Distinct post-translational features of type I collagen are conserved in mouse and human periodontal ligament.

BACKGROUND AND OBJECTIVE: Specifics of the biochemical pathways that modulate collagen cross-links in the periodontal ligament (PDL) are not fully defined. Better knowledge of the collagen post-translational modifications that give PDL its distinct tissue properties is needed to understand the pathogenic mechanisms of human PDL destruction in periodontal disease. In this study, the post-translational phenotypes of human and mouse PDL type I collagen were surveyed using mass spectrometry. PDL is a highly specialized connective tissue that joins tooth cementum to alveolar bone. The main function of the PDL is to support the tooth within the alveolar bone while under occlusal load after tooth eruption. Almost half of the adult population in the USA has periodontal disease resulting from inflammatory destruction of the PDL, leading to tooth loss. Interestingly, PDL is unique from other ligamentous connective tissues as it has a high rate of turnover. Rapid turnover is believed to be an important characteristic for this specialized ligament to function within the oral-microbial environment. Like other ligaments, PDL is composed predominantly of type I collagen. Collagen synthesis is a complex process with multiple steps and numerous post-translational modifications including hydroxylation, glycosylation and cross-linking. The chemistry, placement and quantity of intermolecular cross-links are believed to be important regulators of tissue-specific structural and mechanical properties of collagens. MATERIAL AND METHODS: Type I collagen was isolated from several mouse and human tissues, including PDL, and analyzed by mass spectrometry for post-translational variances. RESULTS: The collagen telopeptide cross-linking lysines of PDL were found to be partially hydroxylated in human and mouse, as well as in other types of ligament. However, the degree of hydroxylation and glycosylation at the helical Lys87 cross-linking residue varied across species and between ligaments. These data suggest that different types of ligament collagen, notably PDL, appear to have evolved distinctive lysine/hydroxylysine cross-linking variations. Another distinguishing feature of PDL collagen is that, unlike other ligaments, it lacks any of the known prolyl 3-hydroxylase 2-catalyzed 3-hydroxyproline site modifications that characterize tendon and ligament collagens. This gives PDL a novel modification profile, with hybrid features of both ligament and skin collagens. CONCLUSION: This distinctive post-translational phenotype may be relevant for understanding why some individuals are at risk of rapid PDL destruction in periodontal disease and warrants further investigation. In addition, developing a murine model for studying PDL collagen may be useful for exploring potential clinical strategies for promoting PDL regeneration.

Evidence for enhanced collagen type III deposition focally in the territorial matrix of osteoarthritic hip articular cartilage.

OBJECTIVE: To determine if type III collagen is concentrated in the chymotrypsin-extractable collagen pool from osteoarthritic articular cartilage to assess its potential as a biomarker of Osteoarthritis (OA) pathogenic mechanisms. METHODS: Full thickness articular cartilage from grossly normal surfaces was analyzed from femoral heads, obtained at hip replacement surgery, from OA (n = 10) and fracture (n = 10) patients. Collagen, extracted by α-chymotrypsin, was characterized by SDS-PAGE/Western blot analysis, ELISA and immunohistochemistry using monoclonal antibodies specific to collagens types II and III. RESULTS: α-Chymotrypsin extracted more collagen from OA than control cartilage. The extractable pool included collagen types II and III from both OA and control hips. Importantly, OA cartilage contained 6-fold more collagen type III than control cartilage, based on ELISA. The estimated total tissue ratio of collagen III/II was in the 1-10% range for individual OA cartilage samples, based on pepsin-solubilized collagen using SDS-PAGE densitometry. Collagen type III N-propeptide trimers were the main molecular fragments seen on Western blot analysis of OA and control extracts. The chymotrypsin-extracted type II collagen gave primarily full-length α1(II) chains and chain fragments of α1(II) on Western blot analysis from both OA and control tissues. Immunohistochemistry showed that type III collagen was more concentrated in the upper half of OA cartilage and in the territorial matrix around individual chondrocytes and chondrocyte clusters. CONCLUSIONS: The findings confirm that collagen type III deposition occurs in adult articular cartilage but significantly more pronounced in osteoarthritic joints, presenting a potential marker of matrix repair or pathobiology.

