Cross-linking connectivity in bone collagen fibrils: the COOH-terminal locus of free aldehyde.

Quantitative analyses of the chemical state of the 16c residue of the alpha 1 chain of bone collagen were performed on samples from fetal (4-6-month embryo) and mature (2-3 year old) bovine animals. All of this residue could be accounted for in terms of three chemical states, in relative amounts which depended upon the age of the animal. Most of the residue was incorporated into either bifunctional or trifunctional cross-links. Some of it, however, was present as free aldehyde, and the content increased with maturation. This was established by isolating and characterizing the aldehyde-containing peptides generated by tryptic digestion of NaB3H4-reduced mature bone collagen. We have concluded that the connectivity of COOH-terminal cross-linking in bone collagen fibrils changes with maturation in the following way: at first, each 16c residue in each of the two alpha 1 chains of the collagen molecule is incorporated into a sheet-like pattern of intermolecular iminium cross-links, which stabilizes the young, nonmineralized fibril as a whole. In time, some of these labile cross-links maturate into pyridinoline while others dissociate back to their precursor form. The latter is likely due to changes in the molecular packing brought about by the mineralization of the collagen fibrils. The resultant reduction in cross-linking connectivity may provide a mechanism for enhancing certain mechanical characteristics of the skeleton of a mature animal.

Amyotrophic lateral sclerosis: unusually low content of collagen in skin.

The skin tissues from patients with amyotrophic lateral sclerosis (ALS) and controls were studied by electron microscopy, and their amino acid compositions were examined. On electron microscopy, the most conspicuous findings in ALS were (1) a marked increase in amorphous material separating collagen bundles, and (2) the smaller diameter of collagen fibrils. These were more marked with longer duration of illness. Amino acid analysis showed that the levels of hydroxyproline, hydroxylysine, and glycine were significantly low (P less than 0.001, P less than 0.01, and P less than 0.001, respectively) in ALS patients as compared with those of controls, and there was a significant negative correlation between the level of hydroxyproline and duration of illness in ALS patients (r = -0.88, P less than 0.01). In addition, the collagen content per dry weight (mg) of the tissues in ALS was significantly smaller (P less than 0.001) than in controls. These results indicate that the metabolism of skin collagen might be affected in the disease process of ALS.

Collagen in the spicule organic matrix of the gorgonian Leptogorgia virgulata.

Decalcification of the calcareous spicules from the gorgonian Leptogorgia virgulata reveals an organic matrix that may be divided into water insoluble and soluble fractions. The insoluble fraction displays characteristics typical of collagen, which is an unusual component of an invertebrate calcium carbonate structure. This matrix fraction exhibits a collagenous amino acid profile and behavior upon SDS-PAGE. Furthermore, the reducible crosslink, dihydroxylysinonorleucine (DHLNL), is detected in this fraction. The composition of the matrix varies seasonally; i.e., the collagenous composition is most prevalent in the summer. These results indicate that the insoluble matrix is a dynamic structure. Potential roles of this matrix in spicule calcification are discussed.

Regional distribution of mineral and matrix in the femurs of rats flown on Cosmos 1887 biosatellite.

We combined biochemical measurements with novel techniques for image analysis in the rat femur to characterize the location and nature of the defect in mineralization known to occur in growing animals after spaceflight. Concentrations of mineral and osteocalcin were low in the distal half of the diaphysis and concentrations of collagen were low with evidence of increased synthesis in the proximal half of the diaphysis of the flight bones. X-ray microtomography provided semiquantitative data in computer-generated sections of whole wet bone that indicated a longitudinal gradient of decreasing mineralization toward the distal diaphysis, similar to the chemistry results. Analysis of embedded sections by backscattered electrons in a scanning electron microscope revealed distinct patterns of mineral distribution in the proximal, central, and distal regions of the diaphysis and also showed a net reduction in mineral levels toward the distal shaft. Increases in mineral density to higher fractions in controls were less in the flight bones at all three levels, with the most distal cross-sectional area most affected. The combined results from these novel techniques identified the areas of femoral diaphysis most vulnerable to the mineralization defect associated with spaceflight and/or the stress of landing.

Bovine dental pulp collagens: characterization of types III and V collagen.

Pulp was essentially solubilized by partial pepsin digestion. The various genetic types of collagens were isolated by differential salt precipitation and extraction. Types I, III and V collagen represented 56, 41 and 2% of the total collagen, respectively. The type V collagen comprised two different molecular species consisting of [alpha 1(V)]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V), the ratio of which was approx. 1:1.3. The major portion of the type III collagen was present as a high molecular weight aggregate which released alpha 1(III) chains upon reduction with 2-mercaptoethanol.

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.

The structure of mineralized collagen fibrils.

