Chemistry of the collagen cross-links. Isolation and characterization of two intermediate intermolecular cross-links in collagen

This paper describes the isolation from reduced collagen of two new amino acids believed to be involved, in their non-reduced form, as intermolecular cross-links stabilizing the collagen fibre. The reduction of intact collagen fibrils with tritiated sodium borohydride was found to stabilize the aldehyde-mediated cross-links to acid hydrolysis and thus allowed their location and isolation from acid hydrolysates on an automatic amino acid analyser. Comparison of the radioactive elution patterns from the autoanalyser of collagen treated in various ways before reduction permitted a preliminary classification of the peaks into cross-link precursors, intramolecular and intermolecular cross-links. The techniques employed to isolate the purified components on a large scale and to identify them structurally are described in detail. Two labile intermolecular cross-links were isolated in their reduced forms, one of which was identified by high-resolution mass spectrometry as N-(5-amino-5-carboxypentyl)hydroxylysine. The structure of this compound was confirmed by chemical synthesis. The cross-link precursor α-aminoadipic δ-semialdehyde was isolated in its reduced form, -hydroxynorleucine, together with its acid degradation product -chloronorleucine. A relatively stable intermolecular cross-link was isolated and partially characterized by mass spectrometry as an aldol resulting from the reaction of the δ-semialdehyde derived from lysine and hydroxylysine.     

The chemistry of the collagen cross-links. Characterization of the products of reduction of skin, tendon and bone with sodium cyanoborohydride.

Reduction of tissues with sodium cyanoborohydride at pH7.4 gave results identical with those obtained by KBH4 treatment. On reduction with sodium cyanoborohydride at pH 4.4, however, a previously undetected basic compound was formed and was identified by mass spectrometry and chemical degradation techniques as dihydrohydroxymerodesmosine. Histidino-hydroxymerodesmosine was not present, and further analysis confirmed that reduced aldol, a mojor product of reduction with KBH4 at the lower pH, was also absent. These results, together with an analysis of the time course of the reduction, support previous assertions that histidino-hydroxymerodesmosine is an artifact [robins *Bailey (1973) Biochem. J. 135, 657-665] and suggests that the non-reduced form of hydroxymerodesmosine probably does not constitute a major intermolecular bond in vivo.     

The chemistry of the collagen cross-links. The absence of reduction of dehydrolysinonorleucine and dehydrohydroxylysinonorleucine in vivo

Two aldimine bonds have been shown to be present as stabilizing cross-links in intact collagen fibres from soft tissues: dehydrohydroxylysinonorleucine as a major component and dehydrolysinonorleucine being present in trace quantities. In the highly insoluble collagens less dehydrohydroxylysinonorleucine is present but the proportion of dehydrolysinonorleucine increases. In elastin the latter aldimine is reduced in vivo to give a more stable cross-link but no comparable reduction could be detected with either of the aldimines present in collagen.     

Reactions of lipid-derived malondialdehyde with collagen.

Malondialdehyde is a product of fatty acid oxidation (e.g. from low density lipoprotein) implicated in the damage of proteins such as collagen in the cardiovascular system (Chio, K. J., and Tappel, A. L. (1969) Biochemistry 8, 2821-2827). Its concentration is raised in diabetic subjects probably as a side effect of increased protein glycation. Collagen has enzyme-catalyzed cross-links formed between its individual molecules that are essential for maintaining the structure and flexibility of the collagen fiber. The cross-link dehydro-hydroxylysinonorleucine reacts irreversibly with 10 mM malondialdehyde at least 3 orders of magnitude faster than glucose reactions with lysine or arginine, such that there is little cross-link left after 1 h at 37 degrees C. Other cross-links and glycated elements of collagen are also vulnerable. Several possible products of malondialdehyde with collagen cross-links are proposed, and the potential involvement of collagenous histidine in these reactions is discussed. We have also isolated Ndelta-(2-pyrimidyl)-L-ornithine from collagenous arginine reacted with malondialdehyde. The yields of this product were considerably higher than those from model reactions, being approximately 2 molecules/collagen molecule after 1 day at 37 degrees C in 10 mM malondialdehyde. Collagenous lysine-derived malondialdehyde products may have been present but were not protected from protein acid hydrolysis by standard reduction techniques, thus resulting in a multitude of fragmented products.     

