Thrombospondin 3 Is a Pentameric Molecule Held Together by Interchain Disulfide Linkage Involving Two Cysteine Residues

Qabar, Aziz N.; Derick, Laura H.; Lawler, Jack; Dixit, Vishva M.

doi:10.1074/jbc.270.21.12725

Cited by 43 publications

(41 citation statements)

References 24 publications

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“…Rotary shadowing has demonstrated that, in the presence of Ca 2ϩ , a globule composed of the C-terminal portion of TSP1 enlarges while the stalk connecting the oligomerization domain to the C-terminal globule shortens (13)(14)(15). This structural change was also observed for TSP3 (16), TSP4 (17), and TSP5/COMP (18). The structure of TSP2 is also Ca 2ϩ -sensitive based on its susceptibility to trypsin proteolysis (19).…”

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confidence: 91%

Disulfide Connectivity of Recombinant C-terminal Region of Human Thrombospondin 2

Misenheimer

Hahr

Harms

et al. 2001

Journal of Biological Chemistry

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The thrombospondin (TSP) family of extracellular glycoproteins consists of five members in vertebrates, TSP1 to -4 and TSP5/cartilage oligomeric matrix protein, and a single member in Drosophila. TSPs are modular multimeric proteins. The C-terminal end of a monomer consists of 3-6 EGF-like modules; seven tandem 23-, 36-, or 38-residue aspartate-rich, Ca 2؉ -binding repeats; and an ϳ230-residue C-terminal sequence. The Ca 2؉ -binding repeats and C-terminal sequence are spaced almost exactly the same in different TSPs and share many blocks of identical residues. We studied the C-terminal portion of human TSP2 from the third EGF-like module through the end of the protein (E3CaG2). E3CaG2, CaG2 lacking the EGF module, and Ca2 composed of only the Ca 2؉ -binding repeats were expressed using recombinant baculoviruses and purified from conditioned media of insect cells. As previously described for intact TSP1, E3CaG2 bound Ca 2؉ in a cooperative manner as assessed by equilibrium dialysis, and its circular dichroism spectrum was sensitive to the presence of Ca 2؉ . Mass spectrometry of the recombinant proteins digested with endoproteinase Asp-N revealed that disulfide pairing of the 18 cysteines in the Ca 2؉ -binding repeats and C-terminal sequence is sequential, i.e. a 1-2, 3-4, 5-6, etc., pattern.

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confidence: 91%

Disulfide Connectivity of Recombinant C-terminal Region of Human Thrombospondin 2

Misenheimer

Hahr

Harms

et al. 2001

Journal of Biological Chemistry

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“…Although the close apposition and head-to-head orientation of Mtx and Thbs3 raise the possibility that the two genes share a bidirectional promoter, several lines of evidence suggest that these genes are regulated independently. First, Mtx mRNA is ubiquitously distributed in the mouse, whereas Thbs3 mRNA is restricted to lung, cartilage, the gastrointestinal tract, and hippocampus (2,8,9). Secondly, the basal elements of the Mtx and Thbs3 promoters can largely be dissociated, which indicates that the Mtx and Thbs3 genes are not regulated by a true bidirectional promoter (7).…”

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confidence: 99%

Metaxin Is a Component of a Preprotein Import Complex in the Outer Membrane of the Mammalian Mitochondrion

Armstrong

Komiya

Bergman

et al. 1997

Journal of Biological Chemistry

125

106

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Metaxin, a novel gene located between the glucocerebrosidase and thrombospondin 3 genes in the mouse, is essential for survival of the postimplantation mouse embryo. In this study, the subcellular location, domain structure, and biochemical function of metaxin were investigated. Anti-recombinant metaxin antibodies recognized 35-and 70-kDa proteins in mitochondria from various tissues; the 35-kDa protein is consistent in size with the predicted translation product of metaxin cDNA. When metaxin cDNA was transfected into COS cells, immunofluorescence staining demonstrated that the protein is located in mitochondria. Metaxin contains a putative mitochondrial outer membrane signal anchor domain at its C terminus, and a truncated form of metaxin lacking this signal anchor domain had a reduced association with mitochondria. In addition, metaxin was highly susceptible to proteases in intact mitochondria. We therefore conclude that metaxin is a mitochondrial protein that extends into the cytosol while anchored into the outer membrane at its C terminus. In its N-terminal region, metaxin shows significant sequence identity to Tom37, a component of the outer membrane portion of the mitochondrial preprotein translocation apparatus in Saccharomyces cerevisiae, but important structural differences, including apparently different mechanisms of targeting to membranes, also exist between the two proteins. Given the similar subcellular locations of metaxin and Tom37, the possible role of metaxin in mitochondrial preprotein import was investigated. Antibodies against metaxin, when preincubated with mitochondria, partially inhibited the uptake of radiolabeled preadrenodoxin into mitochondria. Metaxin is therefore the second mammalian component of the protein translocation apparatus of the mitochondrial outer membrane to be characterized at the molecular level and the first for which an inherited mutation has been described. The early embryonic lethal phenotype of mice lacking metaxin demonstrates that efficient import of proteins into mitochondria is crucial for cellular survival. The characterization of metaxin provides an opportunity to elucidate similarities and possible differences in the mechanisms of protein import between fungi and mammals and in the phenotypes of fungi and mammals lacking mitochondrial import receptors.During studies of thrombospondin 3 (Thbs3), which encodes an extracellular matrix protein (2-4), and glucocerebrosidase (Gba), which encodes a lysosomal enzyme (5), a novel gene, termed metaxin (Mtx), was found to span the interval between Thbs3 and Gba on chromosome 3E3-F1 in the mouse (6). Mtx and Thbs3 are situated in a head-to-head orientation, with the translation start sites 1.4 kilobase pairs apart. Gba and Mtx lie in a tail-totail orientation, with the polyadenylation sites spaced 431 base pairs apart. The Mtx promoter resembles the promoters for housekeeping genes in that it is TATA-less, GC-rich, and regulated by tandem Sp1 elements within 300 base pairs of the transcription start site (7). Although ...

