Elastin Desmosine

After secretion from the cell, certain lysyl residues of tropoelastin are oxidatively deaminated to aldehydes by lysyl oxidase, the same enzyme involved in this process in collagen. However, the major cross-links formed in elastin are the desmosines, which result from the condensation of three of these lysine-derived aldehydes with an unmodified lysine to form a tetrafunctional cross-hnk unique to elastin. Once cross-linked in its mature, extracellular form, elastin is highly insoluble and extremely stable and has a very low turnover rate. Elastin exhibits a variety of random coil conformations that permit the protein to stretch and subsequently recoil during the performance of its physiologic functions. 

Elastin confers extensibihty and elastic recoil on tissues. Elastin lacks hydroxylysine, Gly-X-Y sequences, triple hehcal stmcture, and sugars but contains desmosine and isodesmosine cross-links not found in collagen. 

In addition to the 20 common amino acids, proteins may contain residues created by modification of common residues already incorporated into a polypeptide (Fig. 3-8a). Among these uncommon amino acids are 4-hydroxyproline, a derivative of proline, and 5-hydroxylysine, derived from lysine. The former is found in plant cell wall proteins, and both are found in collagen, a fibrous protein of connective tissues. 6-N-Methyllysine is a constituent of myosin, a contractile protein of muscle. Another important uncommon amino acid is y-carboxyglutamate, found in the bloodclotting protein prothrombin and in certain other proteins that bind Ca2+ as part of their biological function. More complex is desmosine, a derivative of four Lys residues, which is found in the fibrous protein elastin. 

Tropoelastin molecules are crosslinked in the extracellular space through the action of the copper-dependent amine oxidase, lysyl oxidase. Specific members of the lysyl oxidase-like family of enzymes are implicated in this process (Liu etal, 2004 Noblesse etal, 2004), although their direct roles are yet to be demonstrated enzymatically. Lysyl oxidase catalyzes the oxidative deamination of e-amino groups on lysine residues (Kagan and Sullivan, 1982) within tropoelastin to form the o-aminoadipic-6-semialdehyde, allysine (Kagan and Cai, 1995). The oxidation of lysine residues by lysyl oxidase is the only known posttranslational modification of tropoelastin. Allysine is the reactive precursor to a variety of inter- and intramolecular crosslinks found in elastin. These crosslinks are formed by nonenzymatic, spontaneous condensation of allysine with another allysine or unmodified lysyl residues. Crosslinking is essential for the structural integrity and function of elastin. Various crosslink types include the bifunctional crosslinks allysine-aldol and lysinonorleucine, the trifunctional crosslink merodes-mosine, and the tetrafunctional crosslinks desmosine and isodesmosine (Umeda etal, 2001). 

Figure 8.6 Cross-links in elastin involving desmosine and isodesmosine with suggested biosynthetic pathways. Both amino acids contain pyridinium rings, and both contain the elements of four allysine/lysine residues. (Reproduced by permission from Guay M, Lamy F. The troublesome cross-links of elastin 1979. Trends Biochem Sci July, 1979, p. 161.)...

For purposes of this manuscript, we wish to concentrate only on the steps leading to the formation of desmosines, amino acids found predominantly in elastin. With respect to their formation, the following suggests their spontaneous formation from peptidyl lysine and the oxidation product, peptidyl allysine. Narayanan et al. (28,29) have shown that when purified lysyl oxidase and non-crosslined elastin, specifically tropoelastin, are incubated together, the desmosines are formed. Desmosine formation, however, only occurs at temperatures that favor fibrillar arrangements of tropoelastin. Subsequently, it is felt that the maturation of non-crosslinked elastin into cross-linked elastin appears to involve only two major steps, namely insolublization through the formation of fibrils and fixation of the fibrils by crosslinking. 

There is still no way of determining whether or not a given desmosine crosslinks 1, 2, 3, or 4 polypeptide chains of elastin. Based on model studies, however, the most favorable arrangement would be expected if only two chains are crosslinked together by a desmosine (19). This extends from observations that polyalanyl-rich peptides typically favor a-helical conformations and that it is difficult to interconnect more than two polypeptide chains around any given desmosine. With regard to the other amino acids that could potentialy crosslink elastin, the exact number of dehydrolysinonorleucine, dehydromerodesmosine and allysine aldol residues that are involved as intra- or intermolecular crosslinks, and the extent to which these residues may be reduced to form stable crosslinks is not known. 

Further changes in the P1-P5 region, which resulted in improved efficacy for these synthetic substrates, were based on elastin, HLE s natural substrate [36]. This excellent substrate is an insoluble, structural protein, which is primarily composed of hydrophobic amino acid residues. However, it also contains a number of Lys-derived, cross-linked residues, such as desmosine and isodesmosine, that incorporate a positively charged pyridinium ring. In order to model this cross-linking feature, Lys or various amino-protected forms of Lys, were systematically incorporated into the substrate MeO-8uc-Ala-Ala-Pro-Val-NA (4-1). Replacement of any single residue with Lys led to decreased activity, for example, (4-2)-(4-4) Table 2.4). However, the use of side-chain protected Lys derivatives (e.g. the NHj terminus protected with benzyloxycarbonyl or picolinyl) led to increased reactivity to elastase with the optimal position for substitution being P4, see (4-5)-(4-8). 