The stability of collagen cross-links when derived from hydroxylsyl residues

The cross-linked cyanogen bromide peptide, (4×9), previously isolated after reduction of cartilage collagen, has been isolated without prior reduction of the collagen. The unreduced cross-link is cleaved by periodate allowing recovery of the component peptides. When isolated after borotritide reduction of the collagen, (4×9) contains a single residue of radioactive hydroxylysinohydroxynorleucine. Radioactivity in the cross-link remains in the component peptides when the cross-link is cleaved with periodate. Performic acid oxidation removes this radioactivity and produces an additional glutamic acid residue in each peptide. These data indicate that dehydrohydroxylysinohydroxynorleucine undergoes an Amadori rearrangement producing a more stable keto-amine form of the cross-link.     

The influence of 1-hydroxyethane-1,1-diphosphonate and dichloromethanediphosphonate on lysine hydroxylation and cross-link formation in rat bone, cartilage and skin collagen.

The effects in vivo of dichloromethanediphosphonate and 1-hydroxyethane 1,1-diphosphonate on collagen solubility, hydroxylation of lysine and proline and on the formation of collagen intermolecular cross-links were studied by using rat bone, cartilage and skin tissues. Dichloromethanediphosphonate decreased bone collagen solubility both in acetic acid and after pepsin treatment. Although none of the diphosphonates had any effect on the hydroxylation of proline, dichloromethane-diphosphonate, but not 1-hydroxyethane-1,1-diphosphonate, increased the number of hydroxylysine residues in the alpha-chains of bone, skin and cartilage collagen. The stimulatory effect was dose-dependent. The dichloromethanediphosphonate-mediated increase in hydroxylysine residues in bone and cartilage was manifested in an increase of dihydroxylysinonorleucine, the cross-link that is formed by the condensation of two hydroxylysine residues. The cross-link hydroxylysinonorleucine, a condensation product of hydroxylysine and lysine, on the other hand, was decreased. The total number of intermolecular cross-links was not changed by the diphosphonate.     

Effects of ascorbate on insoluble elastin accumulation and cross-link formation in rabbit pulmonary artery smooth muscle cultures

Cultured pulmonary artery smooth muscle cells derived from the medial vessel layer of weanling rabbits were grown in the presence or absence of sodium ascorbate. The connective tissue elements insoluble elastin and collagen were identified and quantified. Formation and accumulation of alpha-aminoadipic acid gamma-semialdehyde (allysine) and the intermolecular cross-links desmosine (Des), isodesmosine (Ides), and aldol condensation product (Aldol) were evaluated from [14C]lysine pulse-chase experiments. [14C]Des, [14C]Ides, peptide-bound [14C]lysine, [14C]allysine, and [14C]Aldol were determined from amino acid analysis. The latter two components were determined after reduction with NaBH4. [14C]Proline conversion to hydroxy[14C]proline and collagenase susceptibility were used to identify and quantify collagen synthesis. Ascorbate dramatically affects insoluble elastin synthesis, accumulation, and cross-link formation. Cells grown in the presence of ascorbate synthesize and accumulate significantly less insoluble elastin than non-ascorbate cultures. Those elastin molecules which do become incorporated into the extracellular matrix in the presence of ascorbate contain a slightly elevated content of hydroxyproline and lysine and, most importantly, are turned over more rapidly.     

Aldol-histidine,a new trifunctional collagen crosslink

A new trifunctional crosslink, 2,10-diamino-5-hydroxymethyl-6-(N-1-histidyl)-undecandioic acid, termed aldol-histidine, was isolated from borohydride reduced cow skin insoluble collagen. The compound was characterized by pmr spectroscopy and mass spectrometry of several volatile derivatives. The crosslink may be derived from Michael addition of a histidine residue to the known aldol crosslink, 2,10-diamino-5-formyl-5-undecandioic acid. This is the first example of a histidine containing crosslink found in structural protein.     

