Immunoreactivity of thymosin beta 4 in human foetal and adult genitourinary tract

Thymosins beta 4 (Tβ4) is a member of the beta-thymosins family, a family of peptides playing essential roles in many cellular functions. Our recent studies suggested Tβ4 plays a key role in the development of human salivary glands and the gastrointestinal tract. The aim of this study was to analyse the presence of Tβ4 in the human adult and foetal genitourinary tract. Immunolocalization of Tβ4 was studied in autoptic samples of kidney, bladder, uterus, ovary, testicle and prostate obtained from four human foetuses and four adults. Presence of the peptide was observed in cells of different origin: in surface epithelium, in gland epithelial cells and in the interstitial cells. Tβ4 was mainly found in adult and foetal bladder in the transitional epithelial cells; in the adult endometrium, glands and stromal cells were immunoreactive for the peptide; Tβ4 was mainly localized in the glands of foetal prostate while, in the adults a weak Tβ4 reactivity was restricted to the stroma. In adult and foetal kidney, Tβ4 reactivity was restricted to ducts and tubules with completely spared glomeruli; a weak positivity was observed in adult and foetal oocytes; immunoreactivity was mainly localized in the interstitial cells of foetal and adult testis. In this study, we confirm that Tβ4 could play a relevant role during human development, even in the genitourinary tract, and reveal that immunoreactivity for this peptide may change during postnatal and adult life.     

Biotechnological production of acetylated thymosin beta4

Thymosin beta4 (43 aa) is a highly conserved acidic peptide which regulates actin polymerization in mammalian cells by sequestering globular actin. Thymosin beta4 is undergoing clinical trials as a drug for the treatment of venous stasis ulcers, corneal wounds and injuries, as well as acute myocardial infarction. Currently, thymosin beta4 is produced with solid-phase chemical synthesis. Biotechnological synthesis of this peptide presents difficulties because N-terminal amino acid residue of thymosin beta4 is acetylated. In this study we propose a method for producing the recombinant precursor of thymosin beta4 and its subsequent targeted chemical acetylation. Desacetylthymosin beta4 was synthesized as a part of a hybrid protein with thioredoxin and a specific TEV (tobacco etch virus) protease cleavage site. The following scheme was developed for the purification of desacetylthymosin beta4: (i) the biosynthesis of a soluble hybrid protein (HP) in Escherichia coli; (ii) isolation of the HP by ion exchange chromatography; (iii) cleavage of the HP with TEVprotease; (iv) purification of desacetylthymosin beta4 by ultra-filtration. N-terminal acetylation of desacetylthymosin beta4 was performed with acetic anhydride under acidic conditions (pH 3). The reaction yield was 55%. Thymosin beta4 was then purified by reverse-phase high performance liquid chromatography. The proposed synthetic approach to recombinant thymosin beta4 is suitable for scale-up and can provide for the medical use of highly purified preparation with a yield of 20 mg from 1 L of culture.     

Differential splicing of thymosin beta 4 mRNA

A cDNA clone was isolated from a mouse pre-B cell line, the sequence of which has a very high homology with rat and human thymosin beta 4 genes. However, the mouse clone has an insertion of 98 bp relative to the published rat and human sequences upstream of the coding region. By isolation of a second set of clones from a different cDNA library and by cloning a PCR amplified region of mouse genomic DNA it was confirmed that the insertion is not a cloning artifact. Furthermore, it was shown by RNase protection assays with RNA from the pre-B cell line that two sizes of thymosin beta 4 mRNA exist, a long form containing the 98 nucleotide insertion, and a short form that corresponds to the known rat and human mRNA. The short form is about 50 times more abundant than the long form. Analysis of genomic DNA by sequencing and Southern blotting revealed that both forms are encoded by a single gene in the mouse. The two forms of mRNA arise by differential RNA splicing; the long mRNA contains three separate exons, whereas the short mRNA is missing exon 2. The long mRNA is present in two different pre-B cell lines, spleen and thymus, but could not be detected in brain, liver, and kidney. It is possible that the longer mRNA, which encodes a hydrophobic NH2-extension of six additional amino acids, plays a role in lymphocyte function or development. In contrast to the mouse which has a single thymosin beta 4 gene, rat and human have multiple homologs. Most or all of these also contain sequences that cross-hybridize with the newly discovered exon 2. A polymorphic thymosin beta 4 gene has been found in human DNA.     

Cell-cycle-regulated expression of thymosin beta 4 in thymocytes.

