Analyses of lysine aldehyde cross-linking in collagen reveal that the mature cross-link histidinohydroxylysinonorleucine is an artifact.

Lysyl oxidase-generated intermolecular cross-links are essential for the tensile strength of collagen fibrils. Two cross-linking pathways can be defined, one based on telopeptide lysine aldehydes and another on telopeptide hydroxylysine aldehydes. Since the 1970s it has been accepted that the mature cross-linking structures on the lysine aldehyde pathway, which dominates in skin and cornea, incorporate histidine residues. Here, using a range of MS-based methods, we re-examined this conclusion and found that telopeptide aldol dimerization is the primary mechanism for stable cross-link formation. The C-telopeptide aldol dimers formed labile addition products with glucosylgalactosyl hydroxylysine at α1(I)K87 in adjacent collagen molecules that resisted borohydride reduction and after acid hydrolysis produced histidinohydroxylysinonorleucine (HHL), but only from species with a histidine in their α1(I) C-telopeptide sequence. Peptide MS analyses and the lack of HHL formation in rat and mouse skin, species that lack an α1(I) C-telopeptide histidine, revealed that HHL is a laboratory artifact rather than a natural cross-linking structure. Our experimental results also establish that histidinohydroxymerodesmosine is produced by borohydride reduction of N-telopeptide allysine aldol dimers in aldimine intermolecular linkage to nonglycosylated α1(I) K930. Borohydride reduction of the aldimine promotes an accompanying base-catalyzed Michael addition of α1(I) H932 imidazole to the α,ß-unsaturated aldol. These aldehydes are intramolecular at the N terminus but at the C terminus they can be both intramolecular and intermolecular according to present and earlier findings.

Molecular insights into prolyl and lysyl hydroxylation of fibrillar collagens in health and disease.

Collagen is a macromolecule that has versatile roles in physiology, ranging from structural support to mediating cell signaling. Formation of mature collagen fibrils out of procollagen α-chains requires a variety of enzymes and chaperones in a complex process spanning both intracellular and extracellular post-translational modifications. These processes include modifications of amino acids, folding of procollagen α-chains into a triple-helical configuration and subsequent stabilization, facilitation of transportation out of the cell, cleavage of propeptides, aggregation, cross-link formation, and finally the formation of mature fibrils. Disruption of any of the proteins involved in these biosynthesis steps potentially result in a variety of connective tissue diseases because of a destabilized extracellular matrix. In this review, we give a revised overview of the enzymes and chaperones currently known to be relevant to the conversion of lysine and proline into hydroxyproline and hydroxylysine, respectively, and the O-glycosylation of hydroxylysine and give insights into the consequences when these steps are disrupted.

2-Oxazoline formation for selective chemical labeling of 5-hydroxylysine.

Hydroxylation of lysine, one of posttranslational modifications of proteins, generates 5-hydroxylysine (Koh) and plays a crucial role in regulating protein functions in cellular activity. We have developed a chemical labeling method of Koh. The 1,2-aminoalcohol moiety of Koh in synthetic peptide sequences was trapped by an alkyne-containing benzimidate to form a 2-oxazoline ring. An additional ammonia treatment process removed the undesirable amidine residue formed between benzimidate and lysine. During the ammonia treatment, the oxazoline residue formed at Koh mainly remained intact, and the ring opening to the amide form was observed for only part of oxazoline, indicating that the chemical labeling is amino acid selective. Azide-substituted biotin or fluorescent dye was attached to the peptide through Huisgen cycloaddition at Koh and converted into an alkyne-labeled oxazoline form. The Koh-labeling assay could provide a platform to enhance proteomic research of lysine hydroxylation.

LC-MS/MS identification of the O-glycosylation and hydroxylation of amino acid residues of collagen α-1 (II) chain from bovine cartilage.