A new model for the structure of mineralized bone collagen is presented which is compatible with neutron diffraction, electromicroscopic, crosslinking, and composition-density data. Mineralized collagen fibrils are comprised of azimuthally oriented, flexible molecules laterally arranged on a superlattice. Four nearest neighbors are longitudinally staggered by 67 nm and two neighbors by 2* 67 nm. In early stages of mineralization the molecules are parallel to the fibril axis with an average interaxis distance of 1.8 nm. In later stages they become flexed away from the fibril axis by an anisotropic lateral compression of molecules to an interaxis distance of 1.3 nm. Three quarters of the mineral in bone is disposed within the fibrils with a symmetry and habit reflecting the above organization of the collagen molecules.

Cross-linking and stereospecific structure of collagen in mineralized and nonmineralized skeletal tissues.

Molecular distributions of the intermolecular cross-links in fetal bovine bone type I collagen fibrils were quantitatively determined and compared with those of periodontal ligament. Results indicated that Hyl and Lys residues in the COOH-terminal nonhelical peptide portions (residues 16C) of both alpha 1 chains were quantitatively converted to aldehydes. These in turn stoichiometrically formed cross-links with residues Hyl-87 on both alpha 1 and alpha 2 chains of neighboring molecules. The ratio of cross-linked alpha 1 to alpha 2 chains was 3.5 to 1 indicating a stereospecific packing of collagen molecules in the fibrils similar manner to periodontal ligament collagen. It was found that there were few aldehyde derived cross-links in the NH2-terminal nonhelical portions of the bone type I collagen. The relative paucity of the cross-links in NH2-terminal region of bone collagens may favor mineralization.

Aging and cross-linking of skin collagen.

This report represents a clear demonstration of a cross-link in collagen whose abundance is related to chronological aging of an organism. Recently its structure was identified as histidinohydroxylysinonorleucine. Quantification of the cross-link in various aged samples of bovine and human skin indicate that it rapidly increases from birth through maturation. Subsequently, a steady increase occurs with aging, approaching 1 mole/mole of collagen. This compound seems to be related to the relative proportions of soluble to insoluble collagen from skin in neutral salt, dilute acid, and denaturing aqueous solvents (higher concentration in the insoluble portion). It is absent from other major collagenous tissues such as dentin, bone and tendon.

Cross-linking and new bone collagen synthesis in immobilized and recovering primate osteoporosis.

Quantification of collagen cross-links of monkey bone (tibia), from various time periods of immobilization (up to 7 months) and their subsequent reambulation (up to 40 months) was determined. Results indicated reducible cross-link concentrations markedly increased and peaked at the seven-month period of immobilization and returned to control values after 40 months of recovery. Chromatographic profiles of the major cross-linked peptides indicated that the increased cross-linking after seven months immobilization occurred between residue Hylald-16c as well as Lysald-16c of the alpha 1 chains and residue 87 Hyl of alpha chains of type I collagen. Mature, stable cross-link concentrations as well as their molecular loci remained constant throughout immobilization and reambulation. These results strongly suggest that rapid new bone collagen synthesis occurred during the osteoporotic state due to immobilization. With long-term recovery, the rate of collagen synthesis apparently returned to match the control bone.

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.

Locus of a histidine-based, stable trifunctional, helix to helix collagen cross-link: stereospecific collagen structure of type I skin fibrils.

The loci of the three amino acid residues that contribute their prosthetic groups to form the stable, nonreducible, trifunctional intermolecular cross-link histidinohydroxylysinonorleucine in skin collagen fibrils were identified. Two apparently homogeneous three-chained histidinohydroxylysinonorleucine cross-linked peptides were chromatographically isolated. They were obtained from a tryptic digest of denatured unreduced 6 M guanidine hydrochloride insoluble bovine skin collagen. Amino acid and sequence analyses demonstrated that the prosthetic groups of alpha 1(I)-chain Hyl-87, alpha 1(I)-chain Lys-16c, and alpha 2(I)-chain His-92 formed the cross-link. The latter results served to define the locus of the stable, nonreducible trifunctional moiety. Identical types of analyses were performed on the three-chained peptides isolated after bacterial collagenase digestion of the cross-linked tryptic peptides. This confirmed the initial identification and location of the three peptides linked by the cross-link. In addition, data reported here provide for a correction of the micromolecular structure for the alpha 2(I) chain. Stereochemical considerations concerning this trifunctional cross-link's specific locus indicate that the steric relationships between the alpha chains of skin and skeletal tissue collagens are fundamentally different and the intermolecular relationships in skin fibrils are specific for skin. The same molecular relationships also indicate that histidinohydroxylysinonorleucine links three molecules of collagen. The stereochemistry of cross-linking for skin collagen is in accordance with and explains the X-ray findings of a 65-nm periodicity found for this tissue [Stinson, R. H., & Sweeny, P. R. (1980) Biochim. Biophys. Acta 621, 158; Brodsky, B., Eikenberry, E. F., & Cassidy, K. (1980) Biochim. Biophys. Acta 621, 162].

