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|Coordinate||5,747,688 bp (GRCm38)|
|Base Change||T ⇒ C (forward strand)|
|Gene Name||latent transforming growth factor beta binding protein 3|
|Chromosomal Location||5,740,904-5,758,532 bp (+)|
|MGI Phenotype||Homozygotes for a targeted null mutation exhibit craniofacial malformations including an overshot mandible and ossification of synchondroses. Mutants develop osteosclerosis of long bones and osteoarthritis, and, in some cases, high corticosterone levels.|
|Amino Acid Change||Cysteine changed to Arginine|
|Institutional Source||Beutler Lab|
C452R in Ensembl: ENSMUSP00000080214 (fasta)
|Gene Model||not available|
|Predicted Effect||probably damaging
PolyPhen 2 Score 0.995 (Sensitivity: 0.67; Specificity: 0.97)
|Phenotypic Category||craniofacial, growth/size, limbs/digits/tail phenotype, skeleton phenotype|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Local Stock||Embryos, gDNA|
|Last Updated||2016-05-13 3:09 PM by Stephen Lyon|
The csp (craniofacial and skeletal malformation with paralysis) phenotype is characterized by watery eyes, hind leg paralysis and craniofacial malformations that are evident two weeks after birth. Kyphosis develops gradually, becoming prominent by the age of six months. Csp homozygotes suffer retarded growth at a young age, but grow normally at later stages. Histological analyses reveal abnormal formation of synchondroses. These phenotypes are observed both on the C57BL/6J background and in mice created by outcrossing to C3H/HeJ and backcrossing to the mutant stock.
The abnormalities observed in csp mice are similar to those of mice with impaired transforming growth factor-β (TGF-β) signaling in skeletal tissue (1).
|Nature of Mutation|
The csp mutation was mapped to Chromosome 19, and corresponds to a T to C transition at position 2343 of the Ltbp3 transcript, in exon 9 of 28 total exons.
The mutated nucleotide is indicated in red lettering, and causes a cysteine to arginine substitution at residue 452 of the LTBP3 protein.
Ltbp3 encodes the latent transforming growth factor-β binding protein-3 (LTBP3), an extracellular matrix (ECM) protein thought to regulate signaling by transforming growth factor-β (TGF-β) by regulating its bioavailability. There are 4 LTBPs, and all possess sequence and structural homology to the fibrillins, secreted ECM proteins (2-4). The 1268 amino acid LTBP3 polypeptide contains several motifs divided between five structurally distinct domains (2). After a 21-amino acid N-terminal signal peptide, Domain 1 is a 28-amino acid basic region. Domain 2 consists of two EGF-like repeats, a 135-amino acid proline- and glycine- rich region, a fibrillin motif, and an 8-Cysteine motif (8-Cys; also called a TGF-bp repeat). Domain 3 is a proline-rich region. Domain 4 consists of twelve EGF-like repeats and two 8-Cys motifs. Domain 5 is a unique 22-amino acid C-terminal segment (2). Of the fourteen total EGF-like repeats in LTBP3, eleven contain a calcium binding consensus sequence (2), which has been suggested to confer resistance to proteolytic cleavage (5).
The 8-Cys motifs are unique to LTBPs, and mediate covalent binding through a disulfide bond to latent TGF-βs (see Background) (6;7). The NMR solution structure of the 8-Cys motif of fibrillin suggests that hydrophobic contacts may be important for LTBP recognition of latent TGF-β (8). As in all the LTBPs, only the third 8-Cys motif of LTBP3 forms disulfide bonds with latent TGF-β1 (9-11). Notably, the second 8-Cys motif has nine cysteine residues instead of eight, but the biological significance of this is unknown (9). A critical cysteine residue (C33) in latent TGF-β1 participates in disulfide bond formation with LTBP3 (6;10;11).
The csp mutation substitutes an arginine for the wild-type cysteine at residue 452 (C452R), in the predicted Domain 4.