O-Glycosylation of collagen is a unique type of posttranslational modifications (PTMs) involving the attachment of galactose (Gal) or glucose-galactose (Glc-Gal) moieties to hydroxylysine (HyK). Also, hydroxyproline (HyP) result from the posttranslational hydroxylation of some proline residues in collagen. Here, LC-MS/MS was effectively employed to identify 23 O-glycosylation sites and a large number of HyP residues associated with bovine type II collagen α-1 chain (CO2A1). The modifications of the 23 O-glycosylation sites varied qualitatively and quantitatively. Both Gal and Glc-Gal moieties occupied 22 of the identified glycosylation sites, while K773 was observed as unmodified. A large number of HyP residues at Yaa positions of Gly-Xaa-Yaa motif were detected. HyP residues at Xaa positions of Gly-HyP-HyP, Gly-HyP-Ala, and Gly-HyP-Val motifs were also observed. Notably, HyP residue of Gly-HyP-Gln motif was detected, which has not been previously reported. Moreover, the deamidation of 8 Asn residues was identified, of which 2 Asp residues were observed at different retention times because of isomerization (Asp vs isoAsp). Partial macroheterogeneities of some CO2A1 glycosylation sites were revealed by LC-MS/MS analysis. ETD experiments revealed partial macroheterogeneities associated with K299-K308, K452-K464, K464-K470, and K857-K884 glycosylation sites. Semiquantitative data suggest that the glycosylation of hydroxylysine residues is site-specific.

Post-translational modifications of collagen upon BMP-induced osteoblast differentiation.

The pattern of collagen cross-linking is tissue specific primarily determined by the extent of hydroxylation and oxidation of specific lysine residues in the molecule. In this study, murine pre-myoblast cell line, C2C12 cells, were transdifferentiated into osteoblastic cells by bone morphogenetic protein (BMP)-2 treatment, and the gene expression of lysyl hydroxylases (LH1, 2a/b, and 3) and lysyl oxidase (LOX)/lysyl oxidase-like proteins (LOXL1-4), and the extent of hydroxylysine were analyzed. After 24h of treatment, the expression of most isoforms were upregulated up to 96h whereas LH2a and LOXL2 decreased with time. In the treated cells, both hydroxyproline and hydroxylysine were detected at day 7 and increased at day 14. The ratio of hydroxylysine to hydroxyproline was significantly increased at day 14. The results indicate that LHs and LOX/LOXLs are differentially responsive to BMP-induced osteoblast differentiation that may eventually lead to the specific collagen cross-linking pattern seen in bone.

Analysis of hydroxyproline isomers and hydroxylysine by reversed-phase HPLC and mass spectrometry.

Collagens, the most abundant mammalian proteins, contain a high content of hydroxylated amino acids, such as, 3- and 4-cis-/trans-hydroxyproline (Hyp) and 5-hydroxylysine (Hyl). Whereas the global content of 4-Hyp was studied by amino acid analysis, no technique to determine all five hydroxyamino acids simultaneously in collagens has been reported. Here, we report the separation of all five hydroxyamino acids as well as two Hyp epimers from all other proteinogenic amino acids after derivatization with N(2)-(5-fluoro-2,4-dinitrophenyl)-l-valine amide (l-FDVA) by RPC-UV-ESI-MS. The general applicability of this method is shown for three Hyp-containing peptides as well as collagen type I.

Properties of collagen in OIM mouse tissues.

The deletion of the alpha2 chain from type I collagen in the oim mouse model of osteogenesis imperfecta has been shown to result in a significant reduction in the mechanical strength of the tail tendon and bone tissue. However, the exact role of the alpha2 chain in reducing the mechanical properties is not clear. We now report that the stabilizing intermolecular cross-links in bone are significantly reduced by 27%, thereby contributing to the loss of tensile strength and the change in stress-strain profile. We also report that, in contrast to previous studies, the denaturation temperature of the triple helical molecule and the intact fibers are 2.6 degrees and 1.9 degrees C higher than the corresponding tail tendon collagen from wild-type mice. The increase in hydroxyproline content accounts, at least in part, for the increase in denaturation temperature. The alpha2 chain clearly plays an important part in stabilizing the type I collagen triple helix and fiber packing, but further studies are required to determine the precise mechanism.

Histomorphometric, biochemical and ultrastructural changes in the aorta of salt-loaded stroke-prone spontaneously hypertensive rats fed a Japanese-style diet.