Dominant mutations in the type II collagen gene, COL2A1, produce spondyloepimetaphyseal dysplasia, Strudwick type.

The chondrodysplasias are a heterogeneous group of disorders characterized by abnormal growth or development of cartilage. Current classification is based on mode of inheritance as well as clinical, histologic, and/or radiographic features. A clinical spectrum of chondrodysplasia phenotypes, ranging from mild to perinatal lethal, is due to defects in the gene for type II collagen, COL2A1. This spectrum includes Stickler syndrome, Kniest dysplasia, spondyloepiphyseal dysplasia congenita (SEDC), achondrogenesis type II, and hypochondrogenesis. Individuals affected with these disorders exhibit abnormalities of the growth plate, nucleus pulposus, and vitreous humor, which are tissues that contain type II collagen. The Strudwick type of spondyloepimetaphyseal dysplasia (SEMD) is characterized by disproportionate short stature, pectus carinatum, and scoliosis, as well as dappled metaphyses (which are not seen in SEDC). The phenotype was first described by Murdoch and Walker in 1969, and a series of 14 patients was later reported by Anderson et al. The observation of two affected sibs born to unaffected parents led to the classification of SEMD Strudwick as an autosomal recessive disorder. We now describe the biochemical characterization of defects in alpha 1(II) collagen in three unrelated individuals with SEMD Strudwick, each of which is due to heterozygosity for a unique mutation in COL2A1. Our data support the hypothesis that some cases, if not all cases, of this distinctive chondrodysplasia result from dominant mutations in COL2A1, thus expanding the clinical spectrum of phenotypes associated with this gene.

Severe osteogenesis imperfecta caused by a small in-frame deletion in CRTAP.

Mutations of proteins involved in posttranslational modification of collagen type I can cause osteogenesis imperfecta (OI) inherited in a recessive pattern. The cartilage-associated protein (CRTAP) is part of a heterotrimeric complex (together with prolyl-3-hydroxylase-1 [P3H1] and cyclophilin B) that 3-hydroxylates the alpha 1 chain of collagen type I at proline residue 986 and plays a collagen chaperon role. CRTAP mutations usually cause severe OI. We report on a patient with OI and a homozygous in-frame deletion in CRTAP and a severe form of OI. The girl was born with markedly deformed long bones. Despite intravenous bisphosphonate treatment, she developed multiple vertebral compression fractures and severe scoliosis and at 4 years of age was able to sit only with support. Although CRTAP transcript levels were normal in the patient's fibroblasts, protein levels of both CRTAP and P3H1 were severely reduced. The degree of 3-hydroxylation at proline residue 986 was also decreased. This report characterizes a patient with a CRTAP small in-frame deletion. We are unaware of prior reports of this finding. We suggest that this deletion affects crucial amino acids that are important for the interaction and/or stabilization of CRTAP and P3H1.

The Helix-II epitope: a cautionary tale from a cartilage biomarker based on an invalid collagen sequence.

OBJECTIVE: An apparent database error in the sequence underlying the Helix-II cartilage biomarker immunoassay was investigated at the protein level. METHODS AND RESULTS: Tandem mass spectrometry established the peptide sequence ERGETGPP*GPA in human type II collagen, not ERGETGPP*GTS used to generate the antibody for the Helix-II assay. CONCLUSIONS: Recent reports in which the Helix-II assay was applied to urine or serum as a marker of cartilage collagen degradation need to be re-evaluated since the epitope does not occur in cartilage type II collagen. Based on collagen sequences and Helix-II epitope properties, type III collagen is one of several candidate sources of the cross-reacting signal in body fluids, but not type II collagen. The findings highlight the need for more stringent scrutiny of the origins and validation of molecular markers in body fluid assays in general.

