The cartilage collagens and joint degeneration.
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Animals↗
Biomedical subjects
D R Eyre
Publications and source records attributed to D R Eyre.
The cartilage collagens: structural and metabolic studies.
The molecular sites of crosslinking between types II and IX collagens of bovine articular cartilage have been defined. All 3 chains of the type IX molecule, alpha 1 (IX), alpha 2 (IX) and alpha 3 (IX), contain an hydroxylysine close to the N-terminus of the central COL2 triple helical domain that can crosslink to an hydroxylysine aldehyde in an alpha 1 (II) N-telopeptide. One chain, alpha 3 (IX), also contains a triple helical hydroxylysine farther into COL2 that crosslinks specifically to an alpha 1 (II)C-telopeptide. The metalloprotease, stromelysin, is capable of depolymerizing the type II-type IX complex by bisecting the type IX collagen molecule and cleaving telopeptides from type II collagen. We discuss the significance of these findings in understanding the structure, growth and remodeling of the heterotypic collagen network of articular cartilage.
Animals↗
Structural and functional characterization of a splicing mutation in the pro-alpha 2(I) collagen gene of an Ehlers-Danlos type VII patient.
A splicing mutation in the pro-alpha 2(I) collagen gene of a patient with Ehlers-Danlos syndrome type VII has been characterized. Protein microsequencing of peptides prepared from the patient's dermal collagen identified an interstitial deletion of 18 residues. The deleted segment corresponds to the amino-terminal telopeptide junction domain encoded by the sixth exon of the pro-alpha 2(I) collagen gene. Sequencing of specifically primed cDNA clones confirmed the presence of two distinct populations of pro-alpha 2(I) mRNAs, a normal one and another which lacks the sequences of exon 6. Limited sequencing of genomic clones showed that one of the pro-alpha 2(I) alleles displays a conservative change in the seventh codon of exon 6 (GAC for GAT), and a base substitution at position +1 of intron 6 (A for G). Since the normal transcript contains the GAT codon, the intronic change was associated with the allele that gives rise to the shortened pro-alpha 2(I) collagen mRNA. The two allelic fragments were subcloned into an expression vector and the pattern of splice-site selection for exons 5-8 was assessed for each of the constructs after transfection into COS cells. This documented skipping of exon 6 sequences only in transcripts of the minigene construct that harbors the G to A transition. Expression of allelic cross-constructs confirmed that the single-base substitution at position +1 of intron 6 is the mutation responsible for the abnormal joining of exons 5 and 7 sequences in the patient's shortened pro-alpha 2(I)mRNA.
Alleles↗
Changes with age in the urinary excretion of lysyl- and hydroxylysylpyridinoline, two new markers of bone collagen turnover.
Two intermolecular cross-linking amino acids, hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), are promising markers in urine of collagen resorption because their levels in urine should reflect only collagen resorption and, unlike hydroxyproline, should not be influenced by degradation of either newly synthesized collagen molecules or noncollagenous proteins. Changes with age in the urinary excretion of HP and LP were studied in 24 h collections of urine from a group of 22 male and 27 female healthy subjects aged from 2 to 70 years. The pyridinolines were quantitated, utilizing their natural fluorescence, after resolution by reversed-phase HPLC. Levels of both pyridinolines were higher in children than in adults, but in adults no evidence of age or sex variations were observed except in the 20-30 year age group. Mean values of HP/Cr and LP/Cr in 37 adults (21-70 years) were 27.2 +/- 1.9 and 8.8 +/- 0.8 mumols/mol, respectively; in the 12 children (2-15 years) the mean values were 14.4 and 12.4 times higher than the respective adult values. Making certain assumptions, the mean amount of bone resorbed in normal adults was tentatively estimated at 1.9 g per 24 h. The finding that differences between children and adults in these relatively specific markers were greater than with hydroxyproline suggests that hydroxyproline values may considerably underestimate the actual amount of bone turnover occurring in growing children or overestimate the adult turnover rate.
Adult↗
Effects of proteoglycan extraction on the tensile behavior of articular cartilage.
