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• W2023702933 abstract "Samples of aggrecan chondroitin sulfate, isolated from normal human knee cartilages of individuals from fetal to 72 years of age, were digested with chondroitin lyases. The products were analyzed by fluorescence-based anion exchange high performance liquid chromatography to separate and quantitate nonreducing terminal structures, in addition to internal unsaturated disaccharide products. The predominant terminal structures were the monosaccharides, GalNAc4S and GalNAc4,6S as they were present on 85–90% of all chains. The remaining chains terminated with the disaccharides GlcAβ1,3GalNAc4S and GlcAβ1,3GalNAc6S. Marked changes in the relative abundance of these terminals were identified in the transition from growth cartilage to adult articular cartilage. First, terminal GalNAc residues were almost exclusively 4-sulfated in aggrecan from fetal through 15 years of age, but were ∼50% 4,6-disulfated in aggrecans from adults (22–72 years of age). Second, the terminal disaccharide GlcAβ1,3GalNAc4S was on ∼7% of chains on aggrecan from fetal through 15 years of age, but on only ∼3% of chains on adult aggrecan. In contrast, the proportion of chains terminating in GlcAβ1,3GalNAc6S, ∼9%, was unchanged from fetal to 72 years of age. This terminal disaccharide is proposed to be recognized by the widely used monoclonal antibody 3B3. However, chemical quantitation of the structure together with solid phase 3B3(−) immunoassay of fetal and adult aggrecans showed that the content of the terminal disaccharide does not necessarily correlate with immunoreactivity of the proteoglycan, as chain density and presentation on the solid phase are critical factors for recognition of chain terminals by 3B3. The quantitative results obtained from chemical analyses of all nonreducing termini of aggrecan chondroitin sulfate chains revealed important changes in chain termination that occur when cellular activities are altered as adult articular cartilage is formed after removal of growth cartilage. These findings are discussed in relation to specific enzymatic steps that generate the nonreducing termini of chains in the biosynthesis pathway of chondroitin sulfate proteoglycans and their modulation in tissue development and pathology. Samples of aggrecan chondroitin sulfate, isolated from normal human knee cartilages of individuals from fetal to 72 years of age, were digested with chondroitin lyases. The products were analyzed by fluorescence-based anion exchange high performance liquid chromatography to separate and quantitate nonreducing terminal structures, in addition to internal unsaturated disaccharide products. The predominant terminal structures were the monosaccharides, GalNAc4S and GalNAc4,6S as they were present on 85–90% of all chains. The remaining chains terminated with the disaccharides GlcAβ1,3GalNAc4S and GlcAβ1,3GalNAc6S. Marked changes in the relative abundance of these terminals were identified in the transition from growth cartilage to adult articular cartilage. First, terminal GalNAc residues were almost exclusively 4-sulfated in aggrecan from fetal through 15 years of age, but were ∼50% 4,6-disulfated in aggrecans from adults (22–72 years of age). Second, the terminal disaccharide GlcAβ1,3GalNAc4S was on ∼7% of chains on aggrecan from fetal through 15 years of age, but on only ∼3% of chains on adult aggrecan. In contrast, the proportion of chains terminating in GlcAβ1,3GalNAc6S, ∼9%, was unchanged from fetal to 72 years of age. This terminal disaccharide is proposed to be recognized by the widely used monoclonal antibody 3B3. However, chemical quantitation of the structure together with solid phase 3B3(−) immunoassay