Collagen: molecular diversity in the body's protein scaffold.

Intensive research in the last decade has revealed a wealth of detail on the mechanism of biosynthesis, molecular structure, and covalent cross-linking of collagen. Tissues of higher animals express a family of at least five genetically distinct types of collagen molecule, each apparently tailored for different construction work outside the cell. Within each genetic type of collagen, further chemical heterogeneity is also evident; the variations in hydroxylation, glycosylation, and cross-linking are dependent, for example, on tissue type, age, and hormonal status. The functional significance of collagen's molecular diversity and its control by different cells and tissues are not yet well understood but abnormalities of collagen in many human diseases keep this protein a focal molecule of medical research.

Molecular basis and clinical application of biological markers of bone turnover.

An ideal battery of tests would include indices of bone resorption and formation. They should be unique to bone, reflect total skeletal activity, and should correlate with traditional measures of bone remodeling activity, such as radiocalcium kinetics, histomorphometry, or changes in bone mass. Factors that confound their measurement, such as circadian rhythms, diet, age, sex, bone mass, liver function, and kidney clearance rates, should be clearly defined (Fig. 9). To date, no bone marker has been established to meet all these criteria, and each marker may have its own specific advantages and limitations. There are still questions that must be answered before there can be complete confidence in the information gained from measurement of any of the bone markers. Furthermore, it should be emphasized that none of the markers are diagnostic for any particular bone disease and cannot be used for this purpose in individual patients. Nevertheless, recent advances in research and development have provided assays with increased specificity, sensitivity, and availability. Because of this, bone markers can be used for a variety of important purposes: as tools for basic bone biology research, for defining general physiological phenomenon in clinical studies or drug trials, and for following individual patients.

Cartilage expression of a type II collagen mutation in an inherited form of osteoarthritis associated with a mild chondrodysplasia.

In a family who expressed severe dominantly inherited osteoarthritis, the underlying mutation was traced by genomic sequencing to a single base change which predicts an amino acid substitution of cysteine for arginine at residue 519 of the triple-helical domain of the type II collagen molecule (Ala-Kokko, L., C. T. Baldwin, R. W. Moskowitz, and D. J. Prockop. 1990. Proc. Natl. Acad. Sci. USA. 87:6565-6568). In the present study we examined whether this predicted protein phenotype was evident in articular cartilage obtained from an affected family member who underwent hip surgery. The cartilage collagen was solubilized by CNBr digestion. Cysteine residues were labeled by reduction and alkylation with 14C-iodoacetate. Collagen CNBr-peptides were fractionated by ion exchange and reverse phase column chromatography. One peptide from the alpha 1(II) chain, alpha 1(II) CB8, was found to be radiolabeled. Tryptic peptides were prepared from it and identified by microsequence analysis. The results show that approximately one-quarter of the alpha 1(II) chains present in the polymeric extracellular collagen of the patient's cartilage contained the Arg519-to-Cys substitution. The protein exhibited other abnormal properties including disulfide-bonded alpha 1(II)-dimers and signs of posttranslational overmodification. The premature cartilage failure and osteoarthritis are presumably a result of the abnormal type II collagen being expressed in the cartilage matrix.

Articular cartilage collagen: an irreplaceable framework?

Adult articular cartilage by dry weight is two-thirds collagen. The collagen has a unique molecular phenotype. The nascent type II collagen fibril is a heteropolymer, with collagen IX molecules covalently linked to the surface and collagen XI forming the filamentous template of the fibril as a whole. The functions of collagens IX and XI in the heteropolymer are far from clear but, evidently, they are critically important since mutations in COLIX and COLXI genes can result in chondrodysplasia syndromes. Here we review what is known of the collagen assembly and present new evidence that collagen type III becomes covalently added to the polymeric fabric of adult human articular cartilage, perhaps as part of a matrix repair or remodelling process.

Collagen polymorphism in extracellular matrix of human osteosarcoma.