During embryonic development, mouse Ltbp3 mRNA is expressed throughout the body, with relatively high expression in the liver at embryonic day 13.5. Expression is also detectable in heart, central nervous system, pancreas, kidney, skin and walls of large arteries (2). In addition, Ltbp3 transcript is found in osteoblasts and periosteal cells of the calvarium (top of skull), mandible, and maxilla, and in cartilage and bone of lower extremities (2). The LTBP3 protein is targeted to the cell surface for secretion, a process that reportedly requires complex formation with TGF-β1 (10;12).
TGF-βs are dimeric polypeptide cytokine growth factors expressed and secreted by virtually every cell type. There are three TGF-β isoforms (1-3), each encoded by a separate gene whose expression is developmentally and spatially regulated. TGF-βs regulate cell growth and differentiation, and the synthesis, degradation, and remodeling ofthe extracellular matrix (ECM). They are expressed during embryogenesis and adulthood, and thus contribute to a wide variety of biological processes, including embryonic development, wound healing, angiogenesis, inflammation, and bone formation [reviewed in (13;14)]. TGF-βs generally promote tissue growth and differentiation in the embryo, but inhibit cellular proliferation in mature tissues (15). Dysregulated TGF-β signaling leads to cancers, particularly pancreatic and colon carcinomas (13;15).
TGF-βs are secreted from cells as latent large molecular mass complexes (LLCs) with three distinct components. TGF-β molecules are initially synthesized as precursor proteins (proTGF-β) comprised of a propeptide and the mature TGF-β (16). Even after cleavage, a non-covalent attachment binds the propeptide to the mature TGF-β, and in this state the propeptide is termed the latency associated peptide (LAP). The LTBPs form disulfide linkages with LAP at specific cysteine residues (6;7). Together, the LAP, LTBP and TGF-β form the LLC, and in this complex, TGF-β possesses no biological activity. LAP functions as an inhibitor of TGF-β, and dissociation from LAP is a crucial regulatory step in TGF-β activation. This dissociation may be stimulated by several proteases (plasmin, MMP-1, MMP-2), thrombospondin-1, integrins, acidic pH conditions, and reactive oxygen species [reviewed in (14)].
The role of LTBPs in TGF-β signaling is not yet clearly defined, although studies of LTBP1 suggest that they serve to localize TGF-β at appropriate sites in the ECM in a regulated manner, thereby modulating TGF-β bioavailability (17). ProTGF-β that fails to bind LTBP-1 is inefficiently secreted, and contains increased numbers of abnormal disulfide bonds, suggesting that LTBP-1 promotes correct folding and subsequent secretion of the LLC (18). Antibodies against LTBP-1 block TGF-β activation in a smooth muscle/endothelial cell coculture system (19), and prevent the TGF-β-stimulated endothelial-mesenchymal transition of mouse heart cultures (20). LTBP-1 may promote TGF-β bioavailability in part through crosslinking of LTBP-1 to the ECM (4;21). Both LTBP-1 and TGF-β are found in ECM microfibrils of fibroblasts (22) and skin cells (23). Moreover, inhibition of transglutaminase, a factor that mediates LTBP1-ECM crosslinking (24;25), reduces production of active TGF-β (26).
Once activated, TGF-β binds to three high-affinity cell surface receptors (types I, II and III) [reviewed in (13)]. The type III receptor is a nonsignaling receptor, and transfers its TGF-β ligand to either type I or type II receptors, which signal through their serine/threonine kinase domains and activate targets including the Smad transcription factors, MAP kinase and stress-activated protein (SAP) kinase pathways (13).
Mice with targeted deletion of LTBP3 have been generated, and like csp mice, Ltbp3-/- mice display defects in skull and long bone development and homeostasis (27;28). Ltbp3-/- mice have craniofacial abnormalities, with a rounded head and shortened snout visible by 10 days of age. Ltbp3-/- mice develop a domed skull, abnormal apposition of upper and lower incisors, and thoracic kyphosis by 2 months of age (27). These defects are caused by premature ossification of the synchondroses of the skull base (which normally never ossify), forcing the membranous bones of the vault to expand outward and upward to accommodate the increasing volume of the developing brain (27).