Fragility and composition of growing rat bone after one week in spaceflight.

To gain some insight into the early effects of spaceflight on skeletal metabolism, we quantified the major chemical constituents and a noncollagenous protein, osteocalcin, in the third-lumbar vertebrae and humeri from 8-wk-old rats that were part of the 7-day NASA Spacelab 3 flight experiments. The ratio of calcium to hydroxyproline in the humeral diaphysis increased from 8.5 in preflight to 9.8 in ground simulation control and only to 8.9 in flight bones. There was no demonstrable change in the fraction of nonmineralized collagen. Osteocalcin content was reduced in the humerus and vertebra. Reduced accumulation of mineral and osteocalcin with no associated decrease in collagen in flight animals suggests that both mineralization and collagen metabolism are impaired in growing animals during spaceflight within a few days after launch. Strength tests of the humeri of flight rats showed substantial deficits that appeared to be related, not only to the reduced bone mass, but also to the composition and quality of new bone formed.

Structural crosslinking of lung connective tissue collagen in the blotchy mouse.

Male mice with the sex-linked mutation Blotchy (Blo) have a defect in copper metabolism which results in deficient activity of a number of copper-containing enzymes. Inbred Blo/y mice spontaneously develop lung abnormalities which resemble emphysema and often die of ruptured aortic aneurysm. Lung, tail tendon, and tibial bone collagens from inbred Blo/y and their normal (+/y) litter mates were reduced with standardized [3H]NaBH4, acid and alkaline hydrolyzed, and chromatographed in order to quantify the aldehydic crosslink precursors, and the labile reducible and nonreducible stable mature covalent intermolecular crosslinks. Reducible lung collagen crosslinks were markedly (60%) decreased in the Blo/y mice and few, if any, mature nonreducible crosslinks were present. Total aldehydes were also decreased (65%) when Blo/y was compared to +/y. In tail tendon and bone, collagen crosslinks were decreased by only 28% and 15%, respectively. Selectively severe lack of activity of the copper-dependent enzyme level oxidase in lung with only partial lack in tendon and bone could account for the results obtained. Alternatively, insufficient reducible crosslinks, coupled with increased collagen turnover in the lung could prevent formation of the more mature stable crosslinks required to provide a proper connective tissue framework for the Blo/y lung.

Nonmineralized and mineralized bone collagen in bone of immobilized monkeys.

Monkey bones from different time periods of immobilization and reambulation of the monkeys were assessed histologically. The bone was also assessed biochemically for nonmineralized collagen in bone. Results indicate that more nonmineralized bone is present in monkeys that have been immobilized, and upon reambulation, these values tend to be normalized to control bone.

Intermolecular cross-linking and stereospecific molecular packing in type I collagen fibrils of the periodontal ligament.

A trypsin digest of denatured NaB3H4-reduced native bovine periodontal ligament was prepared and fractionated by gel filtration and cellulose ion-exchange column chromatography. Prior to trypsin digestion, a complete acid hydrolysate was subjected to analyses for nonreducible stable and reducible intermolecular cross-links. Minute amounts of the former and significant amounts of the reduced cross-links dihydroxylysinonorleucine (1.1 mol/mol of collagen), hydroxylysinonorleucine (0.9 mol/mol of collagen), and histidinohydroxymerodesmosine (0.6 mol/mol of collagen) were found. The covalent intermolecular cross-linked two-chained peptides that were isolated were subjected to amino acid and sequence analyses. The structures for the different two-chained linked peptides were alpha 1CB4-5(76-90)[Hyl-87] X alpha 1CB6-(993-22c)[Lysald-16c], alpha 1CB4-5(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Hylald-16c], alpha 2CB4(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Lysald-16c], and alpha 2CB4(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Hylald-16c]. The cross-link in each peptide was glycosylated. This is the first characterization by sequence analysis of a cross-link involving Hyl-87 in an alpha 2 chain in collagen. A stoichiometric conversion of residue 16c aldehyde to an intermolecular cross-link in each of the COOH-terminal nonhelical peptide regions of both alpha 1 chains in a molecule of type I collagen was found. The ratio of alpha 1 to alpha 2 intermolecularly cross-linked chains involved was 3.3:1, indicating a stereospecific three-dimensional molecular packing of type I collagen molecules in bovine periodontal ligament.

Epidermolysis bullosa acquisita antigen, a new major component of cutaneous basement membrane, is a glycoprotein with collagenous domains.

The epidermolysis bullosa acquisita antigen is a major constituent of the basement membrane zone beneath stratified squamous epithelium. The antigen which is recognized in extracts of skin basement membrane by Western blot analysis with polyclonal or monoclonal antiepidermolysis bullosa acquisita antigen antibodies as 2 chains (a major chain of 290,000 and a minor chain of 145,000) has a native molecular weight over 800,000. Both epidermolysis bullosa acquisita antigen chains contain carbohydrate and the 290K chain is sensitive to collagenase.