The collagenous matrix of human osteosarcoma was characterized biochemically and ultrastructurally. The highly cellular regions of the tumors contained many osteoblast-like cells filled with dilated rough endoplasmic reticulum. The extracellular matrix displayed a random weave of banded collagen fibrils (30--90 nm in diameter) interspersed with thinner, unbanded fibrils (15 nm in diameter). Tumors from 9 patients were analyzed for collagen composition. All gave a similar collagen profile. Three main molecular species of collagen were abundant: type I, type III, and type V, which occurred in the approximate proportions of 65:25:10. A high ratio of alpha 1(I) to alpha 2(I) chains suggested that one-third of the type I collagen was present as a type I trimer molecule. In contrast, normal bone matrix consisted almost exclusively of type I collagen. The fibrillar collagens in the soft tumor seemed unusually rich in hydroxylysine and hydroxylysine glycosides; type I collagen had two to three times the hydroxylysine content of normal bone collagen.

Structural analysis of the extension peptides on matrix forms of type V collagen in fetal calf bone and skin.

The structure of the extension peptides retained on the tissue form of type V collagen molecules was determined. Type V collagen alpha chains containing extension peptides were extracted from fetal calf skin and bone by 4 M guanidine-HCl and 0.5 M acetic acid, respectively. Collagens present in both extracts were fractionated by sodium chloride precipitation. The collagen alpha(V) chains were then resolved by reverse-phase high performance liquid chromatography. The N-terminal extension peptides were characterized by direct sequence analysis after deblocking with pyroglutamate amino-peptidase and analysis of the products of digestion by bacterial collagenase, chymotrypsin, V8 protease and endoproteinase Lys-C. The results showed that the retained extension peptides on type V collagen molecules in the extracellular matrix of skin and bone were amino-propeptides and that the alpha 2(V) chain retains an intact amino-propeptide while the alpha 1(V) chain appears to be partially processed. The extended alpha 1(V) chain isolated from fetal calf bone gave an identical amino-terminal sequence to that of the alpha 1(V) chain isolated from fetal calf skin, suggesting that a specific enzyme may be involved in processing the alpha 1(V) amino-propeptide.

Collagen in the ageing human intervertebral disc: an increase in covalently bound fluorophores and chromophores.

Human disc tissue gradually changes in colour from white in the young to yellow brown in the elderly. It was investigated to what degree this colouration and an associated fluorescence (which are characteristic of the non-enzymic reaction products of sugars or oxidized lipids with proteins), were the result of covalent derivatives on the collagen and other extracellular matrix proteins. Annulus fibrosus was obtained from four subjects aged 19 to 92 years. Papain-solubilized samples of tissue showed increasing yellow colour and the glycation-related fluorescence with age. On chromatography of CNBr-digests of tissue, the yellow colour and fluorescence remained bound to the CNBr-peptide fragments of the collagen and other matrix proteins. One peptide, alpha 1(II)CB12 (from type II collagen), was selected for purification and shown to contain increasing amounts of the characteristic fluorescence with age. Sequencing by Edman degradation over 24 cycles confirmed the identity of the peptide, and by analysis of a portion of the PTH-derivatives showed fluorescence at cycle 11, a lysine residue. The results imply that much of the yellow colour and characteristic fluorescence that accumulate in ageing human discs are contributed by covalent adducts (possibly derived from non-enzymic reactions with carbohydrates or lipids) linked to the collagen and probably to other long-lived matrix proteins. The disc is perhaps particularly susceptible to such protein modifications because, being large and avascular, of its tendency to a low oxygen tension. Such modifications to structural proteins may contribute to the commonly observed degeneration and impaired material function of ageing disc tissue.

The carboxypropeptide trimer of type II collagen is a prominent component of immature cartilages and intervertebral-disc tissue.