Mislocalized and unchecked chondrocyte differentiation may underlie the premature ossification of synchondroses observed in Ltbp3-/- and csp mice (Figure 2). TGF-β1 has been shown to stimulate the expression of parathyroid hormone related protein (PTHrP, an inhibitor of chondrocyte differentiation) and inhibit chondrocyte hypertrophic differentiation (29;30). In Ltbp3-/- synchondroses, PTHrP levels are reduced relative to wild type, while the expression of Indian hedgehog (Ihh, a marker for chondrocytes committed to hypertrophic differentiation) was detected throughout LTBP3-null synchondroses. These data suggest that LTBP3 promotes TGF-β signaling to limit chondrocyte differentiation and synchondrosis ossification (28).
LTBP3-null mice develop age-dependent osteosclerosis with increased bone mass in the long bones and the axial skeleton (28). Similarly, transgenic mice expressing a dominant negative type II TGF-β receptor in osteoblasts acquire age-dependent osteopetrosis (31). Ltbp3-/- mice also develop osteoarthritis, and likewise transgenic mice expressing dominant negative type II TGF-β receptor in skeletal tissue (1), or targeted Smad3 mutants (32). These findings further support the postulate that reduced TGF-β signaling, particularly through Smad3, causes the skeletal phenotypes in Ltbp3-/- and csp mice.
|Primers||Primers cannot be located by automatic search.|
Csp genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide change.
Primers for PCR amplification
Csp(F): 5’-AGAAGCAACTGGGCAGAGGGACATAGC -3’
Csp(R): 5’-TGGTTCTCAACCTTCCCAGTGCCAC -3’
1) 94°C 2:00
2) 94°C 0:15
3) 60°C 0:20
4) 68°C 1:00
5) repeat steps (2-4) 35X
6) 68°C 5:00
7) 4°C ∞
Primers for sequencing
Csp_seq(F): 5’- ACCCTCTGACCACACGCCTAAC -3’
Csp_seq(R): 5’- TGTAGCCCTGGCTGTCCTGGAACTC -3’
The following sequence of 923 nucleotides (from Genbank genomic region NC_000085 for linear DNA sequence of Ltbp3) is amplified:
6446 agaag caactgggca gagggacata gcaatgctca
6481 ggtcctgccc tccactccca cccctgcagc cgacaaacca gaggagaaga gcctgtgttt
6541 ccgccttgtg agcaccgaac accagtgcca gcaccctctg accacacgcc taacccgcca
6601 gctctgctgc tgtagtgtgg gtaaagcctg gggtgcccgg tgccagcgct gcccggcaga
6661 tggtacaggt gaggcagagg cacatcgtgg atgatgtagg gatgggacgg caagctgtgt
6721 acccgtccag gagttcactt gttgtggtgt ctgcatcttg accacagcag ccttcaagga
6781 gatctgcccg gctgggaaag ggtaccatat cctcacctcc caccagacgc tcaccatcca
6841 gggggaaagc gacttctccc tcttcctgca ccccgacggg ccacccaaac cccagcagct
6901 tcctgaaagc cccagccgag caccacccct cgaggacaca gaggaagaga gaggtctggc
6961 ttgatccaat aattccagat ccacagataa aactcagggg ctagccgggc gtggtggcgc
7021 acgcctttaa tcccagcact tgggaggcag aggcaggcgg atttctgagt tcgaggccag
7081 cctggtctac agagtgagtt ccaggacagc cagggctaca cagagaaacc ctgtcttgaa
7141 aaaaaagact catgggctaa ggcagtggtt tgaaacctgt aggttggaac ccctgggggc
7201 agggggtgtc acatatcaga tatcctgcct atcagatatt tacattagga ctcagaacag
7261 tagcaaaatt acagttatga agtagcaatt agatgatttt atggctgggg agtcatcaca
7321 acatgaggaa ctgtagaaaa ggggtggcac tgggaaggtt gagaacca
PCR primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated T is shown in red text.
1. Serra, R., Johnson, M., Filvaroff, E. H., LaBorde, J., Sheehan, D. M., Derynck, R., and Moses, H. L. (1997) Expression of a truncated, kinase-defective TGF-beta type II receptor in mouse skeletal tissue promotes terminal chondrocyte differentiation and osteoarthritis, J Cell Biol. 139, 541-552.
