Elastin crosslinking

The first elastomeric protein is elastin, this structural protein is one of the main components of the extracellular matrix, which provides stmctural integrity to the tissues and organs of the body. This highly crosslinked and therefore insoluble protein is the essential element of elastic fibers, which induce elasticity to tissue of lung, skin, and arteries. In these fibers, elastin forms the internal core, which is interspersed with microfibrils [1,2]. Not only this biopolymer but also its precursor material, tropoelastin, have inspired materials scientists for many years. The most interesting characteristic of the precursor is its ability to self-assemble under physiological conditions, thereby demonstrating a lower critical solution temperature (LCST) behavior. This specific property has led to the development of a new class of synthetic polypeptides that mimic elastin in its composition and are therefore also known as elastin-like polypeptides (ELPs). 

Elastin is a heavily crosslinked biopolymer that is formed in a process named elastogenesis. In this section, the role of elastin and the different steps of elastin production will be described, starting with transcription of the genetic code and processing of the primary transcript, followed by translation into the elastin precursor protein and its transport to the extracellular matrix. Finally, the crosslinking and fiber formation, which result in the transition from tropoelastin to elastin, are described. 

Fig. 4 Structures and formation routes of crosslinks in elastin. In the first step, lysine is catalyti-cally converted to allysine by lysyl oxidase all subsequent condensation steps are spontaneous...

This coacervation process forms the basis for the self-assembly, which takes place prior to the crosslinking. The assembly of tropoelastin is based on an ordering process, in which the polypeptides are converted from a state with little order to a more structured conformation [8]. The insoluble elastic fiber is formed via the enzymatic crosslinking of tropoelastin (described in Sect. 2.1). Various models have been proposed to explain the mechanism of elasticity of the elastin fibers. 

Fig. 15 Amino acid sequences of artificial extracellular matrix (aECM) proteins. Each protein contains a TV tag, a histidine tag, a cleavage site, and elastin-like domains with lysine residues for crosslinking. The RGD cell-binding domain is found in aECM 1, whereas aECM 3 contains the CS5 cell-binding domain. aECM 2 and aECM 4 are the negative controls with scrambled binding domains for aECM 1 and aECM 3, respectively. Reprinted from [121] with permission from American Chemical Society, copyright 2004...

Sallach RE, Cui W, Wen J et al (2009) Elastin-mimetic protein polymers capable of physical and chemical crosslinking. Biomaterials 30 409 22... 

ELASTIN-MIMETIC PROTEIN NETWORKS DERIVED FROM CHEMICALLY CROSSLINKED SYNTHETIC POLYPEPTIDES... 

Elastin-Mimetic Protein Networks Derived from Chemically Crosslinked Synthetic Polypeptides... 

Figure 1 Design of a crosslinkable amino acid sequence based on the elastin-mimetic repeat Lys-25.

Elastin-mimetic protein polymers have been fabricated into elastic networks primarily via y-radiation-induced, radical crosslinking of the material in the coacervate state [10]. Although effective, this method cannot produce polymers gels of defined molecular architecture, i.e., specific crosslink position and density, due to the lack of chemoselectivity in radical reactions. In addition, the ionizing radiation employed in this technique can cause material damage, and the reproducibility of specimen preparations may vary between different batches of material. In contrast, the e-amino groups of the lysine residues in polymers based on Lys-25 can be chemically crosslinked under controllable conditions into synthetic protein networks (vide infra). Elastic networks based on Lys-25 should contain crosslinks at well-defined position and density, determined by the sequence of the repeat, in the limit of complete substitution of the amino groups. 

Nakamura F, Yamazaki K and Suyama K (1992) Isolation and structural characterization of a new crosslinking amino acid, cyclopentenosine, from the acid hydrolysate of elastin. Biochem Biophys Res Comm 186, 1533-1538. 

Suyama K, Yamazaki K and Nakamura P (1995) Gyclopentenosine, trifunctional crosslinking amino acid of elastin and collagen, characterization and distribution. Spec Publ - R Soc Chem 151, 425. 

Although the mode of action is not certain, this compound is an inhibitor of lysyl oxidase essential to the crosslinking of both collagen and elastin. A hereditary defect with a similar effect in the mouse involves a defect in lysyl oxidase. 1... 

Lysine tyrosylquinone (LTQ). Another copper amine oxidase, lysyl oxidase, which oxidizes side chains of lysine in collagen and elastin (Eq. 8-8) contains a cofactor that has been identified as having a lysyl group of a different segment of the protein in place of the - OH in the 2 position of topaquinone.465 Lysyl oxidase plays an essential role in the crosslinking of collagen and elastin. 

Copper was recognized as nutritionally essential by 1924 and has since been found to function in many cellular proteins.470-474 Copper is so broadly distributed in foods that a deficiency has only rarely been observed in humans.4743 However, animals may sometimes receive inadequate amounts because absorption of Cu2+ is antagonized by Zn2+ and because copper may be tied up by molybdate as an inert complex. There are copper-deficient desert areas of Australia where neither plants nor animals survive. Copper-deficient animals have bone defects, hair color is lacking, and hemoglobin synthesis is impaired. Cytochrome oxidase activity is low. The protein elastin of arterial walls is poorly crosslinked and the arteries are weak. Genetic defects in copper metabolism can have similar effects. 

Various natural, chemically modified and mixtures of flavonoids are widely used therapeutically as venous protective or venotonic drugs in chronic venous insufficiency and haemorrhoidal attacks. Flavonoids have been found to inhibit increased vessel wall permeability, fluid changes in the capillary bed and diffusion of plasma proteins. In addition, they may exert a protective effect on the perivascular tissues due to their antihyaluronidase effect and the inhibition of lysine oxidase (producing crosslinks in collagen and elastin) and lysosomal hydrolases (degrade glycosamines). All these effects may account for the venotonic effects of these drugs [5]. However, the venous effects of flavonoids are out of the scope of the present review. 

Copper is an essential component of numerous key metalloenzymes which are critical in melanin formation, myelin formation and crosslinking of collagen and elastin. Copper plays a vital role in hemopoiesis, maintenance of vascular and skeletal integrity, and structure and function of the nervous system. Thus a deficiency of copper can lead to a variety of adverse effects such as increased fragility in bones, aneurysm formation in arteries and a loss of lysyl oxidase activity in cartilage.54 57 Articles on copper also appear in Siget1, volumes 3 and 5, all of volumes 12 and 13, and volume 14,... 

In vivo elastin fiber formation requires the coordination of a number of important processes. These include the control of intracellular transcription and translation of tropoelastin, intracellular processing of the protein, secretion of the protein into the extracellular space, delivery of tropoelastin monomers to sites of elastogenesis, alignment of the monomers with previously accreted tropoelastin through associating microfibrillar proteins, and finally, the conversion to the insoluble elastin polymer through the crosslinking action of lysyl oxidase (Fig. 2). 

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). 

Circular dichroism (CD) studies on a-elastin (Tamburro et al., 1977), K-elastin, bovine, and human tropoelastin (Debelle et al., 1995 Vrhovski et al., 1997) have demonstrated a conformational transition to increased a-helical content with increasing temperature. The a-helical content predicted for tropoelastin is probably confined to the crosslinking domains, as the rest of the molecule is rich in helix breaking proline residues (Muiznieks et al., 2003). 

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