of fetal and adult aggrecans showed that the content of the terminal disaccharide does not necessarily correlate with immunoreactivity of the proteoglycan, as chain density and presentation on the solid phase are critical factors for recognition of chain terminals by 3B3. The quantitative results obtained from chemical analyses of all nonreducing termini of aggrecan chondroitin sulfate chains revealed important changes in chain termination that occur when cellular activities are altered as adult articular cartilage is formed after removal of growth cartilage. These findings are discussed in relation to specific enzymatic steps that generate the nonreducing termini of chains in the biosynthesis pathway of chondroitin sulfate proteoglycans and their modulation in tissue development and pathology. Proteoglycans (PGs) 1The abbreviations used are: PG, proteoglycan; GAG, glycosaminoglycan; CS, chondroitin sulfate; ΔDi0S, 2-acetamido-2-deoxy-3-O-β-d-gluco-4-enepyronosyluronic acid; ΔDi4S, 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyronosyluronic acid)-4-O-sulfo-d-galactose; ΔDi6S, 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyronosyluronic acid)-6-O-sulfo-d-galactose; ΔDiSE, 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyronosyluronic acid)-4,6-di-O-sulfo-d-galactose; GlcA, glucuronic acid; GalNAc4S, N-acetylgalactosamine-4-sulfate; GalNAc6S, N-acetylgalactosamine-6-sulfate; GalNAc4,6S,N-acetylgalactosamine-4,6 disulfate; Δdisaccharide, unsaturated disaccharide; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline. are important components of extracellular matrices. Our current understanding of the structure-function relationships of these macromolecules is well developed with regard to the core proteins, which often confer on the molecule the capacity for specific and ordered distributions of the PGs within the extracellular matrix. However, for all PGs, glycosaminoglycans (GAGs) constitute a significant proportion of their molecular mass. Aggrecan, the major PG in all cartilaginous matrices, contains chondroitin sulfate chains (CS) (about 100 per molecule) as the predominant GAG. They provide a high fixed charge density to the extracellular matrix that maintains hydration and gives cartilage its characteristic ability to withstand compressive loads (1Buschman M.D. Grodzinsky A.J. J. Biomech. Eng. 1995; 117: 179-192Crossref PubMed Scopus (269) Google Scholar, 2Kovack I.S. Biophys. Chem. 1994; 53: 181-187Crossref Scopus (31) Google Scholar). Moreover, distinct fine structures of individual GAG chains, such as chain length (3Roughley P.J. White R.J. J. Biol. Chem. 1980; 255: 217-224Abstract Full Text PDF PubMed Google Scholar, 4Thonar E.J.-M.A. Buckwalter J.A. Kuettner K.E. J. Biol. Chem. 1986; 261: 2467-2474Abstract Full Text PDF PubMed Google Scholar, 5Deutsch A.J. Midura R.J. Plaas A.H.K. J. Orthop. Res. 1995; 13: 230-239Crossref PubMed Scopus (29) Google Scholar) or sulfate ester substitution (5Deutsch A.J. Midura R.J. Plaas A.H.K. J. Orthop. Res. 1995; 13: 230-239Crossref PubMed Scopus (29) Google Scholar, 6Roughley P.J. White R.J. Glant T. Pediatr. Res. 1987; 22: 406-413Crossref Scopus (16) Google Scholar, 7Bayliss, M. T., Davidson, C., Woodhouse, S. M. & Osborne, D. J. (1995) Acta Orthop. Scand. , 66, Suppl. 266, 22–25.Google Scholar), may generate microenvironments that could facilitate or hinder the diffusivity of other macromolecules through tissues such as cartilage (8Snowden F. Maroudas A. Biochim. Biophys. Acta. 1976; 428: 726-740Crossref PubMed Scopus (30) Google Scholar, 9Van den Berg W.P. Van Lent P.L. Van de Putte L.B. Zwarts W.A. Clin. Immunol. Immunopathol. 