Immature bovine cartilages and intervertebral-disc tissue all revealed a prominent protein, not present in the adult tissues, in non-denaturing extracts made with chondroitin ABC lyase (EC 4.2.2.4), Streptomyces hyaluronidase (EC 4.2.2.1) or 1 M NaCl. The protein ran on SDS-polyacrylamide electrophoresis, before disulphide reduction, as a close doublet of bands of apparent molecular weight 110,000 and 105,000. After reduction, they dissociated respectively into two protein bands at 37,000 and 35,000, indicating that the initial molecules were disulphide-bonded trimers. Amino-terminal sequence analysis established the identity of both proteins (Mr 110,000 and Mr 105,000) as forms of the carboxypropeptide of type II collagen. The larger molecule appeared to be the trimer of intact alpha 1(II) carboxypropeptides and the smaller, a version composed of chains that were ten residues shorter at their amino-terminal ends. The material appears to be identical to chondrocalcin, a protein previously found to be enriched in fetal growth plate and named on the basis that it may play a role in cartilage calcification. The present findings, however, indicate that the protein is equally abundant in all type II collagen-synthesizing young cartilages, including nucleus pulposus of the intervertebral disc and other cartilages that never calcify.

Influence of plasma calcium and vitamin D on bone collagen. Effects on lysine hydroxylation and crosslink formation.

In chick bone collagen the degree of lysine hydroxylation and the magnitude of the crosslink ratio dihydroxylysinonorleucine/hydroxylysinonorleucine were both found to be inversely related to the concentration of plasma calcium. Lysine hydroxylation was also affected by a second factor related to vitamin D status.

Collagen type II differs from type I in native molecular packing.

Native molecular packing of types I and II collagens were compared by low-angle X-ray diffraction. Fibers from human intervertebral disc that contained different proportions of types I and II collagens were studied by X-ray diffraction, and were then analyzed biochemically to measure the constituent collagen species. Other cartilages, containing exclusively type I or type II collagen, were also examined. The equatorial diffraction established that in wet, native type II collagen, the molecules are spaced farther apart laterally than in type I collagen under the same conditions. For the disc the average lateral spacing ranged from about 14 A for a fiber from the outer annulus fibrosus containing mostly type I collagen, to 16-17 A for nucleus pulposus that contained all type II collagen. No differences were evident among dried specimens. We have also found that the meridional diffraction pattern from dry fibers of type II collagen differed from that of type I collagen. The findings indicate that under physiological conditions type II collagen fibrils contain more water than type I fibrils. Calculations suggest 50-100% more water. We propose that this difference is an inherent property of type II collagen and that it may be significant for the function of type II collagen in tissues that dissipate compressive forces. The content of glycosylated hydroxylysine residues is the chemical variable likely to be modulating fibrillar hydration.

Acetabular augmentation at six- to 30-year follow-up. A biochemical and histological analysis.

In developmental dysplasia of the hip, a deficient acetabulum may be augmented by placing local autogenous iliac osseous graft, or the ilium itself, over the head of the femur with the expectation that the added bone will function as a bearing surface. We analysed this bone obtained en bloc during subsequent surgery which was performed for degenerative osteoarthritis in three patients at 6, 25 and 30 years after the initial augmentation procedure. In each patient, the augmentation comprised of red cancellous bone covered on its articulating surface by a distinct layer of white tissue. Microscopy of this tissue showed parallel rows of spindle-shaped cells lying between linearly arranged collagen bundles typical of joint capsule. Biochemical analysis showed type I collagen, the principal collagen of joint capsule and bone, with no significant quantity of type II collagen, the principal collagen of cartilage. While the added bone produced by acetabular augmentation was durable, histological and biochemical analyses suggested that it had not undergone cartilage metaplasia. The augmented acetabulum articulates with the head of the femur by means of an interposed hip joint capsule.

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.

Collagen crosslinks and mineral crystallinity in bone of patients with osteogenesis imperfecta.