We undertook an interdisciplinary biomechanical and biochemical study to explore the extent and manner in which the total pool of proteoglycans influences the kinetic and static behavior of bovine articular cartilage in tension. Two biomechanical tests were used: (a) the viscoelastic creep test and (b) a slow constant-rate uniaxial tension test; and two enzymatic proteoglycan extraction procedures were used: (a) chondroitinase ABC treatment and (b) a sequential enzymatic treatment with chondroitinase ABC, trypsin, and Streptomyces hyaluronidase. We found that the viscoelastic creep response of all cartilage specimens may be divided into two distinct phases: an initial phase (less than 15 s), characterized by a rapid increase in strain following load application, and a late phase (15 s less than or equal to t less than 25,000 s), characterized by a more gradual increase in strain. A major finding of this study is that the kinetics of the creep response is greatly influenced by the glycosaminoglycan content of the tissue. For untreated and control specimens, the initial response comprises about 50% of the total strain, while for chondroitinase ABC and sequentially extracted specimens, the initial response comprises up to 83% of the total strain. Furthermore, most untreated and control specimens did not reach equilibrium within the 25,000 s test period, while enzymatically digested specimens often reached equilibrium in less than 100 s. Thus, we conclude that through their physical restraints on collagen, the bulk of proteoglycan present in the tissue acts to retard fibrillar reorganization and alignment under tensile loading, thereby effectively preventing sudden extension of the collagen network. In contrast, the results of our slow constant-rate uniaxial tension experiment show that essentially complete extraction of proteoglycan glycosaminoglycans does not affect the intrinsic tensile stiffness and strength of cartilage specimens or the collagen network in a significant manner. Hence, an important function of the bulk proteoglycans (i.e., the large aggregating type) in cartilage is to retard the rate of stretch and alignment when a tensile load is suddenly applied. This mechanism may be useful in protecting the cartilage collagen network during physiological situations, where sudden impact forces are imposed on a joint.
Animals↗
Degradation of cartilage collagens type II, IX, X and XI by enzymes derived from human articular chondrocytes.
Conditioned culture medium derived from Interleukin-I alpha-activated human articular chondrocytes contained both collagen- and proteoglycan-degrading activities. Preparations of soluble type I collagen and the cartilage collagens type II, IX, X and XI were all degraded when incubated with the conditioned culture medium at 35 degrees C. Fractionation of the enzymic activities using column chromatography with Ultragel AcA 34 and Heparin-Sepharose allowed the separation and identification of neutral proteinase, collagenolytic and proteoglycan-degrading activities. Eluant fractions which contained type I collagenase activity effectively degraded collagen type II, but these fractions did not correspond precisely with those which degraded collagen types IX, X and XI. These observations indicate that chondrocytes have the potential to produce a conventional interstitial type II collagenase together with other enzymes having some specificity for the minor collagens. Thus IL-1-activated chondrocytes produce a range of collagenolytic and proteoglycan-degrading enzymes which can process most of the structural components of the cartilage matrix.
Cartilage, Articular↗
BAPN dose dependence of mature crosslinking in bone matrix collagen of rabbit compact bone: corresponding variation of sonic velocity and equatorial diffraction spacing.
Crosslinking density in demineralized bone matrix collagen was found to depend on the beta-aminopropionitrile (BAPN) dose level for compact bone from rabbit femurs. The dependence was demonstrated for the hydroxypyridinium (HP) concentration, a mature crosslink. A more consistent dependence on BAPN dosage was observed for the fraction of the demineralized bone matrix insoluble in 0.5 M acetic acid (AIF) corresponding to the remaining crosslinked collagen. The average HP concentration in 21 week old controls was 0.24 moles HP/mole collagen which decreased to 0.13 +/- 0.04 for the same age rabbits dosed with 1 gm BAPN/kg/day for 13 wks. The comparable mean AIF values were 0.91 for controls and 0.75 for maximum dose level. Most of the effect of BAPN on crosslinking was observed at the lower dosages below 0.2 g/kg/day. On the other hand, overt osteolathyritic symptoms are seen only for BAPN dosages greater than 0.2 g/kg/day. The mean sonic plesio-velocity was previously found to decrease from 3.4 to 3.03 km/sec as the BAPN dosage was increased. A similar close relationship was discovered for the equatorial diffraction spacing in fully mineralized bone which increased from 1.235 for normal rabbit bone to 1.28 nm for maximum dose. Most of the effect on these physical properties is exhibited at the highest BAPN dosage after much of the decrease in mature crosslinking density has been observed and when the further decrease in mature crosslinking density proceeds very slowly with increased drug dosage. These observations suggest that osteolathyrism does not become manifest until practically all mature crosslinking that can be affected has been inhibited. The mineralization process apparently can be maintained in the newly laid collagen even in the presence of severe osteolathyritic conditions. Intermolecular crosslinking in bone collagen appears to play an important role in the development of bone properties whether by direct or indirect processes. Much of the effects on bone properties occur at the higher BAPN dosages where overt osteolathyrism is observed and where there seem to be only small changes in crosslinking density.