2. Yin, W., Smiley, E., Germiller, J., Mecham, R. P., Florer, J. B., Wenstrup, R. J., and Bonadio, J. (1995) Isolation of a novel latent transforming growth factor-beta binding protein gene (LTBP-3), J Biol. Chem. 270, 10147-10160.
3. Kanzaki, T., Olofsson, A., Moren, A., Wernstedt, C., Hellman, U., Miyazono, K., Claesson-Welsh, L., and Heldin, C. H. (1990) TGF-beta 1 binding protein: a component of the large latent complex of TGF-beta 1 with multiple repeat sequences, Cell 61, 1051-1061.
4. Taipale, J., Miyazono, K., Heldin, C. H., and Keski-Oja, J. (1994) Latent transforming growth factor-beta 1 associates to fibroblast extracellular matrix via latent TGF-beta binding protein, J Cell Biol. 124, 171-181.
5. Colosetti, P., Hellman, U., Heldin, C. H., and Miyazono, K. (1993) Ca2+ binding of latent transforming growth factor-beta 1 binding protein, FEBS Lett 320, 140-144.
6. Saharinen, J., Taipale, J., and Keski-Oja, J. (1996) Association of the small latent transforming growth factor-beta with an eight cysteine repeat of its binding protein LTBP-1, EMBO J 15, 245-253.
7. Gleizes, P. E., Beavis, R. C., Mazzieri, R., Shen, B., and Rifkin, D. B. (1996) Identification and characterization of an eight-cysteine repeat of the latent transforming growth factor-beta binding protein-1 that mediates bonding to the latent transforming growth factor-beta1, J Biol. Chem. 271, 29891-29896.
8. Yuan, X., Downing, A. K., Knott, V., and Handford, P. A. (1997) Solution structure of the transforming growth factor beta-binding protein-like module, a domain associated with matrix fibrils, EMBO J 16, 6659-6666.
9. Yin, W., Fang, J., Smiley, E., and Bonadio, J. (1998) 8-Cysteine TGF-BP structural motifs are the site of covalent binding between mouse LTBP-3, LTBP-2, and latent TGF-beta 1, Biochim. Biophys. Acta 1383, 340-350.
10. Chen, Y., Dabovic, B., Annes, J. P., and Rifkin, D. B. (2002) Latent TGF-beta binding protein-3 (LTBP-3) requires binding to TGF-beta for secretion, FEBS Lett. 517, 277-280.
11. Saharinen, J. and Keski-Oja, J. (2000) Specific sequence motif of 8-Cys repeats of TGF-beta binding proteins, LTBPs, creates a hydrophobic interaction surface for binding of small latent TGF-beta, Mol. Biol. Cell 11, 2691-2704.
12. Penttinen, C., Saharinen, J., Weikkolainen, K., Hyytiainen, M., and Keski-Oja, J. (2002) Secretion of human latent TGF-beta-binding protein-3 (LTBP-3) is dependent on co-expression of TGF-beta, J Cell Sci. 115, 3457-3468.
13. Blobe, G. C., Schiemann, W. P., and Lodish, H. F. (2000) Role of transforming growth factor beta in human disease, N. Engl. J Med. 342, 1350-1358.
14. Annes, J. P., Munger, J. S., and Rifkin, D. B. (2003) Making sense of latent TGFbeta activation, J Cell Sci. 116, 217-224.
16. Dubois, C. M., Laprise, M. H., Blanchette, F., Gentry, L. E., and Leduc, R. (1995) Processing of transforming growth factor beta 1 precursor by human furin convertase, J Biol. Chem. 270, 10618-10624.
17. Oklu, R. and Hesketh, R. (2000) The latent transforming growth factor beta binding protein (LTBP) family, Biochem. J 352 Pt 3, 601-610.
18. Miyazono, K., Olofsson, A., Colosetti, P., and Heldin, C. H. (1991) A role of the latent TGF-beta 1-binding protein in the assembly and secretion of TGF-beta 1, EMBO J 10, 1091-1101.
