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. 

FIGURE 3-8 Uncommon amino acids, (a) Some uncommon amino acids found in proteins. All are derived from common amino acids. Extra functional groups added by modification reactions are shown in red. Desmosine is formed from four Lys residues (the four carbon backbones are shaded in yellow). Note the use of either numbers or Creek letters to identify the carbon atoms in these structures, (b) Ornithine and citrulline, which are not found in proteins, are intermediates in the biosynthesis of arginine and in the urea cycle. 

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). 

Two types of crosslinking domains exist in tropoelastin those rich in alanine (KA) and those rich in proline (KP). Within the KA domains, lysine residues are typically found in clusters of two or three amino acids, separated by two or three alanine residues. These regions are proposed to be Q-helical with 3.6 residues per turn of helix, which has the effect of positioning two lysine sidechains on the same side of the helix, although there is no direct structural evidence (Brown-Augsburger et al., 1995 Sandberg et al, 1971), and facilitating the formation of desmosine crosslinks. Desmosine crosslinks are formed by the condensation of two allysine... 

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.)...

Ala. The action of lysyl oxidase to produce allysine allows three of these modified residues to condense with one lysine residue to form the heterocyclic complex amino acid desmosine. which cross-links two or even three chains. A highly cross-linked network results. 

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. 

To form the desmosines, three peptidyl allysine molecules and a molecule of peptidyl lysine must condense. The steps in condensation probably involve the formation of 1,2-dihydropyridines and 1,4-dihydropyridines as shown in Figure 4 (19-24,46,48). Several kinds of chemical evidence (46,48) suggest that the hydropyridines are easily oxidized under normal oxygen tension to corresponding pyridinium ions, such as the desmosines (isodesmosine or desmosine). The exact pathway by which the desmosines are formed, however, is still not clear. 

Currently, there are at least two views related to the mechanism by which the desmosines are formed (19). These include the direct reaction of the so-called allysine aldol (cf. Figure 4) with dehydrolysinonorleucine to form desmosines, or alternatively, the reaction of dehydromerodesmosine with an allysine residue. 

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. 

Figure 4. A general scheme for the formation of desmosine from lysine. The figure was adapted from W. R. Gray (19). Reactions within the box are considered to be reversible. Steps that result in the formation of lysinonorleucine, merodes-rnosine or desmosine are considered irreversible. Two separate pathways by which desmosine may be formed are indicated by the solid or broken lines.

Concentrations are in residues/1000 amino acid residues. When cross-links are expressed as lysine equivalents, desmosine and isodesmosine each equal four, dehydromerodesmosine and merodesmosine each equal three, and dehydrolysinonorleucine, lysinonorleucine, and the aldol-condensation product each equal two lysine residues. Data taken from Francis et al. (51). 

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). 

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