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“…Pentameric TSPs, including TSP3, form ␣-helical coiled-coil domains in which the axial channel or pore is lined by hydrophobic amino acids (12,22,24). In the case of TSP5/COMP this axial pore is capable of binding physiologically relevant hydrophobic molecules such as vitamin D and all-trans retinol with appropriate affinities (14), and this property is likely to extend to TSP3.…”

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confidence: 99%

“…However, the specificity of the antibody that was used in this study is questionable, since in another study it stained all tissues in some sections indiscriminantly (26). In the chicken, TSP3 mRNA was expressed in embryonic cartilage and in neurons that are actively extending processes (28), and in Xenopus embryos TSP3 mRNA was detected in the notocord, floor plate, sensorial layer of the ectoderm, and sensory epithelia (30).Pentameric TSPs, including TSP3, form ␣-helical coiled-coil domains in which the axial channel or pore is lined by hydrophobic amino acids (12,22,24). In the case of TSP5/COMP this axial pore is capable of binding physiologically relevant hydrophobic molecules such as vitamin D and all-trans retinol with appropriate affinities (14), and this property is likely to extend to TSP3.…”

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confidence: 99%

Mice with a Disruption of the Thrombospondin 3 Gene Differ in Geometric and Biomechanical Properties of Bone and Have Accelerated Development of the Femoral Head

Hankenson¹,

Hormuzdi²,

Meganck

et al. 2005

Molecular and Cellular Biology

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Thrombospondin 3 (TSP3) is structurally similar to cartilage oligomeric matrix protein (COMP/TSP5), but its function is unknown. To determine the functional significance of TSP3, we generated mice with a targeted disruption of Thbs3. TSP3-null mice are viable and fertile and show normal prenatal skeletal patterning, based on Alcian blue/Alizarin red S staining. However, subtle and transient abnormalities were detected in the developing postnatal skeleton. Young adult TSP3-null mice are heavier than controls, and analyses of the geometric and biomechanical properties of long bones show increases in the moments of inertia, endocortical and periostal radii, and failure load. The bones of 9-week-old TSP3-null male mice also have a significantly greater cortical area. Most of these differences were no longer detected in 15-week-old mice. Microcomputed tomography scans showed that the trabecular bone proximal to the femoral head growth plate developed at an earlier time in TSP3-null mice than in wild-type mice. Thus, vascular invasion and ossification start in the femoral heads of TSP3-null mice at 9 weeks, whereas the wild-type femoral head is still composed of hypertrophic chondroctyes in a calcified matrix at 15 weeks. These results provide evidence for a role for TSP3 in the regulation of skeletal maturation in mice.Thrombospondin 3 (TSP3; encoded by Thbs3) was first discovered during sequencing of DNA upstream from the start of transcription of the mouse Muc1 (episialin) gene (31). Subsequent studies showed that these two genes are part of a tightly organized, conserved gene cluster on chromosome 3, E3-F1, which also includes glucocerebrosidase (Gba) and metaxin 1 (mtx1). The latter two genes are transcribed divergently and share a common promoter region of about 1 kb (5, 9, 32). However, despite this genomic arrangement, and the presence of an enhancer of TSP3 in intron 6 of the metaxin gene (10), no metabolic interactions have been identified among any of the products of these four genes.The TSP family consists of five members and can be divided into two groups. TSP1 and TSP2 are trimers with a chain molecular mass of 145 kDa, whereas TSPs 3 to 5 are pentamers with a subunit mass of Ϸ110 kDa. TSPs 3 to 5 differ from TSPs 1 and 2 in that they lack the procollagen domain and type I repeats of the trimeric proteins and contain four rather than three type II (epidermal growth factor-like) repeats (1, 6).The expression of TSP3 mRNA has been studied in the developing mouse (17, 25), chicken (28), Xenopus laevis (30), and adult human tissues (2). Expression was detected in the perichondrium and in the proliferating zone of the growth plate in long bones and vertebrae after day 15, in the dentate gyrus, accessory olfactory bulbs, hippocampus, and choroid plexus of the brain after day 17, and in the terminal bronchi and alveoli of the lung after day 17. Qabar et al. (26), using midline sagittal sections of whole embryos, reported expression in the forming musculature of the body wall and intestine, in the sternum and ve...

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Thrombospondin 3 Is a Pentameric Molecule Held Together by Interchain Disulfide Linkage Involving Two Cysteine Residues

Cited by 43 publications

References 24 publications

Disulfide Connectivity of Recombinant C-terminal Region of Human Thrombospondin 2

Disulfide Connectivity of Recombinant C-terminal Region of Human Thrombospondin 2

Metaxin Is a Component of a Preprotein Import Complex in the Outer Membrane of the Mammalian Mitochondrion

Mice with a Disruption of the Thrombospondin 3 Gene Differ in Geometric and Biomechanical Properties of Bone and Have Accelerated Development of the Femoral Head

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