BACKGROUND AND AIM: It is demonstrated that dietary habits play a role in cardiovascular diseases. In stroke-prone spontaneously hypertensive rats (SHRsp), concomitant salt loading and a Japanese-style diet greatly accelerate hypertension and the appearance of cerebrovascular lesions by directly damaging arterial vessels. A number of studies have characterised medium and small vessel lesions in SHRsp, but little attention has been paid to the changes in the wall structure of large arteries induced by exposure to a salt-enriched diet. The aim of this study was to investigate the effects of a Japanese-style diet and salt loading on the thoracic aorta. METHODS AND RESULTS: Two-month-old SHRsp were kept on a Japanese-style diet with 1% sodium chloride solution replacing tap water. Two months later, they were sacrificed and compared with age-matched or two-month-old control SHRsp kept on a standard diet and tap water in terms of the histomorphometry, ultrastructure and biochemical composition of the thoracic aorta. The vessel was consistently thicker in the four-month-old SHRsp (+20%, p < 0.05 vs two-month-old rats) regardless of diet. The salt-loaded SHRsp showed a significant reduction in elastic fibre density (-20%, p < 0.05 vs two-month-old rats) and an increase in the other matrix components (%), whereas the four-month-old controls showed preserved elastic fibres and a significant increase in the other matrix components (+65%, p < 0.05 vs two-month-old rats). There was a considerable increase in the amounts of 4-OH-proline (+147%), 5-OH-lysine (+174%) and desmosines (+360%) in the four-month-old controls vs their two-month-old counterparts (p < 0.01), but not in the salt-loaded animals. Ultrastructural analysis revealed clear damage and accelerated aging in the thoracic aorta of the salt-loaded SHRsp. CONCLUSIONS: Salt loading and a Japanese-style diet destabilize thoracic aorta architecture in SHRsp after two months of treatment.

The role of the alpha2 chain in the stabilization of the collagen type I heterotrimer: a study of the type I homotrimer in oim mouse tissues.

We have previously reported that the fragility of skin, tendon and bone from the oim mouse is related to a significant reduction in the intermolecular cross-linking. The oim mutation is unlikely to affect the efficacy of the lysyl oxidase, suggesting that the defect is in the molecule and fibre. We have therefore investigated the integrity of both the oim collagen molecules and the fibre by differential scanning calorimetry. The denaturation temperature of the oim molecule in solution and the fibre from tail tendon were found to be higher than the wild-type by 2.6deg.C and 1.9deg.C, respectively. With the loss of the alpha2 chain, the hydroxyproline content of the homotrimer is higher than the heterotrimer, which may account for the increase. There is a small decrease in the enthalpy of the oim fibres but it is not significant, suggesting that the amount of disorder of the triple-helical molecules and of the fibres is small and involves only a small part of the total bond energy holding the helical structure together. The difference in denaturation temperature of the skin collagen molecules (t(m)) and fibres (t(d)) is significantly lower for the oim tissues, 19.9deg.C against 23.1deg.C, indicating reduced molecular interactions and hence packing of the molecules in the fibre. Computation of the volume fraction of the water revealed that the interaxial separation of the oim fibres was indeed greater, increasing from 19.6A to 21.0A. This difference of 1.4A, equivalent to a C-C bond, would certainly decrease the ability of the telopeptide aldehyde to interact with the epsilon -amino group from an adjacent molecule and form a cross-link. We suggest, therefore, that the reduction of the cross-linking is due to increased water content of the fibre rather than a distortion of the molecular structure. The higher hydrophobicity of the alpha2 chain appears to play a role in the stabilisation of heterotrimeric type I collagen, possibly by increasing the hydrophobic interactions between the heterotrimeric molecules, thereby reducing the water content and increasing the binding of the molecules in the fibre.

Alteration of elastin, collagen and their cross-links in abdominal aortic aneurysms.

