Biosynthetic pathway of desmosines in elastin

1. Elastins purified by various methods from the ligamentum nuchae and the lungs of cattle of various ages were analysed for amino acid compositions and lysine-derived cross-links. 2. In fully mature elastins from adults the main cross-links were desmosine, isodesmosine and lysinonorleucine and the so-called aldol-condensation product. Trace amounts of merodesmosine were also found. 3. During the normal maturation of elastins the amounts of desmosine, isodesmosine and lysinonorleucine increased, whereas the aldol-condensation product and intact lysine residues decreased and merodesmosine remained the same. 4. Elastins from young animals contained significant amounts of dehydromerodesmosine whereas elastins from adults contained virtually nil. Evidence is presented which suggests that the biosynthetic pathway of desmosine and isodesmosine proceeds via the aldol-condensation product and dehydromerodesmosine.     

Separation and determination of cross-linking amino acids of elastin by high-voltage paper electrophoresis

A method is described for the separation and quantitative determination of the weakly basic (cross-linking) amino acids of elastin. A 6 N HCl hydrolyzate is submitted to high-voltage electrophoresis at pH 3.8. At least eight spots can be identified in the weakly basic region and quantitated by the ninhydrin-photodensitometric method. Some of these are spot 3 for desmosine + isodesmosine, and 7 for lysinonorleucine. Quantitative data given for two typical elastin preparations are in agreement with direct amino acid analysis.     

A structural model for desmosine cross-linked peptides.

Desmosine-enriched peptides were isolated from a thermolysin digest of bovine ligamentum nuchae elastin and a partial sequence was determined. A 'two-cross-link' model is proposed in which a second cross-link, perhaps lysinonorleucine, joins two peptide chains approx. 35 amino acid residues removed from the desmosine. Implied in this model is a certain asymmetry or directionality which places restrictions on the 'sense' of the peptide chains (either always parallel or anti-parallel) in order to align the cross-linking sites. Imposing such restrictions raises the possibility of specific alignment of elastin precursor molecules by microfibrillar proteins and/or aligning peptides on the precursor molecules themselves.     

Isolation and structural characterization of a new crosslinking amino acid, cyclopentenosine, from the acid hydrolysate of elastin.

A novel polyfunctional crosslinking amino acid, which was developed slower than lysinonorleucine and faster than desmosine on thin layer chromatography, was isolated from the hydrolysate of bovine aorta elastin. Its proposed structure was verified by ultraviolet spectroscopy, fast atom bombardment mass spectroscopy, and nuclear magnetic resonance spectroscopy. The data indicated it to be a trifunctional amino acid with a cyclopentene structure. The mass spectral analysis indicated a parent compound with a mass of 381 (C18H27N3O6). The proposed structure is one derived from the condensation of three allysine residues. Based on the names of other crosslinking amino acids found in elastin, the trivial name of cyclopentenosine is given to this compound.     

Crosslinkage of salt-soluble elastin in vitro.

Radioactively labeled soluble elastin, synthesized $\text{in vitro}$ by viable copper-deficient pig aorta in a culture medium containing L-[4,5-³H] lysine, was incubated with normal newborn pig aorta. The insoluble residue, after extraction of the aorta with cold 0.5M NaCl at pH 7.4, was reduced with NaBH₄. Insoluble elastin, prepared from this by autoclaving after extraction with guanidine, was hydrolyzed with HCl and the hydrolysate was chromatographed on Aminex A-5. Among the radioactive residues eluted in the basic region, four elastin crosslinks (isodesmosine, desmosine, lysinonorleucine and merodesmosine) were identified by comparison with known standards on the Beckman amino acid analyzer. This provides the first direct evidence that soluble elastin is a precursor of insoluble elastin.     

Biosynthesis of lysine-derived elastin crosslinks in aortic cell culture.