1986; 39: 187-197Crossref PubMed Scopus (21) Google Scholar). Immunohistochemical studies with antibodies raised to determinants on CS (10Jenkins R.B. Hall T. Dorfmann A. J. Biol. Chem. 1981; 256: 8279-8282Abstract Full Text PDF PubMed Google Scholar, 11Yamagata M. Kimata K. Oike Y. Tani K. Maeda N Yoshida K. Shinomura Y. Yoneda M. Suzuki S. J. Biol. Chem. 1987; 262: 4146-4152Abstract Full Text PDF PubMed Google Scholar, 12Hardingham T.E. Fosang A.J. Hey N.J. Hazell P.K. Kee W.J. Ewings R.J.F. Carbohydr. Res. 1994; 255: 241-254Crossref PubMed Scopus (23) Google Scholar, 13Karamonos N.K. Hjerpe A. Aletras A. Tsegenidis T. Anastassiou E.D. Antonopoulus C.A. Arch. Biochem. Biophys. 1995; 316: 100-109Crossref PubMed Scopus (9) Google Scholar, 14Sorrell J.M. Carrino D.A. Caplan A.I. Int. J. Dev. Neurosci. 1996; 14: 233-248Crossref PubMed Scopus (18) Google Scholar) provide evidence that tissue specific and developmentally regulated changes occur in fine structures within CS chains. In this regard, much interest has focused on immunological analysis of cartilage with monoclonal antibody 3B3 (15Caterson B. Mohmoodian F. Sorrell J.M. Hardingham T.E. Bayliss M.T. Carney S.L. Ratcliff A. Muir H.M. J. Cell Sci. 1990; 97: 411-417Crossref PubMed Google Scholar) as an indication for the presence of particular structures at the nonreducing termini of CS chains. This IgM monoclonal antibody was raised against chondroitinase-digested rat chondrosarcoma aggrecan (16Christner J.E. Caterson B. Baker J.E. J. Biol. Chem. 1980; 255: 7102-7105Abstract Full Text PDF PubMed Google Scholar) and shows a preference (17Couchman J.R. Caterson B. Christner J.E. Baker J.R. Matrices and Cell Differentiation. A. Liss, New York1984: 31-46Google Scholar), but not an absolute specificity (18Baker J.R Christner J.E. Ekborg S.L. Biochem. J. 1991; 273: 237-239Crossref PubMed Scopus (14) Google Scholar), for the 6-sulfated isomer of the CS Δdisaccharide (ΔDi6S). However, some CS PGs also react with the antibody without prior chondroitinase ABC digestion, and this reactivity is referred to as 3B3(−). Based on the reports that 3B3(−) reactivity is lost after periodate oxidation under conditions that should destroy nonreducing terminal GlcA residues (15Caterson B. Mohmoodian F. Sorrell J.M. Hardingham T.E. Bayliss M.T. Carney S.L. Ratcliff A. Muir H.M. J. Cell Sci. 1990; 97: 411-417Crossref PubMed Google Scholar), it has been proposed that the sequence GlcAβ1,3GalNAc6S (Di6S) at the nonreducing termini of CS chains is the “mimotope” recognized by the antibody (19Ratcliff A. Shurety W. Caterson B. Arthritis Rheum. 1993; 36: 543-551Crossref PubMed Scopus (65) Google Scholar, 20Caterson B. Hughes C.S. Kuettner K.E. Goldberg V.M. Osteoarthritic Disorders. AAOS, Rosemont, IL1995: 315-327Google Scholar). Qualitative analyses of 3B3(−) reactivities have largely been interpreted as demonstrating the presence or absence of Di6S termini on CS PGs at different stages of development, maturity, or pathology of connective tissues (19Ratcliff A. Shurety W. Caterson B. Arthritis Rheum. 1993; 36: 543-551Crossref PubMed Scopus (65) Google Scholar, 20Caterson B. Hughes C.S. Kuettner K.E. Goldberg V.M. Osteoarthritic Disorders. AAOS, Rosemont, IL1995: 315-327Google Scholar, 21Byers S. Caterson B. Hopwood J.J. Foster B.K. J. Histochem. Cytochem. 1992; 40: 275-282Crossref PubMed Scopus (47) Google Scholar, 22Slater R.R. Bayliss M.T. Lachiewicz P.F. Visco D.M. Caterson B. Arthritis Rheum. 1995; 38: 655-659Crossref PubMed Scopus (76) Google Scholar, 23Visco D.M. Johnstone B. Hill M.A. Jolly G.A. Caterson B. Arthritis Rheum. 