Thymosin beta 4 belongs to a family of ubiquitous peptides present at a high cellular content but still with an unknown intracellular function. The expression of this peptide was studied in concanavalin-A-stimulated, proliferating rat thymocytes during cell cycle progression. An early, transient 10-fold increase of the peptide occurred 1 h after stimulation without elevation of the corresponding mRNA level. This increase coincided with that of thymosin beta 4 biosynthesis. The sharp decline of the thymosin beta 4 content was not due to a secretion of the peptide into the medium. During S phase and mitosis, the biosynthetic rates as well as mRNA content, but not the cellular thymosin beta 4 concentration, increased again. After 96 h of culture the values returned to those of quiescent cells.     

Thymosin beta4 and thymosin beta4-derived peptides induce mast cell exocytosis

The peptide thymosin beta4 (Tbeta4) promotes angiogenesis and wound healing. Mast cells are involved in these processes as well and therefore we investigated the effect of Tbeta4 on mast cells. Exposure to 0.2-2000nM Tbeta4 induced mediator release (up to 23%) in murine peritoneal and human HMC-1 mast cells in a concentration-dependent manner. While the peptide AcSDKP, matching the 4 N-terminal amino acid residues of Tbeta4, mediated low but detectable mediator release, peptides corresponding to the Tbeta4 amino acid sequences 16-38 and 17-23 stimulated mast cells mediator release on a level equal to or higher than that observed with native Tbeta4. These observations and certain characteristics of Tbeta4-mediated mast cell activation suggest that the actin-binding motif LKKTET present in Tbeta4 (amino acid 17-22) might be implicated in this process. Thus, Tbeta4 activates mediator release in mast cells by a process that possibly involves an actin-binding motif and this could be important for understanding the mechanisms of Tbeta4-mediated effects in vivo.     

Simultaneous isolation and determination of prothymosin alpha, parathymosin alpha, thymosin beta 4, and thymosin beta 10

A method was described for the isolation of peptides from rat thymus. Frozen, powdered tissue was suspended in boiling buffer to inactivate endogenous proteinases, the suspension was homogenized, and the peptides were isolated by a two-step procedure including gel filtration and purification by HPLC. The recoveries from rat thymus were, in micrograms per gram of whole tissue, 60-80 for prothymosin alpha, 50-80 for thymosin beta 4, and 20-30 for thymosin beta 10. The procedure also yielded smaller quantities of a fourth peptide, designated parathymosin alpha. The quantities of these peptides in vertebrate tissues can be evaluated by applying radioimmunoassays for prothymosin alpha and thymosin beta 4 to the boiled tissue extract.     

Thymosin beta 4Xen: a new thymosin beta 4-like peptide in oocytes of Xenopus laevis

Two new thymosin beta 4-like peptides have been detected in ovaries of Xenopus laevis and Rana esculenta. Previously, it was reported that thymosin beta 4 can be found in various species, from mammals to amphibians, e.g., in X. laevis [S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B.L. Horecker (1983) Arch. Biochem. Biophys. 221, 570-576]. However, oocytes and spleen from R. esculenta contain no thymosin beta 4 but a similar peptide without methionine. The peptide from R. esculenta elutes from a reversed-phase column about 5 min later than thymosin beta 4. The peptide from X. laevis, referred to as thymosin beta 4Xen, can hardly be distinguished from thymosin beta 4 by its retention time on HPLC, by amino acid analysis, its isoelectric point, or tryptic fingerprinting. Amino acid analyses of the tryptic fragments, however, have revealed that thymosin beta 4 and beta 4Xen are different. The amino acid sequence of thymosin beta 4Xen is reported. Thymosin beta 4 and beta 4Xen differ in the amino acid residues at positions 15, 40, and 41. At position 15 serine is replaced by alanine and at 41-42 the sequence is Thr-Ser instead of Ala-Gly. Depending on their size, defolliculated oocytes contain between 2.7 and 52.6 ng thymosin beta 4Xen which is comparable to the amount of histones in oocytes.     