The increase in denaturation temperature following cross-linking of collagen is caused by dehydration of the fibres

Differential scanning calorimetry (DSC) was used to study the thermal stability of native and synthetically cross-linked rat-tail tendon at different levels of hydration, and the results compared with native rat-tail tendon. Three cross-linking agents of different length between functional groups were used: malondialdehyde (MDA), glutaraldehyde and hexamethylene diisocyanate (HMDC). Each yielded the same linear relation between the reciprocal of the denaturation temperature in Kelvin, T(max), and the water volume fraction, epsilon (1/T(max)=0.000731epsilon+0.002451) up to a critical hydration level, the volume fraction of water in the fully hydrated fibre. Thereafter, water was in excess, T(max) was constant and the fibre remained unchanged, no matter how much excess water was added. This T(max) value and the corresponding intrafibrillar volume fraction of water were as follows: 84.1 degrees C and 0.48 for glutaraldehyde treated fibres, 74.1 degrees C and 0.59 for HMDC treated fibres, 69.3 degrees C and 0.64 for MDA treated fibres, and 65.1 degrees C and 0.69 for untreated native fibres. Borohydride reduction of the native enzymic aldimines did not increase the denaturation temperature of the fibres. As all samples yielded the same temperature at the same hydration, the temperature could not be affected by the nature of the cross-link other than through its effect on hydration. Cross-linking therefore caused dehydration of the fibres by drawing the collagen molecules closer together and it was the reduced hydration that caused the increased temperature stability. The cross-linking studied here only reduced the quantity of water between the molecules and did not affect the water in intimate contact with, or bound to, the molecule itself. The enthalpy of denaturation was therefore unaffected by cross-linking. Thus, the "polymer-in-a-box" mechanism of stabilization, previously proposed to explain the effect of dehydration on the thermal properties of native tendon, explained the new data also. In this mechanism, the configurational entropy of the unfolding molecule is reduced by its confinement in the fibre lattice, which shrinks on cross-linking.     

The chemistry of the collagen cross-links. Age-related changes in the reducible components of intact bovine collagen fibres

The change in the amounts of the three major reducible cross-links was followed throughout the bovine-life span. The major reducible cross-link in embryonic skin is 6,7-dehydro-N(epsilon) -(2-hydroxy-5-amino-5-carboxypentyl)hydroxylysine, but this is gradually replaced in the latter stages of gestation or early postnatal growth period by two other Schiff bases, 6,7-dehydro-N(epsilon)-(5-amino-5-carboxypentyl)hydroxylysine and a component not yet identified, designated Fraction C. These latter two Schiff bases increase in amount during the rapid growth period to a maximum, after which they then slowly decrease until at maturity they are virtually absent. The proportion of these Schiff bases closely reflects the rate of growth, i.e. the amount of newly synthesized collagen present at any one time. Similarly, the three Schiff bases present in tendon and the one in cartilage slowly decrease during maturation. No evidence for the possible stabilization of these aldimine bonds during maturation by reduction in vivo was found by three different analytical techniques. Concurrently with the decrease in the proportion of the Schiff bases some new reducible components increased during maturation, but their characterization as N(epsilon)-glycosylamines demonstrated that they were not related to the lysine-derived aldehyde components. The significance of these components in the aging process cannot at present be assessed. As no evidence was obtained for any new reducible cross-links replacing the Schiff bases, it is probable that the latter are intermediate cross-links and that during maturation they are stabilized to some as yet unknown non-reducible cross-link as previously proposed (Bailey, 1968).     

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.     

Cross-linking of connectin, an elastic protein in muscle

Following reduction with NaB³H₄, connectin, an elastic protein prepared from chicken muscle, was found to contain the reducible cross-links derived from lysine and hydroxylysine aldehydes. The aldimine form of lysinonorleucine is the most abundant reducible cross-link in this elastic protein. A smaller proportion of the reduced cross-link, histidino-hydroxymerodesmosine, is also detected. Since collagen contamination in the connectin preparation was if any negligible, it is concluded that connectin and connective tissue proteins, collagen and elastin, share common features of coss-linking.     

