desmosine and delta-hydroxylysylnorleucine

To elucidate the roles of LH2b in collagen cross-linking, MC3T3-E1 cell clones expressing higher (S) or lower (AS) levels of LH2b were established. Compared with controls, the collagen cross-linking pattern was shifted toward hydroxylysine-aldehyde (S clones)- or lysine-aldehyde (AS clones)-derived pathways. The data indicate that LH2b directs collagen cross-linking pathways through its action on telopeptidyl lysine residues.. Lysine (Lys) hydroxylation is a post-translational modification of collagen critical for cross-linking and glycosylation. Currently, three isoforms of lysyl hydroxylase (LH) have been identified, but their specific functions are still not well defined. Recently, we proposed that LH2 might modulate collagen cross-linking pattern through its action on Lys residues located in the telopeptide domains of collagen.. To directly test this hypothesis, several MC3T3-E1 cell-derived clones expressing higher (sense [S]) or lower (antisense [AS]) levels of LH2b, the predominant form of LH2 in this cell line, were established and cultured for 2 weeks, and collagen cross-links and precursor aldehydes in the matrices were analyzed.. In S clones tested, the ratio of dihydroxylysinonorleucine (DHLNL) to hydroxylysinonorleucine (HLNL) was significantly higher than the average of controls (76% and 140% increase, respectively), and the level of pyridinoline (Pyr) was elevated (100% and 150% increase, respectively). In contrast, when MC3T3-E1 cells were transfected with a LH2b antisense construct (AS clones), the DHLNL/HLNL ratios were significantly lower than that of controls (56% and 73% decrease, respectively), and Pyr was not detected. Furthermore, significant amounts of an aldol-derived cross-link, dehydrohistidinohydroxymerodesmosine, were produced ( approximately 0.3 mol/mol of collagen) in AS clones.. The data clearly show a critical role of LH2b in determining collagen cross-linking pathways, most likely through its action on telopeptidyl Lys residues.

Topics: 3T3 Cells; Amino Acids; Animals; Blotting, Western; Collagen; Desmosine; Dipeptides; DNA, Antisense; DNA, Complementary; Gene Expression; Genetic Vectors; Histidine; Immunoprecipitation; Isoenzymes; Lysine; Mice; Osteoblasts; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; Protein Processing, Post-Translational; Reverse Transcriptase Polymerase Chain Reaction; RNA; Transfection

Keloid is a tissue with an excessive accumulation of collagen. In this study, we have partially characterized post-translational modifications of type I collagen in human keloid in order to pursue their potential involvement in this pathology. The levels of lysyl hydroxylation of the helical portions of alpha 1 and alpha 2 chains of type I collagen in keloid were significantly higher than those of normal, while the levels of prolyl hydroxylation were identical between these two groups. The contents of the major reducible cross-links in dermal collagen, dehydro-hydroxylysinonorleucine and dehydro-histidinohydroxymero-desmosine, were both significantly higher in keloids (up to sixfold) than those of normal. In addition, significant amounts of hydroxylysine-aldehyde derived cross-links that are characteristic of skeletal tissue collagens, dehydro-dihydroxylysinonorleucine (about 0.3 mole/mole of collagen) and pyridinoline (about 0.1 mole/mole of collagen), were found in keloids. These results indicate that keloid-forming cells are phenotypically different from those in normal dermis and that the collagen produced is highly cross-linked. The increased cross-linking provides the fibrils with more stability that may result in an accumulation of collagen.

Topics: Adult; Amino Acids; Collagen; Cross-Linking Reagents; Desmosine; Dipeptides; Histidine; Humans; Hydroxylation; Hydroxylysine; Hydroxyproline; Keloid; Middle Aged; Protein Processing, Post-Translational; Protein Structure, Secondary; Skin

Progressive crosslinking of proteins appears to be a general phenomenon in aging cells and tissues. Crosslinked proteins can form insoluble aggregates which become increasingly resistant to proteolysis as more crosslinks form. However, most evidence for progressive crosslinking with age is indirect, and little is known about the chemical mechanisms involved. We have therefore developed a method for detection and isolation of any type of stable covalent crosslink from protein hydrolysates which requires no prior knowledge of the molecular structure of whatever crosslink(s) may be present. It utilizes the specificity of the diphenylborinic acid reagent for alpha-amino acid groups and the chromatographic properties and uv absorbance of the crosslink derivatives. The method is demonstrated using eight different crosslinks from collagen and fibrin, and a general procedure is given for detection of any type of crosslink in a protein hydrolysate.