Acetates↗
Identification of the cartilage alpha 1(XI) chain in type V collagen from bovine bone.
Type V collagen prepared from bovine bone was resolved into three distinct alpha-chains by high performance liquid chromatography and gel electrophoresis. Peptide mapping established two chains as alpha 1(V) and alpha 2(V) as expected and the third as the cartilage alpha 1(XI) chain (previously thought to be unique to cartilage). In adult bone, the type V collagen fraction was richer in alpha 1(XI) chains than in fetal bone (about 1/3 of the chains in the adult). How these polypeptides are organized into native molecules is not yet clear, though the stoichiometry suggests cross-type heterotrimers between the type V and XI chains.
Animals↗
Covalent interactions of type IX collagen in cartilage.
The cross-linking of type IX collagen in fetal bovine cartilage was investigated. The main cross-link was dihydroxy-lysinonorleucine (borohydride-reduced) with a lesser amount of the mature cross-link, pyridinoline. Dihydroxylysinonorleucine was present in all three chains of the COL2 domain of the type IX molecule, but only two of them contained pyridinoline even in mature cartilage. Amino acid sequence analysis of individual tryptic peptides that contained 3H-labeled cross-links showed that they had derived from sites of covalent interaction between type IX collagen and the telopeptide sequences of type II collagen. One two-chained peptide was a helical sequence of alpha 2 (IX) COL2 linked to an alpha 1 (II) N-telopeptide. A second peptide was a different helical sequence from another type IX chain linked to an alpha 1(II) c-telopeptide. This latter helical sequence was also the principal site of pyridinoline cross-linking in type IX collagen.
Animals↗
Bone type V collagen: chain composition and location of a trypsin cleavage site.
The component alpha-chains of type V collagen from bovine bone were isolated and structurally characterized by gel electrophoresis, high performance liquid chromatography (HPLC) and amino acid sequence analysis. Three distinct alpha-chains were identified. Two of these were the well described alpha 1 (V) and alpha 2 (V) chains; the third proved to be identical to the cartilage alpha 1 (XI) chain. In adult bone the ratio between the three chains was about 1:1:1. Native type V collagen was cleaved by trypsin at 33 degrees C or 37 degrees C into 3/5 fragments. Aminoterminal sequence analysis of the alpha 1 (V) and alpha 1 (XI) fragments showed they both resulted from trypsin cleavage between residue 434 and 435. Trypsin apparently cleaves the type V molecule within a relatively unstable domain of the triple helix which presumably may also be a natural site of initial cleavage by a protease in vivo.
Amino Acid Sequence↗
Short rib polydactyly syndrome type I: an autopsy approach to diagnosis of chondrodysplasias.
The autopsy diagnosis of neonates with chondrodysplasias is often difficult due to the rarity of many of these conditions and to a complex classification scheme. Accurate diagnosis is essential for counseling of parents with one or more affected infants. Classification is currently based on radiological appearances and gross morphology. Following examination of two siblings with short rib polydactyly syndrome (SRPS) type I, we undertook to analyze the main problems in clinical pathological classification of chondrodysplasias. The problems identified are: variability in the constellations of morphological features that are used for diagnosis; insufficient radiological data being obtained at the time of autopsy; failure to preserve tissue appropriately for the necessary studies and lack of knowledge of the underlying abnormalities in most chondrodysplastic syndromes. It is anticipated that biochemical and molecular genetic abnormalities will eventually be discovered to reduce diagnostic uncertainty in the chondrodysplasias. Presently the diagnostic process is facilitated when frozen tissue is available for studies such as collagen, proteoglycan, and enzyme analysis and mRNA and DNA analyses.
