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.     

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.     

Chemical compositions of elastins isolated from aortas and pulmonary tissues of humans of different ages

1. Elastins were isolated from the visceral pleuras and parenchymas of lungs of humans of different ages. 2. The elastin content of pleuras increased whereas that of parenchymas remained constant with increasing age. 3. The amino acid compositions and carbohydrate contents of elastins isolated from both pulmonary tissues changed in the same way with increasing age of the subjects. These changes were similar to those observed in elastins isolated from the aorta. 4. Similar glycoproteins were isolated from pleuras and aortas, and were more difficult to extract from the elastins of older subjects. Contamination with these glycoproteins was responsible for the changes in composition of elastin, as the age of the tissue from which it was extracted increased. 5. The amount of the cross-linking amino acids desmosine and isodesmosine was lower in elastins isolated from both aorta and pulmonary tissues of senile subjects than those from younger subjects.     

Rapid procedure for the isolation of polyfunctional amino acids in elastin

A rapid technique for the isolation of the elastin crosslinks, desmosine, isodesmosine, and lysinonorleucine, is reported. Elastin hydrolysates were fractionated by ion-exchange chromatography using volatile buffers. 95% of the desmosine and 86% of the lysinonorleucine were recovered.     

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.     

Studies on the evolution of elastin--I. Phylogenetic distribution.

1. Aortae and other tissues from numerous animals were examined for the presence of the rubbery protein elastin by (a) chemical purification and amino acid analysis, (b) presence of the cross-linking amino acids desmosine and isodesmosine and (c) histological staining. 2. Elastin was found in all vertebrates examined (42 species) with the exception of cyclostomes (3 species). It was absent from all invertebrates tested (14 species). 3. The amino acid compositions of vertebrate elastins showed marked and interesting interspecies variations.     

Photodesmosine,an isomer of desmosine obtained by photolysis of this amino acid in ultraviolet light

Desmosine and isodesmosine are two isomers representing the main cross-links of elastin. We describe a new isomer, photodesmosine, which is produced by the photolysis of desmosine at 254 nm. The mechanism of this photolysis is described and is shown to consist of two competing paths. After opening of the pyridium ring to give a tetrasubstituted aminoketone, this compound can either be hydrolysed to give lysine and a trisubstituted analogue of glutaconic aldehyde or undergo a recyclisation and rearomatisation to give a pyridium compound substituted in positions 1, 2, 3 and 4. An understanding of this mechanism is important in order to use photolysis as a specific method to break elastin cross-links. Although only desmosine and isodesmosine have been reported in purified elastin, the chromatographic properties of photodesmosine suggests that if other natural isomers exist in this protein they could be eluted from an ion-exchange resin at much earlier times than those observed in the case of the two already described cross-links.     

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.     

Influence of D-penicillamine on the formation of elastin and its cross-links.

The amount of insoluble elastin and its content of desmosine cross-links were investigated in aortas of chick embryos, to which D-penicillamine was administered on the 6th or 14th--16th day of incubation. D-Penicillamine was shown to alter the formation and maturation of elastin. Using lower doses (less than 50 mg) the weight of pooled aortic elastin is higher as compared with controls (related to 1 mg of elastin or to total weight of elastin). Increased isodesmosine:desmosine ratio in these samples indicates that this elastin is very young. On the other hand, a high dose of D-penicillamine (100 mg) decreased the content of elastin and also of its desmosine cross-links. The authors explain their findings by counteraction of two factors due to administration of penicillamine: the increased solubility of "insoluble elastin", and the decreased cross-link formation.     

Amino acid sequences C-terminal to the cross-links in bovine elastin.

All the desmosine-containing elastolytic peptides of bovine ligamentum-nuchae elastin have now been examined for amino acid sequences C-terminal to the cross-links. In addition, amino acid residues C-terminal to lysine residues in bovine tropoelastin were also examined. No tyrosine C-terminal to cross-links in bovine elastin or C-terminal to lysine in tropoelastin was detected. Apparently all the tyrosine residues C-terminal to lysine residues in pig tropoelastin are replaced with phenylalanine in bovine tropoelastin. All the data presented are consistent with the scheme proposed for the formation of desmosine and isodesmosine cross-links of elastin by Gerber & Anwar [(1975) Biochem. J. 149, 685--695].     

The collagenous protein with elastin crosslinks from Descemet's membrane is not related to type VIII collagen

A collagen-like insoluble protein containing the elastin cross-links (desmosine and isodesmosine) has been isolated from Descemet's membrane. Recently type VIII collagen (endothelial collagen) has been shown to be a major constituent of this membrane. Biochemical studies suggest that these two proteins are unrelated. The cyanogen bromide peptide maps show negligible similarity. Antiserum raised against oxalic acid digests of elastin (alpha-elastin) did not react against an oxalic acid digests of type VIII collagen but did show some reaction against the cross-linked preparation. Immunofluorescent localization has demonstrated the presence of type VIII collagen in trachea but a desmosine cross-linked collagen could not be isolated from this tissue.     

