of 21.53 ± 0.6 as compared with a turnover of the large monomer, of 5.56 ± 1.58 It is the small molecular species that are in protein that with These turnover products be to or be For the aggrecan species in the the monomer, the d/lAsp ratio remains constant on the other for the A1 as a there is an increase with age in the d/lAsp ratio until a is years of that between and years, the rate of racemization is the rate of years of these are that for the of in disc not to increase age the rate of synthesis of is to increase as disc PG with the rate of of the well which is as the proportion of non-aggregating with suggesting to lower It is the of these small fragments with the of the disc to them by synthesis of aggrecan that in the decrease in disc PG content. not in of the non-aggregating but in the of and protein due to R. Roughley P.J. Biochem. J. PubMed Scopus Google Scholar). due to between accumulation of the in A1 and A1D1, we that the A1 contain with a lower turnover rate and hence longer residence time that of the in the The in and the of buoyant density aggrecan with longer residence time in the It is for these fractions that the ratio d/lAsp with It is from that the turnover rate constant for A1 is lower for for A1D1, the turnover rate constant is found to be which to a half-life of 5.56 ± 1.58 years, for a turnover rate constant of NP and of ± 0.01/year to a half-life of ± 2.0 The that the turnover rate constant, was the same for the NP and AF in both normal and degenerate discs is these tissue have different which different as well as different of the matrix. We have no for this It is important to that in d/lAsp was lower in aggrecan from degenerate tissue as compared with degenerate aggrecan is this is in with published for articular cartilage N. DeGroot J. Bank R. Bayliss M.T. Johannes W.J.B. Lafeber F.P.J.G. Maroudas A. TeKoppele J.M. Matrix Biol. 2001; 20: 409-417Crossref PubMed Scopus (97) Google Scholar). that of turnover and hence lower of accumulation are present in degenerate It is important to that the of the between the molecular of different PG species from the of d/lAsp was on the that the racemization rate constant, ki, of and is the same the protein was by the that there were between the d/lAsp of the and fractions of subjects and in which changes are small and not have the in the racemization rate these been present was to the question the of the small aggregating as well as the non-aggregating in human IVD, using the racemization of aspartic acid as a tool for in turnover rates of different PG in both normal and pathological that the non-aggregating of a normal 24-year-old have residence that are similar to of the aggregating and hence are of similar molecular age that the non-aggregating were synthesized at the same time as aggregating It is that the non-aggregating were synthesized as aggregating that were by The PG fragments that were no longer to but were the then the non-aggregating P.J. J. Google Scholar). of non-aggregating fragments of aggrecan to be to the IVD and not in articular cartilage. the large and avascular of the which of large macromolecules by diffusion. The that the non-aggregating were synthesized as is as there is no to the of We for the in and for We the of the in as well as human will be with

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