Several other types of covalent crosslinks, mostly derived from lysine or 5-hydroxylysine residues (the latter being formed by post-translational modification), are found in collagen and elastin. A few examples are given (5.2-5.7) A6 7-dehy-drolysinonorleucine (5.2), lysinonorleucine (5.3), dehydrohydroxylysinonorleucine (5.4), lysino-5-ketonorleucine (5.5), desmosine (5.6) and isodesmosine (5.7). An intrachain thiol ester loop is present in a2-macroglobulin and proteins of the complement system and consists of a fifteen-membered ring derived from cysteine and glutamic acid (5.8). 

Elastin fibers can be separated into amorphous and fibrillar components. The amorphous component consists of elastin, which is characterized by having 95% nonpolar amino acid residues and two unique lysine-derived amino acid residues, desmosine and isodesmosine. 

Formation of desmosine and isodesmosine covalent cross-links in elastin. Three allysine residues (Ra, R3, and R4) and one lysyl residue (R ) condense to give a desmosine cross-link. The allysine residues ( -aldehydes) are derived from the oxidative deamination of lysyl residues. The isodesmosine cross-link is formed similarly, except that it contains a substitution at position 2 rather than at position 4, along with substitutions at 1,3, and 5 on the pyridinium ring. 

Elastase is the name given to proteinases that possess the ability to hydrolyze mature cross-linked elastin [18]. Elastin is an insoluble structural protein responsible for the elastic properties of the lung, skin, and arteries and is quite resistant to most proteinases. Elastin is high in hydrophobic amino acid residues such as valine, alanine, glycine, and proline [19]. Insoluble elastin fibers contain cross-links usually between four lysine residues, which form a unique cyclic product, desmosine. The presence of soluble desmosine cross-links in plasma can be used as a measure of elastin breakdown. Of all the elastases in humans, neutrophil elastase has received the most attention over the years due to its broad substrate specificity and abundance within the cell. However, neutrophils and macrophages contain several proteinases (Table 1), which are capable of degrading elastin. 

Finally, in recent years analytical techniques have been developed for measuring elastin fragments or desmosine levels in serum (King et al., 1980 ... 

Elastin - Elastin is a highly elastic fiber present in ligaments and arterial blood vessels. The polypeptide is rich in glycine, alanine, and valine. Its secondary structure is the most random of the fibrous proteins described here. Like collagen, elastin contains lysine groups involved in cross-links between the chains. In elastin, however, four lysine chains can be combined to form a desmosine cross-link (see here). Thus, fewer cross-links are needed to provide strength for the chains and a more elastic network is created. 

The polypeptide chain of elastin is rich in glycine, alanine, and valine and is very flexible and easily extended. In fact, its conformation probably approximates that of a random coil, with little secondary structure at all. However, the sequence also contains frequent lysine side chains, which can be involved in cross-links. These cross-links prevent the elastin fibers from being extended indefinitely, causing the fibers to snap back when tension is removed. The cross-links in elastin are rather different from those in collagen, for they are designed to hold several chains together. Four lysine side chains can be combined to yield a desmosine cross-link (see here)... 

Figure I. Elastin cross-links formation. Lysine is converted into allysine via lysyl oxidase, which leads to the formation of lysinonor leucine and allysine aldol (blue). The spontaneous condensation between lysine and allysine will generate tetra-substituted desmosine and isodemosine cross-link structures (red). 

Elastin, a stmctural protein with mbber-like elastic properties. It is the main component of the elastic yellow connective tissue occurring, e.g., in the lungs and aorta. The amount of elastin is rather low in the inelastic white connective tissue of tendons. Elastin consists of 850-870 aa with a high content of Gly (27%), Ala (23%), Val (17%), and Pro (12%). It forms a three-dimensional network of fibers crosslinked by desmosine, lysinonor-leucine, and isodesmosine. It has been reported that elastin has an unanticipated regulatory function during arterial development, controlling the proliferation of smooth muscle and stabilizing arterial structure [L. Robert, W. Hornebeck (Eds.), Elastin and Elastases, Volume 1, CRC Press, Boca Raton, EL, 1989 D. R. Eyre et al, Annu. Rev. Biochem. 1984, 53, 717 D. Y. Li et al., Nature 1998, 393, 276]. 

Isodesmosine, a 1,2,3,5-tetra-substituted pyridinium amino acid, is found in the elastin matrix and is formed in patients suffering from COPD as such, it is of interest for drug discovery and diagnosis (14TL6343). Isodesmosine and desmosine can be prepared via a Pr(OTf) 3-catalyzed Chichib-abin pyridine synthesis (Scheme 7).These materials are sparingly soluble in water the synthesis of the pyridines was heavily influenced by the solubility of the starting materials, a lysine and aldehyde derivative, in the water methanol co-solvent. In fact, as methanol increased, the yield of the desired pyridinium decreased. 

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