Figure 1The domain organization of the human TB domain proteins
All known human TB proteins are shown, with domain symbols summarized in the box at the top of the Figure. Note the high conservation of fibrillin domain organization compared with LTBPs. Some of the alternatively spliced forms of the LTBPs demonstrated to occur in humans are also illustrated .
Figure 2The roles for TB domain proteins in the ECM
(A) Fibrillin microfibrils can be associated with (i) elastin to form elastic fibres in tissues such as the blood vessel wall, lung and skin, or (ii) occur independently, for example in the suspensory ligaments of the eye, peridontal ligament and mesangium of the kidney glomerulus. (B) Some of the key cell matrix interactions mediated by fibrillin microfibrils: LTBPs bind microfibrils and sequester TGFβ in a latent state. A variety of BMP and GDF growth factors bind fibrillin directly and are sequestered to the ECM. Microfibrils direct the formation of elastic fibres by interacting with various proteins including fibulins. Integrins bind microfibrils, contributing to cell adhesion. Microfibrils have also been shown to interact with a variety of other proteins and ECM components (Figure 6) [1–3]. (C) The current model of LTBP organization in the ECM for maintenance of latent TGFβ. The LLC consists of dimeric TGFβ non-covalently bound to its propeptide, and the propeptide is covalently bound to LTBP via its second TB domain [13–16]. The LLC itself is sequestered to the ECM, potentially via interaction of its C-terminus with the N-terminus of fibrillin [11,12] and interactions with unknown matrix components at its N-terminus , which may involve transglutaminase cross-links .
Figure 3The structural features of TB, hybrid and cbEGF domains in the fibrillin LTBP family
(A) A schematic diagram illustrating the pattern of disulfide bonds between cysteine residues (yellow) in each domain type. Regions of variable loop length are represented by a single bracketed circle, with the potential number of residues noted at the bottom right. Also highlighted in green are residues that are conserved in all human domains looked at. Conserved residues in cbEGF domains are based on alignments of human fibrillin cbEGF domains, and conserved residues in hybrid and TB domains are based on alignments of human fibrillin and LTBP domains. *In the human fibrillin-1 TB1 domain the sixth cysteine residue is in an unusual position just before the conserved tryptophan residue, the disulfide arrangement in this domain is unknown. **The fibrillin hybrid 1 domain possesses an additional single cysteine residue (in parentheses). (B) Three-dimensional structures taken from TB4 (PDB code 1UZJ), hybrid 2 (PDB code 2W86) and cbEGF23 (PDB code 1UZJ) of human fibrillin-1. Structures are rainbow coloured with the N-termini in blue through to the C-termini in red. Disulfides are also shown as stick representations with carbon atoms coloured green and sulfur atoms coloured yellow. The calcium atom bound to the cbEGF is shown as a red sphere. (C) A flattened two-dimensional representation of the secondary structure of these domains; a black line represents the backbone, arrows represent β-sheet, and rectangles represent α-helix. Disulfides are represented as yellow lines. A three-dimensional interactive version of this Figure is available at http://www.BiochemJ.org/bj/433/0263/bj4330263add.htm.
Figure 4Domain organization of TB proteins in a variety of organisms and the evolutionary relationships between them
Proteins were detected using the NCBI (National Center for Biotechnology Information) blast tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with the fibrillin-1 TB3 domain as the search query. Further proteins were also discovered by blasting genomes from specific organisms in the JGI (Joint Genome Institute) database (http://www.jgi.doe.gov/). Domains were annotated initially using the SMART web-based domain prediction tool , followed by manual inspection of regions of interest, and location of cysteine residues not associated with domains. It should be noted that many of these protein sequences are predicted from shotgun genome sequences of varying quality and are not backed up by cDNAs, so many may be missing exons or include other errors. These errors probably give rise to the various unrecognized cysteine-containing domains shown in orange. Where possible reprediction of proteins was carried out from genomic DNA, using the WISE2 web-based tool (http://www.ebi.ac.uk/Tools/Wise) with human fibrillin-1 as an example sequence. This gave significant improvements in the protein sequence predictions in some cases, such as in the fibrillin of N. vectensis and Ixodes scapularis. Sequence references are given in Supplementary Table S1 at http://www.BiochemJ.org/bj/433/bj4330263add.htm. The phylogenetic tree shown is based on the Tree of Life web project (http://tolweb.org/tree/).