In cortical bone samples from patients with osteogenesis imperfecta (OI), the concentrations of hydroxypyridinium cross-linking amino acids in collagen were measured by reversed-phase HPLC and the x-axis crystallinity of the apatite mineral phase was determined by x-ray diffraction. Bone samples from three patients with type I, nine patients with type III, and eight patients with type IV OI were analyzed and compared with human bone from nine controls. The concentration of the two chemical forms of the mature collagen crosslinking amino acids, hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), and the ratio HP/LP were found to be alike in bone collagen of OI patients and healthy controls. However, the c-axis crystallinity of the apatite was found to be reduced in the type III and IV OI patients compared with controls. Regression analysis of crosslink concentrations and c-axis crystallinity in OI bones did not show any correlation. Therefore, collagen molecules deposited in the extracellular matrix of OI bone appear to fulfill the structural requirements for the action of the enzyme lysyl oxidase, such that a normal concentration of intermolecular crosslinks is formed compared with healthy bone. Consequently, crosslink formation and apatite crystal growth seem to be regulated independently in OI bone.

Post-translational control of collagen fibrillogenesis in mineralizing cultures of chick osteoblasts.

Cultured osteoblasts from chick embryo calvaria were used as a model system to investigate the post-translational extracellular mechanisms controlling the macroassembly of collagen fibrils. The results of these studies demonstrated that cultured osteoblasts secreted a collagenous extracellular matrix that assembled and mineralized in a defined temporal and spatial sequence. The assembly of collagen occurred in a polarized fashion, such that successive orthogonal arrays of fibrils formed between successive cell layers proceeding from the culture surface toward the media. Mineralization followed in the same manner, being observed first in the deepest and oldest fibril layers. Collagen fibrillogenesis, the kinetics of cross-link formation, and collagen stability in the extracellular matrix of the cultures were examined over a 30 day culture period. Between days 8 and 12 in culture, collagen fibril diameters increased from < 30 nm to an average of 30-45 nm. Thereafter, diameters ranged in size from 20 to 200 nm. Quantitation of the collagen cross-linking residues, hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP), showed that these mature cross-links increased from undetectable levels to concentrations found in normal chick bone. Analysis of the kinetics of their formation by pulse-chase labeling the cultures with [3H]lysine showed a doubling time of approximately 5 days. The relationships between cross-link formation, fibrillogenesis, and collagen stability were examined in cultures treated with beta-aminopropionitrile (beta-APN), a potent inhibitor of lysyl oxidase and cross-link formation. In beta-APN-treated cultures, total collagen synthesis was increased twofold, with no change in mRNA levels for type I collagen, whereas the amount of collagen accumulated in the cell layer was decreased by 50% and mineral deposition was reduced. The rate of collagen retention in the matrix was assessed by pulse-chase analysis of [3H]proline over a 16 day period in control and beta-APN-treated cultures. In control cultures, about 20% of the labeled collagen was lost from the cell layers over a 16 day period compared with > 80% in the presence of beta-APN. The beta-APN-treated cultures also showed a wider diversity of fibril diameters with a median in the > 45-60 nm range. In summary, these data suggest that cross-linking and assembly of collagen fibrils secreted by osteoblasts in vitro occur in a fashion similar to that found in vivo. The rate of cross-link formation is relatively constant and may be correlated with increasing collagen mass.(ABSTRACT TRUNCATED AT 400 WORDS)

Bone resorption assessed by immunoassay of urinary cross-linked collagen peptides in patients with osteogenesis imperfecta.

Urinary excretion of type I collagen cross-linked N-telopeptides was studied in 52 children and adolescents with osteogenesis imperfecta (OI) and found to be above the 75th percentile of controls in 44 of the patients. OI patients suffering from fractures during the preceding 6 months had significantly higher values (p < 0.05). In contrast, patients with better motor performance tended to have lower values (p = 0.059). The concentration of urinary type I collagen cross-linked N-telopeptides was positively correlated with urinary calcium excretion (p < 0.05), which was found to be elevated in 20 of the patients. Our results show that during childhood and adolescence in OI not only the synthesis but also the turnover of mature cross-linked type I collagen is disturbed and provide evidence that bone resorption rates are elevated.