Culture of an established line of aortic medial cells in the presence of L-[¹⁴C] lysine for 72 hours, beginning on the twenty-first day after transfer, has resulted in the incorporation of label into a residue, insoluble after autoclaving. Acid hydrolysates of this residue with or without reduction by NaBH₄ were subjected to ion exchange chromatography. Several radioactive lysine-derived residues were identified, by comparison to standards, as the distinctive crosslinks of elastin, isodesmosine, desmosine, merodesmosine and lysinonorleucine. This confirms the synthesis of elastin in aortic cell culture and establishes the formation of insoluble crosslinked elastin. Differences in the heights of the peaks in the reduced and nonreduced elastin indicate the probable occurrence of dehydromerodesmosine and dehydrolysino-norleucine as well and suggest that these may be intermediates in crosslink formation.     

A model two-component system for studying the architecture of elastin assembly in vitro

Tropoelastin is encoded by a single human gene that spans 36 exons and is oxidized in vivo by mammalian lysyl oxidase at the epsilon amino group of available lysines to give the adipic semialdehyde, which then facilitates covalent cross-link formation in an enzyme-free process involving tropoelastin association. We demonstrate here that this process is effectively modeled by a two protein component system using purified lysyl oxidase from the yeast Pichia pastoris to facilitate the oxidation and subsequent cross-linking of recombinant human tropoelastin. The oxidized human tropoelastin forms an elastin-like polymer (EL) that is elastic, shows hydrogel behavior and contains typical elastin cross-links including lysinonorleucine, allysine aldol, and desmosine. Protease digestion and subsequent mass-spectrometry analysis of multiple ELs allowed for the identification of specific intra- and inter-molecular cross-links, leading to a model of the molecular architecture of elastin assembly in vitro. Specific intra-molecular cross-links were confined to the region of tropoelastin encoded by exons 6-15. Inter-molecular cross-links were prevalent between the regions encoded by exons 19-25. We find that assembly of tropoelastin molecules in ELs are highly enriched for a defined subset of cross-links.     

Characterization of insoluble elastin from copper-deficient pigs. Its usefulness in elastin sequence studies.

Insoluble elastin from copper-deficient animals has an amino acid composition intermediate between mature elastin and salt-soluble elastin (a higher lysine content and correspondingly low number of cross-links relative to the normal protein) and is solubilized by successive treatment with trypsin and chymotrypsin at 4 and 37 degrees C. Small amounts of B3H4 (11 mg--2 g of elastin) reduced allysine, allysine aldol, dehydronorleucine, and dehydromerodesmosine in insoluble elastin from copper-deficient pig aorta. In contrast, desmosine and isodesmosine were reduced only when a large excess of reductant (400 mg borohydride) was included in the reaction mixture. Reduction studies indicated that lysinonorleucine and merodesmosine were present in their dehydro forms to a greater extent in copper-deficient pig elastin than in normal elastin. After reduction with borohydride approximately 35% of the reduced form of the insoluble elastin remained insoluble after digestion with trypsin and chymotrypsin. A peptide containing the aldehyde oxidation product of lysine (allysine) and demonstrating an enrichment in glutamic acid was purified from the reduced form of copper-deficient pig elastin and partially sequenced. Its sequence (Gly-Ala-Glu-allysine-(Glu)...) and amino acid composition suggest: (1) clustering of glutamic acid residues in the elastin molecule, and (2) that allysine residues are not restricted to the alanine-enriched sites described for other elastin cross-links. Insoluble elastin from copper-deficient animals promises to be a useful tool for elastin sequence studies.     

Purification of a lysinonorleucine cross-linked peptide fraction from porcine aorta elastin.

Highly purified elastin from porcine aorta was submitted to elastase digestion. The enzymic products were subjected to gel filtration on Sephadex G 25. The excluded fraction was then submitted to thermolysin digestion and gel filtration. The excluded fraction was submitted to partial acid hydrolysis and gel filtration. Several sub-fractions were obtained. The F3 subfraction containing cross-linking agents (desmosines and lysinonorleucine) was finally subjected to ion exchange chromatography. A highly enriched peptide fraction containing lysinomorleucine was obtained and then purified by preparative electrophoresis. The ratio of enrichment passed from 2 residues of lysinonorleucine (expressed as lysine) from starting material (elastin) to 178 out of 1,000 residues in the final step. In this peptide fraction, if we express in molar ratio and consider the amount of lysinonorleucine is one residue, the following amino-acid composition is Pro:3, Gly:1, Ala:2, LNL:1, Lys:2. No traces of desmosines are detected. The role of lysinonorleucine in bridging functions in elastin is discussed.     