Topics: Animals; Boron Compounds; Cattle; Chromatography, High Pressure Liquid; Collagen; Cross-Linking Reagents; Desmosine; Dipeptides; Fibrin; Fluorescence; Histidine; Hydrolysis; Proteins; Spectrophotometry, Ultraviolet

Collagen cross-links of skin tissue (left upper arm) from 11 patients with amyotrophic lateral sclerosis (ALS) and 9 age-matched control subjects were quantified. It was found that patients with ALS had a significant reduction in the content of an age-related, stable cross-link, histidinohydroxylysinonorleucine, that was negatively correlated with the duration of illness. The contents of sodium borohydride-reducible labile cross-links, dehydro-hydroxylysinonorleucine and dehydro-histidinohydroxymerodesmosine, were significantly increased and were positively associated with the duration of illness (r = 0.703, p less than 0.05 and r = 0.684, p less than 0.05, respectively). The results clearly indicate that during the course of ALS, the cross-linking pathway of skin collagen runs counter to its normal aging, resulting in a "rejuvenation" phenomenon of skin collagen. Thus, cross-linking of skin collagen is affected in ALS.

Topics: Aged; Aging; Amyotrophic Lateral Sclerosis; Arm; Borohydrides; Collagen; Desmosine; Dipeptides; Female; Histidine; Humans; Male; Middle Aged; Muscular Diseases; Nervous System Diseases; Oxidation-Reduction; Skin

Tissue samples from 5 different sites of 6 bovine mandibles were used to quantify collagen cross-links as moles cross-link per mole of collagen. Gingiva contained primarily 3 reducible cross-links, deH-DHLNL, deH-HHMD, and deH-HLNL, and relatively small amounts of non-reducible cross-links. The cross-link density varied from site to site within the same mandible. DeH-DHLNL showed the most significant increase from anterior to posterior; the content in the retromolar region was 3-fold greater than in the anterior lingual site. Histologically, the posterior samples, especially the retromolar gingiva, contained less mature (thinner) collagen bundles. These findings suggest that the maturity of bovine mandibular gingival collagen varies at different sites.

Topics: Animals; Cattle; Chromatography, High Pressure Liquid; Collagen; Connective Tissue; Desmosine; Dipeptides; Gingiva; Histidine; Hydroxyproline; Lysine

The effects of chronic exposure to ozone on lung collagen crosslinking were investigated in two groups of juvenile cynomolgus monkeys exposed to 0.61 ppm of ozone 8 hrs per day for 1 year. One group was killed immediately after the exposure period; the second exposed group breathed filtered air for 6 months after the ozone exposure before being killed. Previous studies of these monkeys had revealed that lung collagen content was increased in both exposed groups (J.A. Last et al., (1984). Toxicol. Appl. Pharmacol. 72, 111-118). In the present study specific collagen crosslinks were quantified in order to determine whether the excess collagen in the lungs of these animals was structurally normal or abnormal. In the group killed immediately after exposure, the difunctional crosslink dehydrodihydroxylysinonorleucine (DHLNL) was elevated, as was the ratio of DHLNL to dehydrohydroxylysinonorleucine (HLNL). Lung content of the mature nonreducible crosslink hydroxypyridinium was also increased in this group. In the group killed after a 6-month postexposure period, lung content of the difunctional crosslinks DHLNL and HLNL was indistinguishable from control values. However, lung hydroxypyridinium content was significantly increased. The changes in collagen crosslinking observed in the group killed at the termination of exposure are characteristic of those seen in lung tissue in the acute stage of experimental pulmonary fibrosis. The changes seen in the postexposure group suggest that while the lung collagen being synthesized at the time the animals were killed was apparently normal, "abnormal" collagen synthesized during the period of ozone exposure was irreversibly deposited in the lungs. This study suggests that long-term exposure to relatively low levels of ozone may cause irreversible changes in lung collagen structure.

Topics: Animals; Chromatography, High Pressure Liquid; Collagen; Desmosine; Dipeptides; Female; Lung; Macaca fascicularis; Male; Pyridines; Sulfuric Acids

Collagen crosslinks in neonatal rats were labelled in vivo by a single intraperitoneal injection of 200 microCi of [14C]lysine. Rats were killed at times ranging from 30 minutes to 10 weeks after injection. Whole skin, tendon, and bone were analyzed, after reduction and hydrolysis, for collagen crosslink content by HPLC. Crosslinks and amino acids were visualized by their incorporation of radioactivity from [14C]lysine and also fluorometrically by post-column derivatization with o-phthalaldehyde. The incorporation of 14C from labelled lysine into the principal difunctional reducible crosslinks, N6.6'-dehydro-5,5'-dihydroxylysinonorleucine and N6.6'-dehydro-5-hydroxylysinonorleucine, increased most rapidly between 4 and 12 hours after injection, results similar to those observed by others studying crosslink biosynthesis in vitro. Incorporation of 14C into the tetrafunctional crosslink histidinohydroxymerodesmosine proceeded more slowly than it did for the difunctional crosslinks. Values for the amount of radioactivity incorporated into the various crosslinks reached an apparent constant value between 3 and 5 days after injection for all three tissues studied. These values remained approximately constant for the duration of the experiment except for HHMD in tendon, which showed an increase in incorporated radioactivity at 8 and 10 weeks after injection. Direct chemical quantification of these same crosslinks by determination of the fluorescence of their o-phthalaldehyde adducts was also performed. We conclude that in vivo labelling of collagen crosslinks can be studied, at least in rapidly growing neonates, after a single injection of radioactive lysine. The results of such studies support previous suggestions by others about the rate of formation of difunctional crosslinks based upon studies using in vitro systems. Our results further suggest that formation of the tetrafunctional reducible crosslink histidinohydroxymerodesmosine proceeds relatively rapidly in vivo. Finally, we conclude that such labelled crosslinks are apparently quite stable after biosynthesis, suggesting the possibility of studies of the metabolic fate of collagen crosslinks over appreciable fractions of the lifetime of a rat.