1993; 12: 1718-1725Crossref Scopus (83) Google Scholar, 24Carlson C.S. Loeser R.F. Johnstone B. Tulli H.M. Dobson D.B. Caterson B. J. Orthop. Res. 1995; 13: 399-409Crossref PubMed Scopus (47) Google Scholar). However, little is known about the quantitative relationship between content of such chain termini in a PG and its reactivity with 3B3(−), and no information is available about the binding affinity of the antibody for the saturated mimotope compared with the affinity for the unsaturated epitope (25Hascall V.C. Midura R.J. Sorrell M.J. Plaas A.H.K. Adv. Exp. Med. Biol. 1995; 376: 205-216Crossref PubMed Scopus (17) Google Scholar). A range of studies have identified distinct nonreducing terminal residues present on CS synthesized in vitro by cells in culture (27Otsu K. Inoue H. Tsusiki Y. Yonkura H. Nakasini Y. Suzuki S. Biochem. J. 1985; 227: 37-48Crossref PubMed Scopus (33) Google Scholar, 28Midura R.J. Calabro A. Yanagishita M. Hascall V.C. J. Biol. Chem. 1995; 270: 8009-8015Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) and in isolated microsomal preparations (29Silbert J.E. J. Biol. Chem. 1978; 253: 6888-6892Abstract Full Text PDF PubMed Google Scholar), but data on the composition of nonreducing termini on CS from proteoglycans extracted from tissues are not available. It is therefore largely unknown if the mechanism for chain termination proposed from biosynthetic experiments in vitro operates in vivo, and whether it is altered in tissue development, maturation, or pathologies. Particularly in this context, it remains to be determined if immunoassays for the nonreducing terminal structures, such as 3B3(−), are reliable for detection of changes in the chain termination reactions. We report here, using a newly developed highly sensitive fluorotag HPLC method (26Plaas A.H.K. Hascall V.C. Midura R.J. Glycobiology. 1996; 6: 823-829Crossref PubMed Scopus (40) Google Scholar) that distinct patterns exist in the nonreducing terminal residues of CS on aggrecan isolated from human cartilages of different ages. Based on these results it is proposed that distinct enzymatic steps operate at the CS chain termini, depending on the maturation stage of the tissue. Since only limited information is available by which quantifiable biochemical parameters can be selected that characterize the changeover from a transient growth cartilage to a stable adult cartilage, the nonreducing terminal structures of CS chains may provide sensitive indicators for modulations in extracellular matrix production during the development and growth of cartilage as well as other connective tissues. β-Glucuronidase (bovine liver), monoclonal antibody 3B3 ascites fluid, and rat anti-mouse IgM were from ICN. Nitrocellulose membranes (0.2 μm) were from Bio-Rad. Hybond N+ membranes (0.2 μm), the ECL Western blotting reagent, and Hyperfilm were obtained from Amersham Corp. All other chemicals were obtained as described in Plaas et al. (26Plaas A.H.K. Hascall V.C. Midura R.J. Glycobiology. 1996; 6: 823-829Crossref PubMed Scopus (40) Google Scholar). Macroscopically normal cartilage was excised from the hips of 59- and 72-year-old women undergoing replacement surgery following femoral neck fractures. Aggrecan was purified by dissociative CsCl density gradient centrifugation from 4 m guanidine HCl extracts of the cartilages. The high buoyant density fractions (ρ > 1.59 g/ml, the D1 fractions) were dialyzed exhaustively against water, lyophilized, dissolved in water, and assayed for dimethylmethylene blue reactivity (30Farndale R.W. Sayers C.A. Barrett A.J. Connect. Tissue Res. 1982; 9: 247-248Crossref PubMed Scopus (1165) Google Scholar). Preparations were stored at −20 °C in 1 mg/ml aliquots. The additional cartilage aggrecan samples were prepared from macroscopically normal cartilage from the femoral condyles of human knee joints, as described previously (3Roughley P.J. White R.J. J. Biol. Chem. 1980; 255: 217-224Abstract Full Text PDF PubMed Google Scholar), and one D1-proteoglycan preparation from fetal cartilage was a gift of Dr. Victor Stanescu (CNRS, Hopital des Enfants Malades, Paris, France). D1-aggrecan, metabolically labeled with 35SO4 (containing ∼1 × 104 cpm/μg of CS), was prepared from monolayer cultures of the Rx rat clonal chondrosarcoma line (26Plaas A.H.K. Hascall V.C. Midura R.J. Glycobiology. 