Enrichment and analysis of desmosine and isodesmosine in biological fluids

A method has been developed for the enrichment and analysis of the elastin crosslinks, desmosine and isodesmosine, in biological fluids and tissues. It is adapted from published methods, offering improved recovery, sensitivity, resolution, and speed of analysis. Samples were hydrolyzed in 6 M HCl, after which the desmosines were enriched by CF1 cellulose chromatography and analyzed by HPLC with a C₁₈ column. Isodesmosine and desmosine were quantitated based on absorbance at 275 nm, with a limit of detection of approximately 30 pmol and recovery of approximately 66% in urine. Their t_R values on our HPLC system were approximately 9 and 12 min, respectively. This method was used to evaluate the daily and weekly variation in the concentrations of desmosine and isodesmosine in human urine. The results suggest that this method can be used to process large numbers of biological samples for analysis of desmosine and isodesmosine.     

Histone H1 is a substrate for lysyl oxidase and contains endogenous sodium borotritide-reducible residues

Incubation of purified bovine aortic lysyl oxidase with rat liver or calf thymus H1 histone results in the catalytic formation of hydrogen peroxide, indicating the substrate potential of H1 for this connective tissue enzyme. Sodium borotritide-reducible residues consistent with aminoadipic semialdehyde and the lysinonorleucine crosslinkage were generated in H1 by incubation with lysyl oxidase. H1 histone also contains endogenous reducible functions including an unidentified prominent tritiated peak eluting near tyrosine as well as other lesser peaks, one of which is consistent with lysinonorleucine.     

Urinary desmosine as a biomarker in acute lung injury.

Acute lung injury (ALI) is a complex disorder associated with an acute inflammatory response thought to contribute to tissue injury. Desmosine, a cross-linking amino acid present in elastin, is released during matrix degradation and cleared by the kidney. Results from animal models and human disease studies have suggested that ALI is associated with the release of desmosine, resulting in increased urinary desmosine. A radioimmunoassay was used to monitor urinary desmosine levels over 10 days in ten patients with ALI. The concentration of desmosine was measured with and without acid hydrolysis. Baseline urinary desmosine was increased in two of ten patients. The concentration of desmosine at baseline did not appear to be related to age, gender, neutrophil elastase (NE)/alpha(1)-antiprotease complex concentration or P(a)O(2)/F(i)O(2) ratio. No meaningful changes in desmosine levels were noted after removal from mechanical ventilation. Baseline desmosine concentrations did not appear to correlate with the risk of death. The limited sensitivity, predictive correlations and dynamic modulation would suggest that urine desmosine has a limited role as a biomarker for ALI. Hydrolysis of urine samples appears necessary for optimal measurement of urine desmosine.     

An enzyme-linked immunosorbent assay to quantitate the elastin crosslink desmosine in tissue and urine samples

An enzyme-linked immunosorbent assay method has been developed for the determination of desmosine. The method is based on an inhibition immunoassay (under nonequilibrium conditions) and uses rabbit antisera directed against a desmosine-bovine serum albumin conjugate and microtiter plates coated with desmosine-gelatin conjugate. The assay quantitates desmosine in the range 2.5-50 pmol in tissue and urine samples. Important applications of this rapid and sensitive assay are in studying elastin metabolism and in screening for monoclonal antibodies against desmosine. Methods are described for obtaining a constant level of substitution of desmosine per molecule of bovine serum albumin and for preparing a desmosine-gelatin coating antigen. Five different antibody preparations directed against desmosine exhibit 15-20% cross-reactivity toward pyridinoline (3-hydroxypyridinium), a nonreducible collagen crosslinking compound also present in urine and many tissue samples.     

