Isolation and characterization of thymosin beta 9 Met from pork spleen

We have identified a new thymosin beta 4-like peptide in pork spleen. The new peptide (12 mg) and thymosin beta 4 (33 mg) were isolated from 230 g of spleen by solid phase extraction, preparative isoelectric focusing, and HPLC. The new peptide was termed thymosin beta 9 Met to indicate its close relationship to thymosin beta 9 from calf. The only difference from thymosin beta 9 is the substitution of leucine by methionine at position 6. This peptide replaces thymosin beta 10 which is the minor thymosin beta 4-like peptide in most mammals, e.g., in man, rat, mouse, cat, and rabbit. The structure was determined by amino acid analysis, tryptic digestion, and carboxypeptidase digestion. Pork spleen contains 192 micrograms of thymosin beta 4 and 117 micrograms of thymosin beta 9 Met per gram of tissue.     

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.     

Molecular cloning of the cDNA for rat spleen thymosin beta 10 and the deduced amino acid sequence

A rat spleen cDNA library was prepared and employed for the molecular cloning of the cDNA for thymosin beta 10, a peptide that previously had been found to accompany the closely related peptide, thymosin beta 4, in several species of mammals (S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B. L. Horecker (1983) Arch. Biochem. Biophys. 225, 407-413). First-round screening with a synthetic oligodeoxynucleotide probe yielded 55 positive clones, and sequence analysis of 11 of these clones revealed that they all coded for a peptide containing the thymosin beta 10 sequence, except for an additional arginyl residue at position 39. This peptide, designated thymosin beta 10arg, had been identified previously in rabbit tissues and reported as a variant of thymosin beta 10 (S. Ruggieri, S. Erickson-Viitanen, and B.L. Horecker (1983) Arch. Biochem. Biophys. 226, 388-392). Analysis of the 55 positive clones using a specific oligodeoxynucleotide probe constructed to correspond to the mRNA sequence, including the codon for Arg39, confirmed that they all coded for the amino acid sequence including Arg39. Based on these results, the existence of a molecular species lacking Arg39 is considered unlikely, and we conclude that thymosin beta 10 contains 43, rather than 42, amino acid residues, with identity to thymosin beta 4 in 32 of the 43 residues. We propose that the name thymosin beta 10 be used to refer to the peptide containing Arg39 and that the designation thymosin beta 10arg be dropped. In the cDNA sequence the codons for Ala1 and Ser43 of thymosin beta 10 are flanked by initiator and terminator codons, respectively; thus, both the thymosin beta 4 and thymosin beta 10, which coexist in mammalian cells and tissues, are synthesized without the formation of larger polypeptide precursors.     

Solid-phase synthesis of thymosin beta 4: chemical and biological characterization of the synthetic peptide

The chemical synthesis of thymosin beta 4 using a solid-phase procedure has been accomplished. The synthetic product was found to be homogeneous on paper electrophoresis at pH 6.5, high-performance liquid chromatography on a reversed-phase column, and isoelectric focusing using polyacrylamide gels. The synthetic material was also shown to be identical with the natural thymosin beta 4 by tryptic peptide mapping, amino acid compositional analyses, and polyacrylamide gel isoelectric focusing. Biologically, synthetic thymosin beta 4 was found to be as active as the natural compound in a terminal deoxynucleotidyltransferase induction assay and in a macrophage migration inhibition assay. The proposed structure of the peptide hormone was thus confirmed by a chemical synthesis.     

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.     

Thymosin beta 4 and Fx, an actin-sequestering peptide, are indistinguishable

At least 50% of the actin in resting human platelets is unpolymerized, and the bulk of this unpolymerized actin is complexed with a recently identified acidic, heat-stable 5-kDa peptide, named "Fx." Purified Fx binds stoichiometrically to muscle G-actin, forming a complex identifiable by nondenaturing polyacrylamide gel electrophoresis. Formation of the complex inhibits salt-induced polymerization of G-actin. Here we report that Fx is indistinguishable from thymosin beta 4, an acidic, heat-stable 5-kDa peptide first isolated from calf thymus and thought to be a thymic hormone. The complete amino acid sequence of Fx was determined and was found to be identical with that of thymosin beta 4. Authentic thymosin beta 4 is functionally equivalent to Fx, forming a 1:1 complex with actin monomers and inhibiting polymerization. The widespread distribution and high intracellular concentration of thymosin beta 4 (Fx) strongly suggest that it plays a significant role in regulating actin polymerization in many cell types.     