Cartilage, Articular↗
Substitution of cysteine for glycine within the carboxyl-terminal telopeptide of the alpha 1 chain of type I collagen produces mild osteogenesis imperfecta.
We have characterized a mutation that produces mild, dominantly inherited osteogenesis imperfecta. Half of the alpha 1 (I) chains of type I collagen synthesized by cells from an affected individual contain a cysteine residue in the 196-residue carboxyl-terminal cyanogen bromide peptide of the triple-helical domain (Steinmann, B., Nicholls, A., and Pope, F. M. (1986) J. Biol. Chem. 261, 8958-8964). Unexpectedly, sequence determined from a proteolytic fragment of the alpha 1 (I) chain derived from procollagen molecules synthesized in the presence of both [3H]proline and [35S]cysteine indicated that the cysteine is located at the third residue carboxyl-terminal to the triple-helical domain, normally a glycine. The nucleotide sequence of a fragment amplified from genomic DNA confirmed the location of the cysteine residue and showed that the mutation was a single nucleotide change in one COL1A1 allele. This represents a new class of mutations, point mutations outside the triple-helical domain of the chains of type I collagen, that produce the osteogenesis imperfecta phenotype.
Alleles↗
Susceptibility of cartilage collagens type II, IX, X, and XI to human synovial collagenase and neutrophil elastase.
The action of purified rheumatoid synovial collagenase and human neutrophil elastase on the cartilage collagen types II, IX, X and XI was examined. At 25 degrees C, collagenase attacked type II and type X (45-kDa pepsin-solubilized) collagens to produce specific products reflecting one and at least two cleavages respectively. At 35 degrees C, collagenase completely degraded the type II collagen molecule to small peptides whereas a large fragment of the type X molecule was resistant to further degradation. In contrast, collagen type IX (native, intact and pepsin-solubilized type M) and collagen type XI were resistant to collagenase attack at both 25 degrees C and 35 degrees C even in the presence of excess enzyme. Mixtures of type II collagen with equimolar amounts of either type IX or XI did not affect the rate at which the former was degraded by collagenase at 25 degrees C. Purified neutrophil elastase, shown to be functionally active against soluble type III collagen, had no effect on collagen type II at 25 degrees C or 35 degrees C. At 25 degrees C collagen types IX (pepsin-solubilized type M) and XI were also resistant to elastase, but at 35 degrees C both were susceptible to degradation with type IX being reduced to very small peptides. Collagen type X (45-kDa pepsin-solubilized) was susceptible to elastase attack at 25 degrees C and 35 degrees C as judged by the production of specific products that corresponded closely with those produced by collagenase. Although synovial collagenase failed to degrade collagen types IX and XI, all the cartilage collagen species examined were degraded at 35 degrees C by conditioned culture medium from IL1-activated human articular chondrocytes. Thus chondrocytes have the potential to catabolise each cartilage collagen species, but the specificity and number of the chondrocyte-derived collagenase(s) has yet to be resolved.
Arthritis, Rheumatoid↗
Collagen cross-linking in human bone and articular cartilage. Age-related changes in the content of mature hydroxypyridinium residues.
The concentration in collagen of hydroxypyridinium cross-linking amino acids was measured in samples of bone and cartilage from human subjects aged from 1 month to 80 years. Cortical and cancellous bone samples were dissected and analysed separately. In both bone and cartilage, the content of this mature form of cross-link reached a maximum by 10-15 years of age (the amount in cartilage being 5-10 times that in bone), then stayed essentially in the same range throughout adult life. In bone the ratio of the two chemical variants of the mature cross-link, hydroxylysylpyridinoline to lysylpyridinoline, was constant throughout adult life at 3.5:1, whereas in cartilage it was always greater than 10:1. The ratio of hydroxypyridinium cross-links to borohydride-reducible keto-amine cross-links also changed with age. The reducible cross-links in bone collagen decreased steeply in content between birth and 25 years, but persisted in significant amounts throughout adult life. Reducible cross-links had virtually disappeared from cartilage by 10-15 years of age, being replaced by hydroxypyridinium residues, their maturation products. Cancellous and cortical bone collagens showed similar trends with age in their content of mature cross-links, though for each subject the concentration in cancellous bone was always lower than in cortical bone, presumably reflecting the higher turnover rate and hence the more immature state of cancellous bone.