1996; 6: 823-829Crossref PubMed Scopus (40) Google Scholar). CS contents of purified aggrecan were determined by digesting 10-μg portions with 5 milliunits each of chondroitinase ABC and ACII, and quantitating unsaturated disaccharides (ΔDi0S, ΔDi4S, and ΔDi6S) by capillary zone electrophoresis (5Deutsch A.J. Midura R.J. Plaas A.H.K. J. Orthop. Res. 1995; 13: 230-239Crossref PubMed Scopus (29) Google Scholar). From such analyses, the values for micrograms of CS Δdisaccharides/μg of dimethylmethylene blue reactivity were 0.91 ± 0.01 in the fetal and young juvenile preparations (<15 yr) and 0.68 ± 0.02 in the adult preparations (>15 yr). Aggrecan CS chains were prepared by digestion of samples (200-μg portions) with 1 milliunit each, keratanase II and endo-β-galactosidase. Chains were then liberated by β-elimination in 50 mm NaOH, 1 m NaBH4 for 24 h at 45 °C (5Deutsch A.J. Midura R.J. Plaas A.H.K. J. Orthop. Res. 1995; 13: 230-239Crossref PubMed Scopus (29) Google Scholar). GAG peptides for immunoassays were prepared by papain digestion (1 μg of enzyme/500 μg of GAG) in 50 mm sodium acetate, pH 6.0, 1 mm cysteine at 60 °C for 16 h. Papain was inactivated by addition of iodoacetamide to a final concentration of 10 mm. Nonreducing terminal residues of CS chains were identified in chondroitinase digests of aggrecan after reductive amination of products with 2-aminopyridine and borane dimethylamine with subsequent HPLC separation and quantitation of fluorescent products as described elsewhere (26Plaas A.H.K. Hascall V.C. Midura R.J. Glycobiology. 1996; 6: 823-829Crossref PubMed Scopus (40) Google Scholar). In some cases, β-glucuronidase digestion of intact aggrecan was done prior to assays for nonreducing termini. These digestions were done with 50-μg portions of aggrecan samples in 100 μl of 50 mm sodium acetate, pH 5.0, 1 mmEDTA, 1 μg/ml each of pepstatin and leupeptin, 1 mm N-ethylmaleimide, and 1 unit of enzyme, for 2 h at 37 °C. The solutions were then mixed on ice with 100 μl of Dowex AGX8 H+ and immediately collected by centrifugation through microfiltration units (Millipore Ultrafree MC, 0.45 μm) into 100 μl of 0.5 m ammonium acetate, pH 7.5. Volatile salts and buffer were removed in vacuo, and residues were stored at −20 °C until assay for CS composition and for 3B3(−) immunoreactivity (see below). The β-glucuronidase digestion caused no detectable change in the size or the sulfation patterns of the CS chains, as assessed by Superose 6 chromatography and capillary zone electrophoretic analyses of chondroitinase digests (5Deutsch A.J. Midura R.J. Plaas A.H.K. J. Orthop. Res. 1995; 13: 230-239Crossref PubMed Scopus (29) Google Scholar) (data not shown). Mercuric acetate treatment was performed as described previously and is essential to accurately quantitate all saturated disaccharide nonreducing termini (26Plaas A.H.K. Hascall V.C. Midura R.J. Glycobiology. 1996; 6: 823-829Crossref PubMed Scopus (40) Google Scholar). Quantitation of aggrecan bound to nitrocellulose and nylon N+ membranes was done as follows. 