New pepsin-solubilized low molecular weight collagenous component possibly unique to periodontal ligament.

Limited pepsin digestion of bovine periodontal ligament releases genetic types I, III, and V collagen and a high cystine containing low molecular weight collagenous component. Salt fractionation and molecular sieve chromatography allowed the isolation of the latter as an apparently pure homogeneous moiety which had an approximate molecular mass of 30 000 daltons. Reduction with mercaptoethanol yielded a single 10 000-dalton band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This led us to conclude that the newly isolated low molecular weight collagen fragment consists of three similar molecular weight chains. Unreduced collagen-like glycoprotein (CGP) [Jander, R., Troyer, D., & Rauterberg, J. (1984) Biochemistry 23, 3675-3681] after extraction from tissues with collagen denaturing solvents yields the GP140 glycoprotein upon reduction and does not release any collagen fragment below 90 000 daltons upon mild or vigorous pepsin digestion. The GP140 glycoprotein [Heller-Harrison, R. A., & Carter, W. G. (1984) J. Biol. Chem. 259, 6858-6864] isolated by extraction under reducing and collagen denaturing solvent conditions did not yield a collagen fragment below 40 000 daltons after pepsin treatment. It was clearly shown that both CGP and GP140 yield type VI collagen fragments in the above-cited reports. Since this report demonstrates that the Mr 30 000 collagen fragment is only released by pepsin treatment of nondenaturing solvent treated periodontal ligament and that only very small peptides are found in denaturing solvent treated tissue after pepsin digestion, it is concluded that the newly isolated Mr 30 000 collagen fragment reported here is not derived from type VI collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

[14C]acrylonitrile: preparation via a stable tosylate intermediate and quantitative reaction with amine residues in collagen.

A simple, convenient synthetic procedure for [14C]acrylonitrile is described. Na14CN is used as the radioactive starting material. Small (milligram) amounts are converted to 3-[14C]Hydroxypropionitrile by a substitution reaction with 2-chloroethanol. 3-[14C]Hydroxypropionitrile is then tosylated, and the specific activity of this intermediate product is easily determined using its uv extinction coefficient and scintillation counting. [14C]Acrylonitrile is obtained rapidly on distillation by heating the tosylate in the presence of a high boiling tertiary amine base catalyst. The tosylate intermediate can be stored, in contrast to radioactive acrylonitrile, which is unstable. The reaction of acrylonitrile with lysine, hydroxylysine, and histidine residues in human Achilles tendon collagen, as well as chromatographic separation and identification of the carboxyethyl derivatives of these amino acids, is also described.

The involvement of collagenolysis in ovulation in the rat.

Collagenolytic activity in ovarian follicles was previously demonstrated by using synthetic peptides and reconstituted collagen fibers. However, attempts to demonstrate degradation of ovarian collagen and to correlate collagenase activity with ovulation were not successful. By administration of L-(5-3H) proline, we have labeled ovarian and follicular collagen and followed collagenolytic activity by separation of 3H-hydroxyproline (3H-Hyp) from acid hydrolyzates of ovarian tissue by HPLC. The level of ovarian and follicular 3H-Hyp decreased by about 40% on the afternoon of proestrus or after exogenous stimulation of ovulation by human CG (hCG), and this decrease was abolished by blocking the surge of gonadotropins with Nembutal. To verify that the observed reduction in 3H-Hyp was due to the action of a typical collagenase, the collagenous fraction was prepared from ovarian tissue and from preovulatory follicles before and after the ovulatory stimulus. The extracts were treated with trypsin (25 min, 25 C, 0.01 mg/ml) plasmin and p-amino-phenyl-mercuric acetate to fully activate the collagenase extracted along with collagen. Both, enzymatic and chemical activation of collagenase in vitro resulted in degradation of collagen. This degradation could be inhibited by cysteine and EDTA; both are classic inhibitors of mammalian collagenases. The activity of ovarian collagenase increased within 3 h after hCG-stimulation, peaked at 5-fold 6 h after hCG, and declined afterwards. Administration of cysteine (0.001-0.01 mmol) into the bursal cavity of proestrous rats blocked ovulation and breakdown of ovarian collagen in a dose-dependent manner. Cysteine effectively inhibited ovulation even when injected 7 h after the hCG stimulus. Inhibitors of arachidonic acid metabolism prevent ovulation. Indomethacin (inhibitor of cyclooxygenase) and nordihydroguaiaretic acid (inhibitor of lipoxygenase) blocked ovulation and inhibited hCG-induced ovarian collagenolysis. Collectively, these results corroborate the essential role of collagenolysis in follicular rupture in mammals.