Structure and formation of a stable histidine-based trifunctional cross-link in skin collagen

A stable nonreducible trifunctional cross-linking amino acid has been isolated from mature bovine skin collagen fibrils. Previous cross-link peptide isolations and amino acid analyses indicate the compound has properties identical with those of hydroxyaldolhistidine. Its newly proposed structure was verified using fast atom bombardment mass spectrometry, and 1H and 13C nuclear magnetic resonance. The data indicated that the cross-link consists of the prosthetic groups from one residue each of histidine, hydroxylysine, and lysine. The 1H and 13C nuclear magnetic resonance data indicated that imidazole C-2 of histidine is linked to C-6 of norleucine (epsilon-deaminated lysine residue) which in turn is linked to the C-6 amino group of hydroxylysine. Based on the trivial names for other cross-linking residues found in collagen and elastin it was given the name histidinohydroxylysinonorleucine. In vitro incubation studies for up to 24 weeks, in aqueous solution at physiological pH and ionic strength, using 6-month-old bovine embryo skin demonstrated a one-to-one stoichiometric relationship between the disappearance of the labile reducible bifunctional cross-link dehydrohydroxylysinonorleucine and the appearance of histidinohydroxylsinonorleucine. These results can partially explain the previously observed disappearance of dehydrohydroxylysinonorleucine with chronological age.     

Lysyl oxidase dependent synthesis of a collagen cross-link containing histidine

To date, collagen appears unique among proteins in that it contains histidine in certain of its cross-links. Synthesis of histidine containing collagen cross-links was studied in vitro with lathyritic L-¹⁴C histidine or L-¹⁴C lysine labelled bone collagen fibrils and purified lysyl oxidase. Synthesis of the tetrafunctional cross-link, dehydrohistidinohydroxymerodesmosine occurred with lysyl oxidase and was inhibited by β-aminopropionitrile. Synthesis began after a lag period of sixteen hours and then proceeded linearly for four days. These data indicate that enzyme dependent synthesis of dehydrohistidinohydroxymerodesmosine occurs in vitro in a biochemically defined system. Biosynthesis in vivo might occur under similar conditions.     

The collagen of heart valve.

A hydroxylysine-rich type I collagen has been isolated from pepsin-digested porcine heart valve. The ratio of alpha1 to alpha2 in the isolated molecule was 2:1. The component alpha chains exhibited unusual chromatographic behavior in comparison to corresponding chains from human dermis and lathyritic rat skin collagen. The composition of component cyanogen bromide peptides identified the alpha chains as authentic type I chains and demonstrated hydroxylysine enrichment throughout the length of the chain. delta6-Dehydro-5,5'dihydroxylysinonorleucine, a collagen cross-link derived from two hydroxylysyl residues and ordinarily found in hard tissue collagens was found to be the predominant cross-link in heart valve.     

The interaction of collagen with alpha1-acid glycoprotein.

The influence of alpha1-acid glycoprotein on the formation of fibrous long spacing fibers of collagen has been investigated. It was observed that addition of the glycoprotein to dialyzed collagen solutions caused a significant decrease in the intensity of the circular dichroic spectrum of collagen. This phenomenon, which displays an optimum with respect to glycoprotein, is consistent with previous observations of fibrous long spacing fiber formation. Changes in viscosity of collagen initially dissolved in acetic acid were monitored during dialysis. It was found that a significant increase in viscosity must occur during dialysis of collagen before fibrous long spacing formation could take place. This increase in viscosity can be related directly to removal of acetic acid from the collagen solution. Removal of all sialyl residues from the alpha1-acid glycoprotein with neuraminidase prevents fibrous long spacing formation while removal of up to 35% of the sialyl residues has no effect on the interaction of glycoprotein with collagen. Amino acid composition and radioactivity studies suggest that 45-55% of the insoluble fibrous long spacing fibers is glycoprotein. In contrast to native collagen fibers, reduced fibrous long spacing fibers do not contain histidinohydroxymerodesmosine or hydroxylysinonorleucine. Instead, they contain significant quantities of allysine aldol and epsilon-hydroxynorleucine.     