Thymosin beta arg10, a major variant of thymosin beta 10 in rabbit tissues

Two homologous peptides, designated thymosin beta 4 and thymosin beta 10, respectively, have been shown to be widely distributed in mammalian cells and tissues (S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B.L. Horecker (1983) Arch. Biochem. Biophys. 221, 570-576; S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B.L. Horecker, (1983) Arch. Biochem. Biophys. 225, 407-413). In the rabbit, thymosin beta 4 is replaced by a variant, thymosin beta ala4, that contains alanine in place of serine at the blocked NH2-terminus. It is reported that in rabbit tissues thymosin beta 10 is also replaced by a variant, designated thymosin beta arg10, that contains an additional amino acid, arginine, inserted following lysine-38. The rabbit tissues analyzed also differ from those of other mammals in the relative quantities of thymosin beta ala4 and beta arg10, which are nearly equal, compared to tissues from other mammals where the quantities of thymosin beta 10 are only one-third to one-tenth those of thymosin beta 4.     

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.     

Xenopus laevis integrins. Structural conservation and evolutionary divergence of integrin beta subunits

We report the sequences of cDNA clones for two different integrin beta subunits isolated from a Xenopus laevis neurula cDNA library. mRNAs corresponding to both genes are first detected at gastrulation. We show that these two beta subunits are very highly related (98% identity in amino acid sequence) and probably arose at the time of tetraploidization of the X. laevis genome around 50 million years ago. Comparison of these sequences with those of various other vertebrate integrin beta subunit establishes that all species analyzed to date contain a highly conserved integrin beta subunit (beta 1). The interspecies homologies within this class of integrin beta subunits (82-86% identity in amino acid sequence) are much greater than those among the three different beta subunits which are known in humans (40-48% identity in amino acid sequence). Analysis of the homologies clearly indicates duplication and divergence of this multigene family more than 500 million years ago prior to the appearance of the vertebrates. We also observe cross-hybridization between cDNA probes for chicken integrin beta subunits and genomic DNAs of several invertebrate species. Despite the divergence in sequence among different integrin beta subunits, certain features of their structure are remarkably conserved.     

Primary structure of thymosin beta 12, a new member of the beta-thymosin family isolated from perch liver.

A new polypeptide termed thymosin beta 12 has been isolated from perch liver and its primary structure elucidated. This polypeptide contains 43 amino acid residues with a molecular weight of 4822 Da. The content of thymosin beta 12 from perch liver has been determined as 43 micrograms/g of tissue. The amino-terminal end of this polypeptide is blocked by an acetyl group as deciphered by fast-atom bombardment mass spectrometric analysis. Sequence analysis reveals that thymosin beta 12 is 79% homologous to thymosin beta 4, an immunomodulator which was originally isolated from calf thymus. Thymosin beta 12 also shows 84% sequence homology to thymosin beta 11, a beta 4 analog which replaces beta 4 in two species of bony fish, oscar and rainbow trout. The evolutionary implication of such results will be discussed. The isolation of a new beta 4-related peptide from perch liver which differs from beta 11 indicates that beta-thymosin peptides are widely distributed in lower vertebrate classes.     

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.     

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.     

Solid phase synthesis of thymosin beta 9

Thymosin beta 9, a 41 residue thymic polypeptide, has been synthesized by a solid phase method. A modification of the low HF method was used to deprotect and cleave the peptide from the resin. Thymosin beta 9 was then obtained in analytically pure form by a one-step purification procedure in 32% yield. The activity of thymosin beta 9 in the terminal deoxynucleotidyl transferase assay was greater than calf thymus fraction 5, but comparable to thymosin beta 4. In contrast to thymosin alpha 1, neither beta 4 nor beta 9 was active in the rosette inhibition assay.     

Investigation of the epitopic structure of thymosin beta10 by epitope mapping experiments.