Adolescent↗
Quantitation of hyaluronic acid in tissues by ion-pair reverse-phase high-performance liquid chromatography of oligosaccharide cleavage products.
A method for quantifying hyaluronic acid in biological tissues and fluids is described. The assay uses ion-pair HPLC to resolve and quantify the oligosaccharide end products of Streptomyces hyaluronidase digestion. Tissue samples were solubilized by papain, and the nondiffusate after dialysis was exhaustively digested with Streptomyces hyaluronidase. The resulting tetrasaccharide and hexasaccharide cleavage products were resolved by reverse-phase high-performance liquid chromatography in the presence of the ion-pairing agent, tetrabutylammonium phosphate. The saccharides were detected and quantified by their absorbance at 232 nm due to the alpha, beta-unsaturated carboxyl group generated by the eliminase reaction. In control experiments 93 +/- 3% of a hyaluronic acid standard so treated was reproducibly recovered as its tetra- and hexasaccharide cleavage products. As little as 0.5 microgram of the oligosaccharides could be quantified with no interference from a vast excess of chondroitin sulfate or other tissue components. The assay was applied to various types of human, bovine, and rabbit cartilage and to samples of other tissues including nucleus pulposus, annulus fibrosus, skin, aorta, cervix, cockscomb, synovial fluid, and vitreous humor. Results on human articular cartilage showed a linear increase in the content of hyaluronate from 0.1 to 0.5% of tissue dry weight between birth and 80 years of age.
Adolescent↗
Fine powdering exposes the mineral-protected collagen of bone to protease digestion.
The method of heat-denaturation and trypsin digestion was used to dissect bone biochemically into mineral-protected and mineral-unprotected pools of collagenous matrix. It was found that varying the particle size of the bone powder had a profound effect on the results. Using mature bovine cortical bone, the observed pool of "unmineralized" (mineral-unprotected) collagen could be varied from 2% to more than 60% of the total bone collagen simply by decreasing the particle size of the bone sample from greater than 1 mm to less than 38 micron. No major differences were seen in the contents of hydroxypyridinium cross-links between the collagens of the trypsin-soluble and trypsin-insoluble pools from the fine powders, contrary to earlier reports. A trend to a higher content of these cross-links was evident, however, in the very small collagen pool extracted from the coarsest bone particles. Similar extraction differences were noted using bacterial collagenase to probe for mineral-protected vs. mineral-unprotected domains of bone collagen. In summary, the biochemical dissection results appear largely to be an artifact of the powdering technique, the shear energies of which presumably destroy the intimate physical relationship between the mineral crystallites and the collagen fibrils at the fractured surfaces of the bone particles. As the fractured surface area increases with decreasing particle size so the fraction of protease degradable collagen increases. Since powdering is routinely adopted for many structural studies on both the mineral and organic phases of bone, the findings on finely powdered bone should be interpreted cautiously.
Animals↗
Collagen cross-linking: distribution of hydroxypyridinium cross-links among invertebrate phyla and tissues.
1. Using a specific HPLC assay, a wide variety of marine invertebrate connective tissues was screened for the 3-hydroxypyridinium amino acids that are prominent intermolecular cross-linking residues in the collagens of many vertebrate connective tissues. 2. One or both of the two structural forms that exist, hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP), was found in organisms from the following phyla: coelenterata, Annelida, Echinodermata, Mollusca and Arthropoda. 3. Neither amino acid was found in tissues from representative species of Porifera and Chordata. 4. Of special note was an unusually high ratio of LP to HP in Limulus polyphemus gill cartilage.
Animals↗
Type II collagen deficiency in achondrogenesis (Langer-Saldino).
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Cartilage↗