Figure 5Alignments of selected regions of functional interest in fibrillins and LTBPs
Alignments of selected fibrillin (A, B and C) and LTBP (D and E) regions are shown. Alignments were prepared using the ClustalW program  using matrix BLOSUM45. Amino acids are highlighted using the following scheme: red text on yellow background, residue absolutely conserved; black text on light blue background, conservation of specific residue type; black text on light green background, conservation of similar residue types. The starting human residue number is noted at the top of each alignment. (A) Alignment of the fibrillin EGF3 and hybrid 1 sequences. The absolutely conserved single cysteine residue (Cys204) is boxed. The Asn164 residue which is important for the fibrillin–LTBP and the fibrillin–fibulin interaction is also highlighted; note this is only conserved in species with LTBPs. (B) Alignment of the C-terminal region indicating the conserved CXXC motif in all species for which sequences of this region are available, from humans to the jellyfish Clytia hemisphaerica. (C) Alignment of a subsection of the fibrillin TB4 domain, showing that the integrin-binding RGD motif is restricted to vertebrates. (D) Alignment of the second TB domain of various LTBP-1-like proteins showing conservation of a two amino acid insert and various residues implicated in binding the TGFβ propeptide . The two amino acid insert extends a β-hairpin, placing the 2–6 disulfide in a surface-exposed position so that it can react with the TGFβ propeptide . Human LTBP-2, -3 and -4 are also shown below (coloured as the alignment above), demonstrating the absence of insert and other features in LTBP-2, which does not bind the TGFβ propeptide. (E) Alignment of the C-termini of LTBP-1-like proteins, demonstrating high conservation of EGF3 sequences compared with other regions. Human LTBP-2, -3 and -4 are also shown. Note the poor sequence conservation of this region in LTBP-3, which does not bind fibrillin. A. mellifera, Apis mellifera; A. pisum, Acyrthosiphon pisum; C. familiaris, Canis familiaris; C. teleta, Capitella teleta; D. rerio, Danio rerio; D. pulex, Daphnia pulex; E. caballus, Equus caballus; G. aculeatus, Gymnopilus aculeatus; G. gallus, Gallus gallus; H. sapiens, Homo sapiens; L. gigantea, Lottia gigantea; M. domestica, Monodelphis domestica; M. musculus, Mus musculus; O. anatinus, Ornithorhynchus anatinus; O. latipes, Oryzias latipes; P. humanus, Pediculus humanus; T. castaneum, Tribolium castaneum; X. tropicalis, Xenopus tropicalis.
Figure 6Known protein and proteoglycan interactions of fibrillin-1 with various other ECM components
Yellow bars represent the regions of fibrillin to which the interactions have been mapped. References are as follows: BMP7 prodomain , GDF8 and 5, and BMP2, 4, 7 and 10 , MAGP2 , MAGP1 , fibulin-2, -4 and -5 [12,83], LTBP-2 , LTBP-1 and -4 [11,12], decorin , perlecan domains 1 and 2 , versican and aggrecan , integrins [47,52–56] and fibronectin .
Figure 7Possible stages in the evolution of TB domain protein function
(A) The first unknown organism with TB domain proteins: TB proteins evolved first with either a structural and/or growth-factor-binding role. (B) The common ancestor of humans and jellyfish: fibrillin forms microfibrils; suitable BMP7-like growth factors are present, but binding to fibrillin is not determined. (C) The common ancestor of humans and sea urchin: LTBP and TGFβ–like proteins first appear; no elastic fibre components. (D) The common ancestor of humans and bony fish: elastin and other elastic fibre components emerge; LTBP- and integrin-binding sites also appear; elastin-independent role for microfibrils is maintained. An animated version of this Figure is available at http://www.BiochemJ.org/bj/433/0263/bj4330263add.htm.
Table 1Genetic diseases associated with TB domain proteins and related pathologies
The main features of each disease are summarized, along with the gene involved and the effect on TGFβ signalling where relevant.
Role of TGFβ
Elongation of the long bonesArachnodactyly (long fingers)Pectus carinatum/ pectus excavatumAbnormal joint flexibilityHigh arched palateEctopia lentis and early onset glaucomaAortic aneurysm and dilatationMitral valve prolapse
Excessive free TGFβ seen in mouse models. Increased levels of phosphorylated Smad2 seen in Marfan syndrome aorta
Craniosynostosis (early fusion of skull bones)
Arachnodactyly (long fingers) and other Marfan-like skeletal features
Mitral valve prolapse may occur, but aortic dilatation rare
Stiff skin syndrome
Tight and thick skinLimited joint mobilityShort stature
Increase in phosphorylated Smad2 in dermis, indicative of TGFβ signalling
Brachydactyly (short fingers)
Myopia, glaucoma and ectopia lentis
Contractural arachnodactyly (long fingers bent in at the joints) and contractures of various other joints
Primary congenital glaucoma, or secondary glaucoma with megalocornea and spherophakia
Oligodontia, failure of six or more teeth to develop
Severe developmental defects in the pulmonary, gastrointestinal, genitourinary, musculoskeletal and dermal systems
TGFβ signalling shown to be significantly higher when patient fibroblasts co-cultured with reporter cells
Cleft palate or bifid uvulaAbnormally long limbs and fingers
TGFBR1 (dominant)TGFBR2 (dominant)
Increased levels of phosphorylated Smad2 in aorta
Contractures of fingers and toes
Abnormal joint flexibility
Widely spaced eyes
Aortic aneurysm and aortic dissection with tortuosity
Table 2Parallel evolution of fibrillin-interacting proteins and TGFβ
Proteins detected by searching the NCBI and JGI databases with the relevant human sequence using the BLAST tool. The identities of detected proteins were then confirmed by using them in a BLAST search back against the human genome and checking the original search protein was the highest scoring result returned. Any unexpected protein appearances were further checked by domain prediction using the SMART web program. A, aggrecan; V, versican.