Topics: Amino Acids; Animals; Animals, Newborn; Bone and Bones; Chromatography, High Pressure Liquid; Collagen; Desmosine; Dipeptides; Histidine; Hydroxylysine; Kinetics; Lysine; Macromolecular Substances; Rats; Rats, Inbred Strains; Skin; Tendons

Reconstruction of the anterior cruciate ligament (ACL) with patellar tendon (PT) is a common procedure for the symptomatic ACL-deficient knee. Questions regarding graft incorporation, viability, and nutrition of the transplanted tissue are of concern. This relates to the graft's response to its new intrasynovial milieu and new physical forces. These factors were studied in a rabbit model of ACL reconstruction using PT and were evaluated with histological and biochemical parameters with respect to time. A histological and biochemical metamorphosis of the grafted PT occurred in this study. Autografts demonstrated a gradual assumption of the microscopic properties of normal ACL; by 30 weeks postoperatively, cell morphology was ligamentous in appearance. Normally, type III collagen is not observed in PT, however, a gradual increase in its concentration was seen in the grafts; by 30 weeks its concentration (10%) was the same as in normal ACL. Similarly, glycosaminoglycans content increased from its normally low level in PT to that found in native ACL. Collagen-reducible crosslink analysis demonstrated that grafted tissue changed from the normal PT pattern of low dihydroxylysinonorleucine (DHLNL) and high histidinohydroxymerodesmosine (HHMD) to the pattern seen in normal ACL (high DHLNL and low HHMD) by 30 weeks. These data suggest that when PT is placed in the anatomic and environmental milieu of the ACL, a "ligamentization" of the grafted tissue results; also the autograft initially depends on synovial fluid nutrition, as revascularization occurs after 6 weeks.

Topics: Animals; Collagen; Cross-Linking Reagents; Desmosine; Dipeptides; Glycosaminoglycans; Histidine; Histocytochemistry; Knee Joint; Ligaments, Articular; Male; Rabbits; Tendons; Transplantation, Autologous

Differential scanning calorimetry (DSC) has been applied to the study of connective tissue to evaluate the denaturation process of collagen. We have applied this technique to the study of the ageing of rat skin. We have tried to correlate the variations of the parameters measured by DSC and the modifications of collagen crosslinks with ageing. The thermograms obtained are composed of one main peak located between two shoulders. The relative size of each peak varies with time: the first peak diminishes regularly from 2 to 20 months whilst, at the same time, the third peak increases; the recovery temperature increases with age (+ 16 degrees C between 2 and 20 months); the total denaturation enthalpy does not vary: the main value obtained is 5.9 X 10(-2) J/mg collagen. On the other hand, the assay of reducible collagen crosslinks in rat skin, over the same age range, shows a decrease of heat-labile aldimine crosslink (essentially hydroxylysinonorleucine). These results and the study of thermograms obtained with altered rat skin (animals treated with beta-aminopropionitrile or skin reduced with NaBH4) allow us to conclude that heat-labile and heat-stable crosslinks account for a collagen thermal stabilization which can explain the delay of denaturation characterized by the third peak of DSC thermograms.

Topics: Aging; Animals; Calorimetry, Differential Scanning; Collagen; Desmosine; Dipeptides; Histidine; Male; Protein Denaturation; Rats; Rats, Inbred Strains; Skin Physiological Phenomena; Thermodynamics

Biopsies of a cutaneous osteoma and of normal-looking skin from a 1-year-old girl were studied for histological appearance and collagen biochemistry. The mineralized tissue contained a matrix similar to bone: Only type I collagen, with a hydroxylysine content (0.48%) higher than in the skin (0.35%) and dihydroxylysinonorleucine as the major reducible crosslink. As expected, the normal skin adjacent to the lesions contained type I and type III collagen and as major crosslinks hydroxylysinonorleucine and histidinohydroxymerodesmosine. Histological studies showed the presence of woven bone with very little trabeculation. Numerous active osteoblasts were laying down a rapidly calcified non-lamellar matrix. Osteocytes and multinucleated osteoclasts were also noted. The study demonstrates the osseous nature of the lesion and suggests that an abnormal cell differentiation is associated with this form of osteoma.

Topics: Bone and Bones; Collagen; Desmosine; Dipeptides; Female; Histidine; Humans; Hydroxylysine; Infant; Infant, Newborn; Osteoma; Skin Neoplasms