35S-Labeled rat Rx chondrosarcoma aggrecan was diluted with Tris-buffered saline to final concentrations of 0.78–100 μg/ml of total GAG. Aliquots (25 μl) of each dilution were applied to membranes in a 96-well dot-blot apparatus (Bio-Rad). Macromolecules were adsorbed to the membranes for 1 h at room temperature before removing buffer and any unbound aggrecan by vacuum filtration. Individual dots were punched out with a dermal puncher and washed with 5% (w/v) milk powder in PBS. Total radioactivity bound to the membranes was determined by scintillation counting. At concentrations between 0.78 and 50 μg/ml, a constant proportion of the applied sample (30 ± 5%), remained immobilized to either nitrocellulose or nylon N+. However, at higher concentrations, both membranes became saturated with a maximum of 350–400 ng of CS immobilized per dot area. The binding of papain generated CS peptides and of intact aggrecans to membranes was determined with a quantitative toluidine blue assay (31Cardoso L.E.M. Erlick R.B. Peracoli J.C. Mourao P.A.S. Lab. Invest. 1992; 67: 588-595PubMed Google Scholar). For nylon N+ membranes, CS chains and aggrecans gave almost identical staining intensities at each concentration, for the entire concentration range used for immunoassay. On the other hand, only aggrecan, but not CS peptides, bound to nitrocellulose. Four dots for each concentration of immobilized 35S-labeled rat chondrosarcoma aggrecan (nitrocellulose and nylon N+) or papain generated 35S-labeled CS peptides (nylon N+) were incubated for 6 h at 37 °C in 200 μl of 50 mm sodium acetate, pH 7.5, with 1 milliunit each of chondroitinase ABC and ACII. The amounts of radioactivity released by the enzymes were determined by scintillation counting. More than 90% of the radioactivity was released as Δdisaccharides from nitrocellulose membranes, and under identical conditions, only 30–40% of the radioactivity was removed from nylon N+ membranes. Moreover, papain-generated CS peptides immobilized on nylon N+ were essentially insensitive to chondroitinase digestion, with only ∼5% of bound radioactivity released. This low yield probably reflects the immobilization of the CS chain on the cationic membrane surface resulting in only limited access to the enzyme, or alternatively, some of the digestion products may remain bound to the cationic membrane. Intact aggrecan or CS peptides can be removed from the cationic membrane after a brief incubation with 2m guanidinium HCl, but were not further analyzed, as recovery of 35S macromolecules after desalting was less that 10%. Fetal and adult aggrecans were immobilized on nitrocellulose at saturating concentrations, and four dots were digested with chondroitinase as described above. The Δdisaccharide products were analyzed by capillary zone electrophoresis. Each dot contained 350 ± 34 ng or 310 ± 21 ng of CS from fetal or adult aggrecan, respectively. Further, the percentage disaccharide compositions (ΔDi0S:ΔD4S:ΔD6S) of the bound CS were 10:60:30 for fetal and 1:7:92 for adult aggrecan, and these values were indistinguishable from those obtained by analyses of the preparations prior to application to the nitrocellulose (see Table I). Nonreducing terminals were determined in such digests by fluorescent derivatization and HPLC analyses (26Plaas A.H.K. Hascall V.C. Midura R.J. Glycobiology. 1996; 6: 823-829Crossref PubMed Scopus (40) Google Scholar).Table ICompositional analyses of internal Δdisaccharides in chondroitinase digests of human aggrecanAge1-aThe values shown for the individual ages represent the means of three independent analyses for each sample.nPercent of digestion products1-bDetermined by fluorescent derivitization and anion exchange HPLC (26).Percent of ΔDiOS1-cDetermined by capillary zone electrophoresis (5).