One-step procedure for the determination of thymosin beta 4 in small tissue samples and its separation from other thymosin beta 4-like peptides by high-pressure liquid chromatography

Thymosin beta 4 has been determined by a simple and fast one-step procedure in different tissues of rats. The tissues (1 to 40 mg) were disintegrated and deproteinized by homogenization in perchloric acid. After neutralization by potassium hydroxide the supernatant solution was used for determining thymosin beta 4 by reverse-phase HPLC without further manipulations. Not only does this procedure avoid artificial proteolysis as effectively as extraction of tissues by guanidinium chloride or boiling buffer, but it offers two further advantages. First, no additional steps--as for example desalting--are necessary prior to HPLC and thus the risk of losing thymosin beta 4 is eliminated. Using this procedure thymosin beta 4 is recovered quantitatively. The method is linear over the range 0.04 to 1.13 nmol and thymosin beta 4 is well separated from other thymosin beta 4-like peptides known to be present in mammals; i.e., thymosin beta Ala4, thymosin beta 9, thymosin beta 10, and thymosin beta Arg10. Second, the acid-insoluble pellet of the same extract can be used to determine the DNA content of the sample. Thus it is possible to relate thymosin beta 4 to DNA, which then allows comparing cells of different tissues and cell lines to one another. This procedure is also applicable to small peptides soluble in perchloric acid.     

Biosynthesis rates and content of thymosin beta 4 in cell lines

The content and relative biosynthetic rates of thymosin beta 4 have been determined in 28 different cell lines. The highest content of thymosin beta 4 as well as the highest rate of biosynthesis was observed in Epstein-Barr virus-transformed human B-cell lines. The levels observed in these cells are 1 pg thymosin beta 4 per cell, which is three times higher than that in rat peritoneal macrophages. Thus, these B-cell lines have the highest content of thymosin beta 4 of any cell type yet described. Since all of the Epstein-Barr virus-transformed B-cells described here grow in suspension, it is unlikely that the presence of thymosin beta 4 is related to anchorage in these cells. Thymosin beta 4 is not secreted by viable Epstein-Barr virus-transformed B cells in culture, suggesting some intracellular function of the peptide. These results indicate that these B-cell lines may be suitable for the study of thymosin beta 4 function.     

Functional and profiling studies prove that prostate cancer upregulated neuroblastoma thymosin beta is the true human homologue of rat thymosin beta15

A peptide with a sequence identical to rat thymosin beta(Tb)15 was reported to be upregulated in human prostate cancer. However, in this report we provide evidence that TbNB, initially identified in human neuroblastoma, is the only Tb isoform upregulated in human prostate cancer and that the Tb15 sequence is not present herein. In addition, we demonstrate that human TbNB has a higher affinity for actin in comparison to Tb4 and promotes cell migration. In combination, this experimentally validates TbNB as functional homologue of rat Tb15 in the human organism and clarifies the current composition of the human Tb family.     

Zyxin is upregulated in the nucleus by thymosin beta4 in SiHa cells

Thymosin beta4 is a 43-amino acid actin-binding protein that promotes cell migration and is important in angiogenesis, wound healing, and tumor metastasis. We searched for genes upregulated by thymosin beta4 and identified zyxin as increased in SiHa cells in the presence of exogenously added thymosin beta4 and when thymosin beta4 is overexpressed using adenoviral vectors. Both zyxin and thymosin beta4 show increased localization in the nucleus. We conclude that thymosin beta4 may exert some of its migration promoting activity via increased zyxin expression.     

Structure and immunological properties of thymosin beta 9 Met, a new analog of thymosin beta 4 isolated from porcine thymus

During the course for the studies of thymosin beta 4 and prothymosin alpha from porcine thymus, a new analog of thymosin beta 4 has been identified. This peptide consists of 41 amino acid residues. The amino terminus is blocked by an acetyl group as revealed by fast atom bombardment mass spectrometric analysis. Amino acid sequence studies disclosed that this peptide is identical to bovine thymosin beta 9 except that leucine at position 6 in beta 9 is substituted by methionine. Thus, this new peptide has been termed thymosin beta 9 Met. The recoveries of beta 9 Met, beta 4, and prothymosin alpha in porcine tissues have been determined (in micrograms/g tissue) as follows: thymus (43, 85, 133); spleen (68, 203, 37); liver (10, 31, 27); heart (1.5, 10, 0); kidney (5, 51, 37); brain (0.8, 31, 5). Biologically, thymosin beta 9 Met was found to be more active than beta 4 in enhancing gamma-interferon production in cord blood lymphocytes. However, beta 4 appeared to stimulate higher amounts of interleukin 2 and tumor necrotic factor. The significance for the coexistence of two homologous peptides with similar functions in the thymus and a number of other organs is not clear, and deserves further investigation.     