Urinary desmosine as a biomarker in acute lung injury

Acute lung injury (ALI) is a complex disorder associated with an acute inflammatory response thought to contribute to tissue injury. Desmosine, a cross-linking amino acid present in elastin, is released during matrix degradation and cleared by the kidney. Results from animal models and human disease studies have suggested that ALI is associated with the release of desmosine, resulting in increased urinary desmosine. A radioimmunoassay was used to monitor urinary desmosine levels over 10 days in ten patients with ALI. The concentration of desmosine was measured with and without acid hydrolysis. Baseline urinary desmosine was increased in two of ten patients. The concentration of desmosine at baseline did not appear to be related to age, gender, neutrophil elastase (NE)/α₁-antiprotease complex concentration or P_aO₂/F_iO₂ ratio. No meaningful changes in desmosine levels were noted after removal from mechanical ventilation. Baseline desmosine concentrations did not appear to correlate with the risk of death. The limited sensitivity, predictive correlations and dynamic modulation would suggest that urine desmosine has a limited role as a biomarker for ALI. Hydrolysis of urine samples appears necessary for optimal measurement of urine desmosine.     

Lysinonorleucine cross-link formation in alpha amino heptenoic acid-substituted peptide derivatives

Studies on the cross-linking of a tripeptide (t-butyloxycarbonyl-L -alanyl-D ,L -2-amino-6-heptenoyl-L -alanine methyl ester) have shown that it is possible to form specific cross-links in good yields through Schiff base formation of the ε amino group of lysine. The heptenoic acid residue has been ozonized to an aldehyde and condensed with the ε amino of lysine in the compounds alpha-t-butyloxycarbonyl-L -alanyl-L -lysine methyl ester and alpha-t-butyloxycarbonyl-L -lysine methyl ester to form the cross-link, lysinonorleucine. This compound has been stabilized by reduction with sodium borohydride and quantitated on the amino acid analyzer. This technique converts from 60 to 98% of the available aldehyde to lysinonorleucine.     

Higher urine desmosine levels are associated with mortality in patients with acute lung injury

Desmosine is a stable breakdown product of elastin that can be reliably measured in urine samples. We tested the hypothesis that higher baseline urine desmosine would be associated with higher mortality in 579 of 861 patients included in the recent Acute Respiratory Distress Syndrome Network trial of lower tidal volume ventilation (1). We also correlated urine desmosine levels with indexes of disease severity. Finally, we assessed whether urine desmosine was lower in patients who received lower tidal volumes. Desmosine was measured by radioimmunoassay in urine samples from days 0, 1, and 3 of the study. The data were expressed as a ratio of urine desmosine to urine creatinine to control for renal dilution. The results show that higher baseline (day 0) urine desmosine-to-creatinine concentration was associated with a higher risk of death on adjusted analysis (odds ratio 1.36, 95% confidence interval 1.02-1.82, P=0.03). Urine desmosine increased in both ventilator groups from day 0 to day 3, but the average rise was higher in the 12-ml/kg predicted body weight group compared with the 6-ml/kg predicted body weight group (P=0.053, repeated-measures model). In conclusion, patients with acute lung injury ventilated with lower tidal volumes have lower urine desmosine levels, a finding that may reflect reduced extracellular matrix breakdown. These results illustrate the value of evaluating urinary biological markers that may have prognostic and pathogenetic significance in acute lung injury.     