ΔDi6S/ΔDi4S1-bDetermined by fluorescent derivitization and anion exchange HPLC (26).,1-cDetermined by capillary zone electrophoresis (5).mean ± S.D.Fetal298.31 ± 0.2111.0 ± 1.40.73 ± 0.03Juvenile (newborn to 1 year)397.67 ± 0.357.33 ± 0.580.77 ± 0.0715 yr195.82.02317 yr194.32.031Adult (22–72 years)493.33 ± 0.331.50 ± 0.5825.75 ± 2.751-a The values shown for the individual ages represent the means of three independent analyses for each sample.1-b Determined by fluorescent derivitization and anion exchange HPLC (26Plaas A.H.K. Hascall V.C. Midura R.J. Glycobiology. 1996; 6: 823-829Crossref PubMed Scopus (40) Google Scholar).1-c Determined by capillary zone electrophoresis (5Deutsch A.J. Midura R.J. Plaas A.H.K. J. Orthop. Res. 1995; 13: 230-239Crossref PubMed Scopus (29) Google Scholar). Open table in a new tab Aggrecan was immobilized to membranes by dot-blot as described above. Membranes were treated in succession with 5% (w/v) milk powder in PBS (4 h at 37 °C), 3B3 ascites fluid, diluted 1:10,000 in 1% (w/v) milk powder in PBS (4 h at 4 °C), and rat anti-mouse IgM, diluted 1:50,000 in 1% (w/v) milk powder in PBS. Membranes were then washed in PBS containing 0.05% (w/v) Tween, incubated with ECL detection reagent, and exposed to preflashed ECL Hyperfilm for 30 s, 1 min, or 2 min. After development, films were scanned using a Hewlett Packard Scan Jet 3C/T. Immunoreactivity was expressed as gray scale units of pixel density per dot as determined with the NIH 1.57 Image Analyses software. The film response was linear between 10 and 150 units for all exposure times. The 30-s exposure time was chosen to give the optimum range for assaying aggrecan populations of low and high 3B3(−) reactivity. CS chains, released from aggrecans from normal knee joint cartilages, were chromatographed on Superose 6 (Fig.1). Chains from adult (>22 years) aggrecan were hydrodynamically smaller (K av0.62, molecular mass 2Calculated using an equation derived on Superose 6 calibrated with hyaluronan oligosaccharides of defined sizes (36Wasteson A. J. Chromatogr. 1971; 59: 87-97Crossref PubMed Scopus (292) Google Scholar). ∼18 kDa) than those from fetal (K av 0.40, molecular mass ∼45 kDa) or juvenile (newborn to 1 year) (K av 0.46, molecular mass ∼35 kDa) aggrecans. Moreover, CS chains from the 15- and 17-year-old donors (shown for the 17-year sample, Fig. 1) eluted as a broader peak, indicating a greater heterogeneity in hydrodynamic sizes of the chain population in cartilages of individuals who are in the final stages of skeletal maturation. We examined the possibility that the marked change in the average hydrodynamic size of CS on aggrecan deposited into the cartilages at different stages of development and maturation may be accompanied by changes in the composition of the nonreducing chain terminals. Ion exchange HPLC analyses of fluorotagged chondroitinase digestion products was done under conditions in which all internal Δdisaccharides and nonreducing terminal monosaccharides and disaccharides are measured (26Plaas A.H.K. Hascall V.C. Midura R.J. Glycobiology. 1996; 6: 823-829Crossref PubMed Scopus (40) Google Scholar). The majority of the fluorescent products in the digests of human aggrecan represented the sulfated Δdisaccharides (Table I), and in agreement with previous reports (3Roughley P.J. White R.J. J. Biol. Chem. 1980; 255: 217-224Abstract Full Text PDF PubMed Google Scholar, 6Roughley P.J. White R.J. Glant T. Pediatr. Res. 1987; 22: 406-413Crossref Scopus (16) Google Scholar, 7Bayliss, M. T., Davidson, C., Woodhouse, S. M. & Osborne, D. J. (1995) Acta Orthop. Scand. , 66, Suppl. 