Recombinant thymosin beta 4 can promote full-thickness cutaneous wound healing

Human thymosin beta 4 (TB4) is a small acidic peptide involved in angiogenesis, wound healing, cancer metastasis and cardiac repair. Currently human TB4 is synthesized chemically for research and this is costly. In order to obtain sufficient biologically active human TB4 economically, we cloned and overexpressed this protein in an Escherichia coli system. We also developed a one-step affinity purification method to purify this fusion protein. After the fusion tag was removed from the fusion protein through autohydrolysis by dithiothreitol (DTT), the biological activity and function of this recombinant human TB4 was evaluated by cell proliferation assay using prepared spleen cells and wound assay using a mouse model, respectively. Our data demonstrated that human recombinant TB4 can promote lymphocyte proliferation and differentiation. Further, it can also promote full-thickness cutaneous wound healing in BALB/c mice. To our knowledge, this is the first report of recombinant human TB4 with the ability to promote wound healing.     

Structural requirements for thymosin beta4 in its contact with actin. An NMR-analysis of thymosin beta4 mutants in solution and correlation with their biological activity.

We examined the conformational preferences of mutants of thymosin beta4, an actin monomer sequestering protein by NMR spectroscopy in 60% (v/v) trifluoroethanol. Under these conditions, the wild-type thymosin beta4 conformation consists of an alpha-helix (helix I) extending from residues 5-16 with a more stable fragment from lysine 11 to lysine 16 and a second alpha-helix (helix II) encompassing residues 31-39. The point mutations studied here are located in helix I or in the LKKTET segment (residues 17-22) that form the two main entities of interaction with the actin molecule. The alpha-1H conformational shifts allow us to investigate the helicity of the polypeptides at the residue level and to correlate these structures with their biological activity. We determine that an extension of helix I at its C-terminal end over the LKK-segment results in loss of activity. The correct termination of this helix is connected to a specific orientation of the polypeptide essential for a cooperative action of the thymosin beta4 binding entities required for full activity.     

Sequence of a cloned 523-bp cDNA for thymosin beta 4

The sequence of a 523-bp cDNA, isolated from a clone bank prepared from partially purified rat spleen mRNA coding for thymosin beta 4, was described. The 3' sequence extended through the poly(A) segment and the 5' sequence included 36 bp preceding the translated sequence. The putative amino acid sequence coded by this segment possesses some of the features of a signal peptide.     

Widely distributed residues in thymosin beta4 are critical for actin binding

We have investigated the contributions of hydrophobic residues, the conserved and variable proline residues, and the conserved lysine residues to the affinity and kinetics of thymosin beta4 (Tbeta4) binding to MgATP-actin monomers. Pro4, Lys18, Lys19, Pro27, Leu28, Pro29, and Ile34 were substituted with alanine residues. Mutagenesis of Pro4 or Pro27 has little effect (or=10-fold, but the kinetic basis of the lower stability varies among the mutants. Substitution of the conserved lysine residues weakens the affinity by slowing association and accelerating dissociation. Substitution of hydrophobic residue Leu28 or Ile34 weakens the affinity by accelerating dissociation. These results favor a reaction mechanism in which Tbeta4 binds actin monomers following a two-step mechanism in which the formation of a bimolecular complex is followed by isomerization to a strong binding state that is coupled to the formation of widely distributed hydrophobic contacts. The isomerization equilibrium is slowed by mutagenesis of Pro29, as revealed by the double-exponential time course of association. Mutagenesis of Pro4 or Pro27 accelerates binding and dissociation but minimally affects the binding affinity (相似文献   

Rapid induction of thymosin beta 4 in concanavalin A-stimulated thymocytes by translational control

The expression of thymosin beta 4, an ubiquitous peptide of high cellular content, was studied in concanavalin A-stimulated rat thymocytes within the first 3 h after activation of the cells. An early 6.3-fold increase of the peptide occurred after 1 h of stimulation amounting to 0.4% of the total cellular protein. This increase coincided with that of thymosin beta 4 biosynthesis measured by [35S]methionine incorporation. The share of thymosin beta 4 synthesis in total protein synthesis 1 h after addition of concanavalin A amounts to 1% but no elevation of the corresponding mRNA was observed. These data suggest that a translational control mechanism is involved in this rapid induction. Consequently, actinomycin D did not inhibit thymosin beta 4 induction in contrast to cycloheximide. The peaks of maximal thymosin beta 4 levels and biosynthesis were followed by rapid decreases of these parameters suggesting a function of thymosin beta 4 in the early phase of T cell activation.