We present here a study on the epitopic structure and the immunochemical characteristics of thymosin beta10 (Tbeta10), a 43 aminoacid peptide involved in important cellular mechanisms, by using the epitope mapping Multipin method. Octapeptides overlapping by one amino acid so as to represent the whole sequence of Tbeta10 were synthesized on polystyrene pins and screened, using an ELISA method, with a polyclonal antiserum raised against intact recombinant Tbeta10. The octapeptides were also tested with anti-peptide oligoclonal antisera raised against the synthetic fragments Tbeta10[1-16] and Tbeta10[31-43], with polyclonal antisera raised against natural thymosin gamma4 (Tbeta4) or thymosin beta9 (Tbeta9), and with anti-peptide oligoclonal antisera raised against various fragments of Tbeta4 (i.e. Tbeta4[1-11], Tbeta4[30-43] and Tbeta4[16-38]). Four distinct epitopic fragments were revealed, namely the sequences 1-13, 19-30, 29-40 and 36-43. Among them, the sequence 36-43 appears to offer unique immunochemical characteristics to the Tbeta10 molecule.     

Coupling of folding and binding of thymosin beta4 upon interaction with monomeric actin monitored by nuclear magnetic resonance

Thymosin beta4 is a major actin-sequestering protein, yet the structural basis for its biological function is still unknown. This study provides insight regarding the way this 43-amino acid peptide, mostly unstructured in solution, binds to monomeric actin and prevents its assembly in filaments. We show here that the whole backbone of thymosin beta4 is highly affected upon binding to G-actin. The assignment of all amide protons and nitrogens of thymosin in the bound state, obtained using a combination of NMR experiments and selective labelings, shows that thymosin folds completely upon binding and displays a central extended region flanked by two N- and C-terminal helices. The cleavage of actin by subtilisin in the DNase I binding loop does not modify the structure of thymosin beta4 in the complex, showing that the backbone of the peptide is not in close proximity to segment 42-47 of actin. The combination of our NMR results and previously published mutation and cross-link data allows a better characterization of the binding mode of thymosins on G-actin.     

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.     

The early induction of the actin-sequestering peptide thymosin beta 4 in thymocytes depends on the proliferative stimulus

The expression of the actin-sequestering peptide, thymosin beta 4, was analyzed in proliferating rat thymocytes, activated by diverse stimuli, during the early G1 phase and the S phase. In the presence of concanavalin A a 6.3-fold increase of thymosin beta 4 occurred already after 1 h of stimulation without elevation of the corresponding mRNA level. In contrast, during the S phase the increase of thymosin beta 4 (2.5-fold) was accompanied by a higher mRNA level, but did not exceed the growth related increase of total protein. Stimulation with a crosslinked antibody against rat T cell antigen receptor or stimulation with phorbol 12-myristate 13-acetate (PMA) and Ca(2+)-ionophore A23187, separately or in combination, did not lead to the marked increase of the thymosin beta 4 concentration in the early G1 phase but resulted in elevated thymosin beta 4 peptide and mRNA levels during the S phase. It therefore appears that protein kinase C activation and a rise in cytoplasmic Ca(2+)-concentration are not exclusively responsible for the stimulation of thymosin beta 4 specific translation in thymocytes. This assumption was reinforced by the observation that inhibition of the protein kinase C activity by 1-(5-isoquinolinylsulfony)-2-methylpiperazine (H-7) did not affect the cellular thymosin beta 4 content 1 h and 48 h after concanavalin A (Con A) stimulation.     

Synthesis of a new biological response modifier thyrnosin beta(4) analogue and its restorative effect on depressed

Thymosin beta(4) is a polypeptide isolated from thymosin fraction 5. This peptide exhibits important activities in the regulation and differentiation of thymus-dependent lymphocytes. An analogue of thymosin beta(4), [Phe(4F)(12)] deacetyl- thymosin beta(4), was synthesized by a solution method, followed by deprotection with 1 M trifluoromethanesulphonic acid (TFMSA)-thioanisole (molar ratio, 1:1) in trifluoroacetic acid (TFA) in the presence of dimethlselenium. Finally, the deprotected peptide was incubated with dithiothreitol to reduce sulphoxide on the methionine side chain. The synthetic [Phe(4F)(12)]deacetyl-thymosin beta(4) was found to have a restoring effect on the impaired blastogenic response of T-lymphocytes isolated from uraemic patients with recurrent infectious diseases. This analogue exhibited stronger restorative activity than that of our synthetic deacetyl-thymosin beta(4).