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.     

Elevation of histidinoalanine in calcified human aortas

The cross-links histidinoalanine (HA); pyridinoline (Pyr); desmosine (Des); and isodesmosine (Ides) in human atherosclerotic aortas were studied. Only HA showed a significant increase in calcified aortas, with a high concentration in the insoluble "mineralized" fraction, which was separated out after treatment of tissues with pronase E. The cross-links composition was similar among "mineralized" fractions prepared from tissues of varying degrees of calcification: values were 2.40; 0.10; 0.17; and 0.16 moles per 1000 moles of amino acid residues for HA; Pyr; Des; and Ides, respectively. The findings suggest that the HA-containing peptide may play an important role in the calcification process of aortic tissues.     

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.     

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.     

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.     

Photolysis of desmosine and isodesmosine by ultraviolet light.

1. Desmosine and isodesmosine were separated by ion-exchange and paper chromatography, after acid hydrolysis of purified elastin from beef ligamentum nuchae. The fractions obtained by ion-exchange chromatography were clearly mixtures of related compounds. The desmosine fraction could be resolved into seven compounds and the isodesmosine into four by paper chromatography. 2. Desmosine was maximally degraded by irradiation at 274 nm and isodesmosine at 285 nm. These wavelengths did not correspond to the absorption maxima of the cross links, but to shoulders of the main absorption peaks. 3. When irradiated at their optimum wavelengths, but at various pH, both desmosine and isodesmosine seemed quite stable at pH greater than 8.5. Between pH 8 and 5, the photolytic rate was maximum and decreased slightly at more acidic pH. Below pH 4.0, one of the products of photolysis was free lysine. 4. In analogy to the mechanism of the photolytic degradation of N-methyl pyridinium chloride, it appears that the (iso)desmosines were degraded via the formation of an open amino aldehyde, which was hydrolysed at acid pH to give free lysine and a substituted glutaconic aldehyde.     

High-performance liquid chromatographic quantitation of desmosine plus isodesmosine in elastin and whole tissue hydrolysates

Quantitation of desmosine and isodesmosine, the major crosslinks in elastin, has been of interest because of their uniqueness and use as markers of that protein. Accurate measurement of these crosslinks may allow determination of elastin degradation in vivo and elastin content in tissues, obviating lengthy extraction procedures. We have developed a method of quantitating desmosine plus isodesmosine in hydrolysates of tissue and insoluble elastin using high-performance liquid chromatographic separation and absorbance detection that is rapid (21-35 min) and sensitive (accurate linearity from 100 pmol to 5 nmol). This method has been used to quantitate desmosines in elastin from bovine nuchal ligament and lung and in whole aorta from hamster. The ability to completely separate [3H]lysine from desmosine plus isodesmosine allows the method to be used to study incorporation of lysine into crosslinks in elastin.     

The presence in elastin of possible cyclic precursors of desmosine and isodesmosine

Mass spectral evidence is presented that the elastin of bovine ligamentum nuchae contains, in addition to desmosine and isodesmosine, larger levels of their respective cyclic dehydrodesmopiperidine precursors. In addition to being precursors of the desmosines, such compounds are most likely cross-links in themselves. In fact, the occurrence of these compounds, now isolated as their reduced derivatives, may clarify the discrepancies in the stoichiometry of the conversion of lysine residues to cross-linking components as noted by several workers (1,2,3). With this study the occurrence of all of the tetrafunctional amino acid cyclic structures related to the desmosines is now confimed by mass spectrometry.     

Liberation of desmosine and isodesmosine as amino acids from insoluble elastin by elastolytic proteases

The development of atherosclerotic lesions and abdominal aortic aneurysms involves degradation and loss of extracellular matrix components, such as collagen and elastin. Releases of the elastin cross-links desmosine (DES) and isodesmosine (IDE) may reflect elastin degradation in cardiovascular diseases. This study investigated the production of soluble elastin cross-linking structures by proteinases implicated in arterial diseases. Recombinant MMP-12 and neutrophil elastase liberated DES and IDE as amino acids from insoluble elastin. DES and IDE were also released from insoluble elastin exposed to monocyte/macrophage cell lines or human primary macrophages derived from peripheral blood monocytes. Elastin oxidized by reactive oxygen species (ROS) liberated more unconjugated DES and IDE than did non-oxidized elastin when incubated with MMP-12 or neutrophil elastase. These results support the exploration of free DES and IDE as biomarkers of elastin degradation.