The Evolution of Extracellular Fibrillins and Their Functional Domains

Fibrillins constitute the major backbone of multifunctional microfibrils in elastic and non-elastic extracellular matrices, and are known to interact with several binding partners including tropoelastin and integrins. Here, we study the evolution of fibrillin proteins. Following sequence collection from 39 organisms representative of the major evolutionary groups, molecular evolutionary genetics and phylogeny inference software were used to generate a series of evolutionary trees using distance-based and maximum likelihood methods. The resulting trees support the concept of gene duplication as a means of generating the three vertebrate fibrillins. Beginning with a single fibrillin sequence found in invertebrates and jawless fish, a gene duplication event, which coincides with the appearance of elastin, led to the creation of two genes. One of the genes significantly evolved to become the gene for present-day fibrillin-1, while the other underwent evolutionary changes, including a second duplication, to produce present-day fibrillin-2 and fibrillin-3. Detailed analysis of several sequences and domains within the fibrillins reveals distinct similarities and differences across various species. The RGD integrin-binding site in TB4 of all fibrillins is conserved in cephalochordates and vertebrates, while the integrin-binding site within cbEGF18 of fibrillin-3 is a recent evolutionary change. The proline-rich domain in fibrillin-1, glycine-rich domain in fibrillin-2 and proline-/glycine-rich domain in fibrillin-3 are found in all analyzed tetrapod species, whereas it is completely replaced with an EGF-like domain in cnidarians, arthropods, molluscs and urochordates. All collected sequences contain the first 9-cysteine hybrid domain, and the second 8-cysteine hybrid domain with exception of arthropods containing an atypical 10-cysteine hybrid domain 2. Furin cleavage sites within the N- and C-terminal unique domains were found for all analyzed fibrillin sequences, indicating an essential role for processing of the fibrillin pro-proteins. The four cysteines in the unique N-terminus and the two cysteines in the unique C-terminus are also highly conserved

ClustalX alignment of the regions containing cysteines in the unique A) N- and B) C-terminal domains.

<p>Note the high conservation of the six cysteine residues and the surrounding sequences in both domains.</p

Maximum likelihood phylogenetic tree generated from seven concatenated TB domains of each fibrillin.

<p>The fibrillin protein family is grouped into four separate clades (from bottom to top): single fibrillin (Fbn), fibrillin-1 (Fbn-1), fibrillin-2 (Fbn-2), and fibrillin-3 (Fbn-3). Bootstrap values shown on each node represent the percentage of trees (out of 1,000) yielding the same two-set partition of sequences. The Jones-Taylor-Thornton model with invariant sites and a gamma distribution with four discrete categories was used. The tree was re-rooted around the base of the single fibrillin clade. The scale bar indicates the estimated average number of expected substitutions per site. Analyses of full length fibrillins or of full length fibrillins lacking the unique region resulted in similar phylogenetic trees.</p

Excerpt from the ClustalX alignment of full length fibrillin focusing on the unique regions and the subsequent EGF4 domain.

<p>The end of TB1, the unique region, and the EGF4 domain found characteristically in mammalian fibrillins are indicated on the bottom. For invertebrate organisms, the cbEGF-like (purple box), and the new cbEGF domain (orange box) are highlighted, and cysteine residues are circled in red in these domains. The relative numbers of the cysteine residues within the respective EGF domain is indicated on top. Note that the unique region does not exist in invertebrate fibrillin proteins, and is instead replaced by a cbEGF-like domain. The non-calcium binding EGF4 domain that typically follows the unique region is replaced in invertebrate fibrillins by a cbEGF domain.</p

Comparison of bootstrap values.

<p>Bootstrap values, that is, the percentage of trees yielding the same two-set partition of sequences for a given node, are indicated in percentages (two top rows). Bootstrap values were generated from 1,000 data sets. Distance-based phylogenetic trees were generated with individual TB domains (TB1–TB7). Distance based (DB) and maximum likelihood (ML) based methods were generated with all TB domains of a fibrillin protein concatenated together and with the entire fibrillin sequences as indicated. The numbers of fibrillin sequences included in the respective analyses are indicated in the bottom row.</p

ClustalX alignment of RGD cell interaction sites in A) TB3 and B) TB4.

<p>Only the relevant loop containing the RGD sequence is shown. RGD sequences are highlighted by a box and an arrow. The relative numbers of the cysteine residues within the respective TB domain is indicated on the bottom. Note for TB3 the presence of a RGD integrin-binding site in sea lamprey, ray-finned fish fibrillin-1 and fibrillin-2/3, all fibrillin-2 species and several tetrapod fibrillin-3 sequences. For TB4, the RGD integrin-binding site is present in all vertebrate fibrillins, with the exception of armadillo fibrillin-2. Invertebrate fibrillin sequences do not contain an integrin-binding site in TB4.</p

ClustalX alignment of the second hybrid domain in arthropods.

<p>Relative numbering of the cysteine residues in the hybrid 2 domain as identified in human fibrillins is indicated on the bottom. In arthropods, this domain has two additional cysteine residues (black boxes indicated by arrows) annotated here as cysteine 4a and 6a.</p