19. Flaumenhaft, R., Abe, M., Sato, Y., Miyazono, K., Harpel, J., Heldin, C. H., and Rifkin, D. B. (1993) Role of the latent TGF-beta binding protein in the activation of latent TGF-beta by co-cultures of endothelial and smooth muscle cells, J Cell Biol. 120, 995-1002.
20. Nakajima, Y., Miyazono, K., Kato, M., Takase, M., Yamagishi, T., and Nakamura, H. (1997) Extracellular fibrillar structure of latent TGF beta binding protein-1: role in TGF beta-dependent endothelial-mesenchymal transformation during endocardial cushion tissue formation in mouse embryonic heart, J Cell Biol. 136, 193-204.
21. Oklu, R., Metcalfe, J. C., Hesketh, T. R., and Kemp, P. R. (1998) Loss of a consensus heparin binding site by alternative splicing of latent transforming growth factor-beta binding protein-1, FEBS Lett 425, 281-285.
22. Taipale, J., Saharinen, J., Hedman, K., and Keski-Oja, J. (1996) Latent transforming growth factor-beta 1 and its binding protein are components of extracellular matrix microfibrils, J Histochem. Cytochem. 44, 875-889.
23. Raghunath, M., Unsold, C., Kubitscheck, U., Bruckner-Tuderman, L., Peters, R., and Meuli, M. (1998) The cutaneous microfibrillar apparatus contains latent transforming growth factor-beta binding protein-1 (LTBP-1) and is a repository for latent TGF-beta1, J Invest Dermatol. 111, 559-564.
24. Nunes, I., Gleizes, P. E., Metz, C. N., and Rifkin, D. B. (1997) Latent transforming growth factor-beta binding protein domains involved in activation and transglutaminase-dependent cross-linking of latent transforming growth factor-beta, J Cell Biol. 136, 1151-1163.
25. Verderio, E., Gaudry, C., Gross, S., Smith, C., Downes, S., and Griffin, M. (1999) Regulation of cell surface tissue transglutaminase: effects on matrix storage of latent transforming growth factor-beta binding protein-1, J Histochem. Cytochem. 47, 1417-1432.
26. Kojima, S., Nara, K., and Rifkin, D. B. (1993) Requirement for transglutaminase in the activation of latent transforming growth factor-beta in bovine endothelial cells, J Cell Biol. 121, 439-448.
27. Dabovic, B., Chen, Y., Colarossi, C., Obata, H., Zambuto, L., Perle, M. A., and Rifkin, D. B. (2002) Bone abnormalities in latent TGF-[beta] binding protein (Ltbp)-3-null mice indicate a role for Ltbp-3 in modulating TGF-[beta] bioavailability, J Cell Biol 156, 227-232.
28. Dabovic, B., Chen, Y., Colarossi, C., Zambuto, L., Obata, H., and Rifkin, D. B. (2002) Bone defects in latent TGF-beta binding protein (Ltbp)-3 null mice; a role for Ltbp in TGF-beta presentation, J Endocrinol. 175, 129-141.
29. Pateder, D. B., Ferguson, C. M., Ionescu, A. M., Schwarz, E. M., Rosier, R. N., Puzas, J. E., and O'Keefe, R. J. (2001) PTHrP expression in chick sternal chondrocytes is regulated by TGF-beta through Smad-mediated signaling, J Cell Physiol 188, 343-351.
30. Serra, R., Karaplis, A., and Sohn, P. (1999) Parathyroid hormone-related peptide (PTHrP)-dependent and -independent effects of transforming growth factor beta (TGF-beta) on endochondral bone formation, J Cell Biol. 145, 783-794.
31. Filvaroff, E., Erlebacher, A., Ye, J., Gitelman, S. E., Lotz, J., Heillman, M., and Derynck, R. (1999) Inhibition of TGF-beta receptor signaling in osteoblasts leads to decreased bone remodeling and increased trabecular bone mass, Development 126, 4267-4279.
|Science Writers||Eva Marie Y. Moresco|
|Authors||Xin Du, Koichi Tabeta, Bruce Beutler|
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