Urinary desmosine excretion is inversely correlated with the extent of emphysema in patients with chronic obstructive pulmonary disease

An enhanced proteolysis of lung interstitium is key event in the pathogenesis of emphysema, a major constituent of chronic obstructive pulmonary disease. To assess whether urinary desmosine and/or hydroxyproline may be used as a marker of lung destruction we studied urinary excretions of these products in 20 patients with chronic obstructive pulmonary disease and in 19 appropriate controls in 24h urine collection samples. For desmosine measurements, we developed a new indirect competitive enzyme-linked immunosorbent assay. The extent of emphysema was measured in high resolution computed tomography (CT) scans, by considering lung area with CT numbers <-950 Hounsfield units (HU).Urinary desmosine excretion was significantly higher in patients with chronic obstructive pulmonary disease than in controls (294+/-121 microg versus 183+/-93 microg, P=0.003), and was unrelated with both age and smoking habits. In patients with no evidence or only mild emphysema, desmosine excretion values were significantly higher (P=0.006) than those of patients with moderate to severe emphysema. In patients with chronic obstructive pulmonary disease, urinary hydroxyproline excretion was positively correlated with urinary desmosine excretion but on the average, it was not different from that of controls.These data indicate that urinary desmosine is a sensitive biological marker of lung elastin catabolism. The relatively low levels of urinary desmosine observed in patients with severe emphysema may be accounted for a decrease in elastin catabolism due to reduced lung elastin mass. Urinary desmosine may be used to identify subjects at risk of developing emphysema and to assess the efficacy of therapeutic interventions.     

Human red cell galactose 1-phosphate uridylyltransferase: effects of site-specific reagents on catalytic activity

Egg shell membrane protein was found to contain the crosslinking amino acids desmosine and isodesmosine. Of particular interest, the desmosine and isodesmosine content was increased severalfold when the egg shell membrane protein was subjected to autoclaving. The major protein in membranes, which contains the crosslinking amino acids desmosine and isodesmosine, differs greatly from elastin in amino acid composition and is resistant to digestion with elastase. It is concluded that this protein component is not elastin but contains desmosine isomers. Further, its amino acid composition does not resemble those reported for other fibrous proteins such as keratin, connectin, collagen, or microfibrillar protein.     

Comparative studies of the cross-linked regions of elastin from bovine ligamentum nuchae and bovine, porcine and human aorta.

1. The preparative Edman degradation of desmosine-containing peptides permitted the isolation of peptides C-terminal to the desmosine cross-links in bovine, porcine and human aortic elastin as well as bovine ligamentum nuchae elastin. This identifies the lysines in the tropoelastin which give rise to the desmosine cross-links. 2. The sequences from bovine aortic elastin were identical with those obtained from bovine ligamentum nuchae elastin but differed from those obtained from the other species. The most striking difference involves the occurrence of phenylalanine in bovine elastin and tyrosine in porcine and human elastin C-terminal to the desmosine cross-links. 3. The sequences of the C-terminal peptides were found to fall into two distinct classes, one starting with hydrophobic residues, the other starting with alanine. It is proposed that thehydrophobic residue prevents the enzymic oxidative deamination of the adjacent lysine e-amino group and this then contributes the nitrogen to the pyridinium ring of the cross-links.     

A possible role for dehydrodihydroxylysinonorleucine in collagen fibre and bundle formation.

The concentrations of NaB3H4-reducible collagen cross-links were determined at the time when collagen fibres and bundles are observed in electron micrographs of connective tissue developing around the implanted Ivalon sponge in adult male rats. The highest radioactivity occurs with hydroxylysinonoreleucine and histidinohydroxymerodesmosine, and the lowest with lysinonorleucine, the reducible amounts of these cross-links remaining relatively constant as fibres and bundles appear. On the other hand, dihydroxylysinonorleucine amounts are low during the initial stages of connective-tissue formation and rise sharply as collagen fibres and bundles develop and collagen matures, as shown by increased resistance of insoluble collagen to digestion with bacterial collagenase. The bulk of hydroxylysinonorleucine and dihydroxylysinonorleucine is glycosylated, the former with galactosyl or glucosylgalactosyl residues and the latter with glucosylgalactosyl residues. The changing relationships between the amounts of 3H-labelled hydroxylysinonorleucine, glucosylgalactosyldihydroxylysinonorleucine and non-glycosylated dihydroxylysinonorleucine as fibres and bundles appear suggest three post-translational steps involving lysyl-derived cross-links in the organization of collagen into fibres and bundles.