266, 22–25.Google Scholar), the ratio of ΔDi6S:ΔDi4S was about 0.75 in aggrecan CS from cartilages up to 1 year of age, but increased to a much higher level, ∼25, in samples from individuals older than 15 years of age. The nonreducing termini of CS chains (GalNAc4S, GalNAc4,6S, GlcA-GalNAc4S (Di4S) and GlcA-GalNAc6S (Di6S), Fig.2), identified in the digests, constituted about 1.5, 2.3, and 6.7% of the total chondroitinase digestion products in fetal, young (newborn to 1 year), and adult aggrecan samples (22–72 years), respectively. The number averaged repeating disaccharides for each chain population was calculated from the ratio of interior Δdisaccharides to nonreducing termini (Tables Iand II). This indicated that fetal CS chains contained ∼66, young juvenile chains (newborn to 1 year) ∼42, and adult chains (22–72 years) of ∼14 repeats. The number averaged molecular masses 3Calculated from the number average repeating disaccharide units, corrected for nonsulfated Δdisaccharides (molecular mass 396 Da), and sulfated Δdisaccharides (molecular mass 476 Da) plus the molecular mass for the nonreducing termini and the linkage region (1136 Da) as described previously (26Plaas A.H.K. Hascall V.C. Midura R.J. Glycobiology. 1996; 6: 823-829Crossref PubMed Scopus (40) Google Scholar, 28Midura R.J. Calabro A. Yanagishita M. Hascall V.C. J. Biol. Chem. 1995; 270: 8009-8015Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). calculated from these results were ∼32 kDa for fetal, ∼20 kDa for juvenile, and ∼8 kDa for adult CS. These number averaged molecular mass values were smaller than molecular masses estimated by theK av elution positions on gel filtration chromatography (∼45, 35, and 18 kDa, respectively) (Fig. 1). It is interesting to note that Midura et al. (28Midura R.J. Calabro A. Yanagishita M. Hascall V.C. J. Biol. Chem. 1995; 270: 8009-8015Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) reported for the analyses of metabolically labeled CS chains, isolated from rat chondrosarcoma aggrecan, an average molecular mass of chains determined after gel chromatography of ∼24 kDa, compared with the calculated number averaged molecular mass from chemical analyses of ∼18 kDa. In the current study, the discrepancy in size estimations may be explained, in part, if the dimethylmethylene blue dye accentuates detection of long chains, such that the contribution of shorter chains to the chromatographic profile would be underscored. In addition, the hydrodynamic size of CS on gel filtration columns was found to be influenced by sulfation isomer composition of the chains (5Deutsch A.J. Midura R.J. Plaas A.H.K. J. Orthop. Res. 1995; 13: 230-239Crossref PubMed Scopus (29) Google Scholar). Therefore, uniform sulfation, as seen with adult CS, may result in a different conformation than the copolymeric 4- and 6-sulfation, seen in CS from younger subjects.Table IICompositional analyses of nonreducing terminal residues of human aggrecan CSAge2-aThe values shown for the individual ages represent the means of three independent analyses for each sample.nPercent of (Di4S+Di6S)2-bDetermined by fluorescent derivitization and anion exchange HPLC (26).,2-cThis represents the abundance of GlcA nonreducing terminal residues as a proportion of all detected terminal residues.Di6S/Di4SGalNAc4,6S/4S2-bDetermined by fluorescent derivitization and anion exchange HPLC (26).mean ± S.D.Fetal214.5 ± 0.700.92 ± 0.010Juvenile (newborn to 1 year)314.0 ± 1.00.89 ± 0.08015 yr115.02.9017 yr111.03.20.7Adult (22–72 years)49.50 ± 0.583.02 ± 0.171.80 ± 0.052-a The values shown for the individual ages represent the means of three independent analyses for each sample.2-b Determined by fluorescent derivitization and anion exchange HPLC (26Plaas A.H.K. Hascall V.C. Midura R.J. Glycobiology. 1996; 6: 823-829Crossref PubMed Scopus (40) Google Scholar).2-c This represents the abundance of GlcA nonreducing terminal residues as a proportion of all detected terminal residues. Open table in a new tab" @default.
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