OBJECTIVES: although the mechanism of arterial dilation and aneurysm development has not been clarified, the degradation of elastin and collagen plays undoubtedly a critical role. We evaluated the elastin and collagen content through the detection of their cross-links in aneurysmal and non-aneurysmal abdominal aortic walls. MATERIALS AND METHODS: in 26 human abdominal aortic aneurysm specimens obtained during surgery and in 24 autopsy control samples of non-aneurysmal abdominal aorta the tissue content of elastin and collagen cross-links were measured by HPLC. Collagen was also detected by evaluating two characteristic amino acids, 4-hydroxyproline (4-hypro) with a colorimetric method and 5-hydroxylysine (5-hylys) by gas chromatography. RESULTS: significantly fewer elastin cross-links were found in aneurysm samples compared to controls (desmosines and isodesmosines: 90% reduction; p<0.01). The opposite was true for pyridinoline collagen cross-links (350% increase) and deoxypyridinolines (100% increase, p=0.01). Tissue content of 5-hylys, 4-hypro and total amino acids were reduced significantly by 50% in aneurysmal samples. CONCLUSIONS: beside confirming decreased elastin content in aneurysmal walls, these results show a concurrent increase of collagen cross-links. Since total collagen markers were decreased (decreased 4-hypro and 5-hylys) it is reasonable to suggest that in aneurysmal aortic walls old collagen accumulates cross-links while new collagen biosynthesis is somehow defective.

Development of biochemical heterogeneity of articular cartilage: influences of age and exercise.

The objective of this study was to document the development of biochemical heterogeneity from birth to maturity in equine articular cartilage, and to test the hypothesis that the amount of exercise during early life may influence this process. Neonatal foals showed no biochemical heterogeneity whatsoever, in contrast to a clear biochemical heterogeneity in mature horses. The process of formation of site differences was almost completed in exercised foals age 5 months, but was delayed in those deprived of exercise. For some collagen-related parameters, this delay was not compensated for after an additional 6 month period of moderate exercise. It is concluded that the functional adaptation of articular cartilage, as reflected in the formation of biochemical heterogeneity in the horse, occurs for the most part during the first 5 months postpartum. A certain level of exercise seems essential for this process and withholding exercise in early life, may result in a delay in the adaptation of the cartilage.

Effects of phosphoric acid and glutaraldehyde-HEMA on dentin collagen.

The purpose of this study was to evaluate the effects of phosphoric acid (PA) and a proprietary glutaraldehyde-HEMA aqueous solution (Gluma Desensitizer; GD) on dentin collagen. Specimens of demineralized bovine dentin collagen were treated with either 37% or 50% PA for 1 or 5 min. An additional set of specimens was treated with 37% PA for 1 min followed by GD for 1 min. All specimens were washed with distilled water, lyophilized. reduced with standardized NaB3H4, hydrolyzed with 6 M HCl and subjected to amino acid and cross-link analyses. The results demonstrated that the treatment of demineralized dentin with PA under the conditions tested did not significantly alter the collagen cross-links. The GD-treated samples showed reduction of free lysine (Lys) and hydroxylysine (Hyl) residues, as well as a decrease in the levels of collagen reducible cross-links. In addition, unidentified reducible compounds were detected by high-performance liquid chromatography (HPLC) analysis. These compounds may be derived from cross-links formed between GD-derived aldehyde and Lys/Hyl of collagen. The findings indicate that PA treatment does not significantly affect dentin collagen amino acid and cross-link composition, and that GD treatment affects dentin collagen amino acid and cross-link composition.

A rapid and simple method for quantitation of urinary hydroxylysyl glycosides, indicators of collagen turnover, using liquid chromatography/tandem mass spectrometry.

Some glycosides of hydroxylysine, viz., alpha-1, 2-glucosylgalactosyl-O-hydroxylysine and beta-1-galactosyl-O-hydroxylysine, appear to be good indicators of collagen turnover. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method is proposed for measuring these analytes in urine, with no sample preparation except for a dilution step. Quantitation is performed using external calibration with no internal standard. A preliminary survey indicates good intra- and inter-day reproducibility (better than 5 and 8%, respectively). With the present method, the estimated limits of detection (S/N > 3) in urine are 0.8 and 0.5 microM/L for beta-1-galactosyl-O-hydroxylysine and alpha-1,2-glucosylgalactosyl-O-hydroxylysine, respectively. The method is proposed as a robust tool for a large-scale research investigation on collagen turnover.

The influence of strenuous exercise on collagen characteristics of articular cartilage in Thoroughbreds age 2 years.