Simultaneous determination of the reducible and nonreducible cross-links of connective tissue. Analysis of mineralized and nonmineralized bone collagen

Secondary amine cross-links occur in collagen and elastin from a number of tissue sources. Quantification of these cross-links by amino acid analysis is complicated by the problem of separating cross-links, which are often minor components, from the more common amino acids and also because relatively large amounts of a cross-link are required to determine a color factor. A specific radioactive labeling method has been developed and used to quantify cross-links in bone collagen. Primary amines such as lysine and hydroxylysine are first guanidinated with 3,5-dimethylpyrazole-1-carboxamidine nitrate (DMPC). Secondary amines, which are unreactive with DMPC, are then quantitatively cyanoethylated with [14C]acrylonitrile. This procedure can be used to detect any secondary amine cross-link, with higher sensitivity than ninhydrin analysis, in peptide form as well as in acid hydrolysates. It is applied here in conjunction with [3H]NaBH4 reduction to simultaneously quantify Schiff base cross-links and amounts of in vivo reduction of Schiff bases in mineralized versus nonmineralized bovine bone.     

Phenotypic expression of osteoblast collagen in osteoarthritic bone: production of type I homotrimer.

The metabolism and total amount of the collagen of subchondral bone are increased several fold in osteoathritic femurs compared with controls. We now report for the first time that the quality of the collagen is modified by the formation of type I homotrimer. The homotrimer fibre has been reported to possess a reduced mechanical strength and mineralisation in bone. The presence of the latter therefore accounts for narrower disorganised collagen fibres and decreased mineralisation, and a reduction in mechanical stability of the osteoarthritic femoral head. These changes in the subchondral bone are likely to be of considerable importance in the pathogenesis of osteoarthritis.     

The identity of a cyanogen bromide fragment of bovine dentin collagen containing the site of an intermolecular cross-link.

A peptide fraction isolated from a cyanogen bromide digest of bovine dentin collagen had a molecular weight of 46000. Its size and amino acid composition indicated that it could not consist of peptides derived from the cleavage of a single alpha chain. On reduction with tritiated sodium borohydride, radioactivity was incorporated primarily into 5, 5'-dihydroxylysinonorleucine without degradation at the peptide backbone. Periodate cleavage of the reduced or nonreduced peptide fraction generated one fragment of molecular weight 28000 and one of 18000 completely accounting for the size of the parent peptide. On amino acid analysis the constituent single-chain peptides were determined to be alpha2CB4 and alpha1CB6. Both peptides isolated after periodate oxidation of the tritiated borohydride reduced cross-link peptide were found to contain (3H)hydroxynorvaline. These data show that some hydroxylysine of alpha2CB4, a helical region peptide, was present in aldehyde form and could act as the aldehyde donor icross-link, Schiff's base formation. The only cross-linkage of this alpha2CB4 acting as an aldehyde donor peptide to alpha1CB6 would be a helical region to helical region bond, perhaps accounting for the unusual stability and low solubility of dentin collagen.     

Reduced collagen and ascorbic acid concentrations and increased proteolytic susceptibility with prelabor fetal membrane rupture in women

Prelabor rupture of the fetal membranes affects approximately 10% of women at term, resulting in an increased risk of maternal and neonatal infection. Evidence suggests that membrane rupture is related to biochemical processes involving the extracellular matrix of the membranes. We tested the hypothesis that prelabor ruptured membranes are characterized by reduced collagen concentrations, altered collagen cross-link profiles, and increased concentrations of biomarkers of oxidative damage. We also set out to determine whether these effects are modulated by ascorbic acid status. In a case-control study, we explored the role that ascorbic acid, oxidative stress, collagen, and collagen cross-links play in determining membrane integrity and developed a functional assay to assess membrane proteolytic susceptibility. Prelabor ruptured membrane had a reduced ascorbic acid concentration in comparison with controls while protein carbonyl and malondialdehyde concentrations were increased. Collagen concentrations were also reduced in prelabor ruptured membrane, and while the concentration of collagen cross-links was not significantly different between prelabor and timely ruptured membrane, there was a regional variation in cross-link ratio within the amniotic sac. Proteolytic resistance in vitro was reduced in prelabor ruptured membrane and also exhibited regional variation within the amniotic sac. Our findings are strongly supportive of a role for the enhanced degradation of membrane collagen in the determination of prelabor rupture of fetal membranes. The formation of the rupture initiation site is a function of a regional variation in collagen cross-link ratio. Tissue ascorbic acid status may be an important mediator of these processes.