In order to assess the influence of strenuous exercise on collagen characteristics of articular cartilage, the response of the collagen network was studied in seven 2-year-old Thoroughbreds subjected to strenuous exercise compared to 7 nontrained individuals. After 13 weeks, the animals were subjected to euthanasia, fetlock joints of the forelimbs were scored macroscopically after Indian Ink staining, and articular cartilage from different locations of the articular surface of the proximal first phalanx was sampled and analysed for water content, collagen content, hydroxylysine content and amount of hydroxylysylpyridinoline (HP) crosslinks. Gross lesions were significantly more severe in the exercised than in the nonexercised group. In the control animals, the characteristic site-specific differences in collagen parameters were found as described earlier, but in the strenuously exercised animals this physiological biochemical heterogeneity had disappeared. In the exercised animals, an increase in water content and a sharp decrease in HP crosslinking was found that was correlated with the presence of wear lines. It is concluded that the strenuous exercise provoked significant alterations in the characteristics of the collagen network of the articular cartilage of the fetlock joint which were suggestive of microdamage and loosening of the collagen network. The collagen component of cartilage, in contrast to the proteoglycan component, is known to have a very limited capacity for repair and remodelling due to an extremely low turnover rate. Therefore, alterations within the articular collagen network might be expected to play an important role in the pathophysiology of degenerative joint disorders.

Pyridinium cross-links in bone of patients with osteogenesis imperfecta: evidence of a normal intrafibrillar collagen packing.

The brittleness of bone in patients with osteogenesis imperfecta (OI) has been attributed to an aberrant collagen network. However, the role of collagen in the loss of tissue integrity has not been well established. To gain an insight into the biochemistry and structure of the collagen network, the cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) and the level of triple helical hydroxylysine (Hyl) were determined in bone of OI patients (types I, III, and IV) as well as controls. The amount of triple helical Hyl was increased in all patients. LP levels in OI were not significantly different; in contrast, the amount of HP (and as a consequence the HP/LP ratio and the total pyridinoline level) was significantly increased. There was no relationship between the sum of pyridinolines and the amount of triple helical Hyl, indicating that lysyl hydroxylation of the triple helix and the telopeptides are under separate control. Cross-linking is the result of a specific three-dimensional arrangement of collagens within the fibril; only molecules that are correctly aligned are able to form cross-links. Inasmuch as the total amount of pyridinoline cross-links in OI bone is similar to control bone, the packing geometry of intrafibrillar collagen molecules is not disturbed in OI. Consequently, the brittleness of bone is not caused by a disorganized intrafibrillar collagen packing and/or loss of cross-links. This is an unexpected finding, because mutant collagen molecules with a random distribution within the fibril are expected to result in disruptions of the alignment of neighboring collagen molecules. Pepsin digestion of OI bone revealed that collagen located at the surface of the fibril had lower cross-link levels compared with collagen located at the inside of the fibril, indicating that mutant molecules are not distributed randomly within the fibril but are located preferentially at the surface of the fibril.

Biological variability in assessing the clinical value of biochemical markers of bone turnover.

Analytical and biological variability of three bone markers, deoxypyridinoline (DPD), CrossLaps (CTx) and galactosylhydroxylysine (GHYL) were compared. From 14 healthy subjects (six women, eight men; age 29-44 years) recruited from our laboratory staff, two sets of samples of early morning urine were obtained - four samples taken weekly for 4 weeks (all subjects) and three samples taken monthly for 3 months from five subjects. Data were expressed as the ratio to creatinine concentration. All the methods met the analytical goals (CV(A)< or =1/2CV(I(within-subject))) DPD 0.06, CTx 0.05 and GHYL 0.07 with CV(I(within-subject)) being 0.22, 0.19 and 0.38, respectively. The reference values were of limited usefulness particularly for CTx and GHYL, the index of individuality (II) being 0.50 and 0.48 respectively. As the index of heterogeneity (IH) was not significant, being 0.23 for CTx, 0.28 for DPD and 0.46 for GHYL, which are all <1.71 (1+2S.D.), within-subject variances can be used to calculate the reference change value (RCV): 0.58 for DPD, 0.54 for CTx and 1. 08 for GHYL. Moreover, we found constant variations in DPD and CTx, week to week and month to month. Our findings suggest that DPD and CTx provide more reliable results than GHYL, showing a lower within-subject variation, a lower and time-constant RCV allowing reliable monitoring without regard for timing.

Functional adaptation of equine articular cartilage: the formation of regional biochemical characteristics up to age one year.

Biochemical heterogeneity of cartilage within a joint is well known in mature individuals. It has recently been reported that heterogeneity for proteoglycan content and chondrocyte metabolism in sheep develops postnatally under the influence of loading. No data exist on the collagen network in general or on the specific situation in the horse. The objective of this study was to investigate the alterations in equine articular cartilage biochemistry that occur from birth up to age one year, testing the hypothesis that the molecular composition of equine cartilage matrix is uniform at birth and biochemical heterogeneity is formed postnatally. Water content, DNA content, glycosaminoglycan content (GAG) and biochemical characteristics of the collagen network (collagen content, hydroxylysine content and hydroxylysylpyridinoline [HP] crosslinks) were measured in immature articular cartilage of neonatal (n = 16), 5-month-old foals (n = 16) and yearlings (n = 16) at 2 predefined differently loaded sites within the metacarpophalangeal joint. Statistical differences between sites were analysed by ANOVA (P<0.01), and age correlation was tested by Pearson's product moment correlation analysis (P<0.01). In neonatal cartilage no significant site differences were found for any of the measured biochemical parameters. This revealed that the horse has a biochemically uniform joint (i.e. the cartilage) at birth. In the 5-month-old foals and yearlings, significant site differences, comparable to those in the mature horse, were found for DNA, GAG, collagen content and hydroxylysine content. This indicates that functional adaptation of articular cartilage to weight bearing for these biochemical parameters takes place during the first months postpartum. Water content and HP crosslinks showed no difference between the 2 sites from neonatal horses, 5-month-old animals and yearlings. At both sites water, DNA and GAG decreased during maturation while collagen content, hydroxylysine content and HP crosslinks increased. We propose that a foal is born with a uniform biochemical composition of cartilage in which the functional adaptation to weight bearing takes place early in life. This adaptation results in biochemical and therefore biomechanical heterogeneity and is thought to be essential to resist the different loading conditions to which articular cartilage is subjected during later life. As collagen turnover is extremely low at mature age, an undisturbed functional adaptation of the collagen network of articular cartilage at a young age may be of significant importance for future strength and resistance to injury.

Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint.

The aim of this study was to evaluate topographical differences in the biochemical composition of the extracellular matrix of articular cartilage of the normal equine fetlock joint. Water content, DNA content, glycosaminoglycan (GAG) content and a number of characteristics of the collagen network (total collagen content, levels of hydroxylysine- (Hyl) and the crosslink hydroxylysylpyridinoline, (HP) of articular cartilage in the proximal 1st phalanx (P1), distal 3rd metacarpal bone (MC), and proximal sesamoid bones (PSB) were determined in the left and right fetlock joint of 6 mature horses (age 5-9 years). Twenty-eight sites were sampled per joint, which included the clinically important areas often associated with pathology. Biochemical differences were evaluated between sampling sites and related with the predisposition for osteochondral injury and type of loading. Significant regional differences in the composition of the extracellular matrix existed within the joint. Furthermore, left and right joints exhibited biochemical differences. Typical topographic distribution patterns were observed for each parameter. In P1 the dorsal and palmar articular margin showed a significantly lower GAG content than the more centrally located sites. Collagen content and HP crosslinks were higher at the joint margins than in the central area. Also, in the MC, GAG content was significantly lower at the (dorsal) articular margin compared with the central area. Consistent with findings in P1, collagen and HP crosslinks were significantly lower in the central area compared to the (dorsal) articular margin. Biochemical and biomechanical heterogeneity of articular cartilage is supposed to reflect the different functional demands made at different sites. In the present study, GAG content was highest in the constantly loaded central areas of the joint surfaces. In contrast, collagen content and HP crosslinks were higher in areas intermittently subjected to peak loading which suggests that the response to a certain type of loading of the various components of the extracellular matrix of articular cartilage are different. The differences in biochemical characteristics between the various sites may help to explain the site specificity of osteochondral lesions commonly found in the equine fetlock joint. Finally, these findings emphasise that the choice of sampling sites may profoundly influence the outcome of biochemical studies of articular cartilage.