Thymosin alpha 11: a peptide related to thymosin alpha 1 isolated from calf thymosin fraction 5.

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192

158

A polypeptide containing -112 amino acid residues, with the thymosin a, sequence at its NH2 terminus, has been isolated from rat thymus by using a radioimmunoassay with an antibody prepared against synthetic thymosin ap. The new polypeptide, named "prothymosin a," was found to be the major substance crossreacting with thymosin a, antiserum in rat thymus extracts; peptides corresponding to thymosin a, or thymosin a,, were not detected. In gel filtration at pH 2.8, prothymosin a emerged as a single symmetrical peak corresponding to an apparent molecular weight of 32,000, approximately 3 times larger than the minimum molecular weight calculated from its amino acid composition. Thymosin a,, a peptide containing 28 amino acid residues, was isolated by Goldstein and coworkers (1) from thymosin fraction 5, a mixture of peptides from calf thymus (2). Thymosin fraction 5 had been reported earlier to restore parameters of immunocompetence in neonatally thymectomized mice (3). Thymosin a1 was found to be active in some of the in vitro tests used for thymosin fraction 5 (4), and it was considered to be one of the factors that modulated steps in the maturation of T cells (5).We have reported (6) our inability to detect thymosin a1 in guanidinium chloride extracts of calf thymus. The suggestion that thymosin a1 might represent a proteolytic fragment of a larger native polypeptide was supported by the finding that preparations of calf thymosin fraction 5 contained at least two other related peptides (7). One of these, designated des-(25-28)-thymosin a1, contained only the first 24 amino acid residues; the other, named thymosin all, contained the sequence of thymosin a1 plus seven additional residues at the COOH terminus.In an effort to isolate the native thymic polypeptide from which these fragments appeared to be derived, we developed a radioimmunoassay based on an antibody prepared against synthetic thymosin a1. With this assay and a procedure designed to eliminate any possibility of proteolytic modification, we isolated a major polypeptide, =112 amino acid residues long, that contains the thymosin a1 sequence at its NH2 terminus. We named this polypeptide prothymosin a because it appears to be the source of the thymosin a1-related peptide fragments found in preparations of thymosin fraction 5. MATERIALS AND METHODSRat thymuses from male Charles River CD rats, 5 weeks old, were excised immediately after sacrifice of the animals by decapitation, quickly frozen in liquid nitrogen, and stored at -700C. Synthetic thymosin a, (8) was provided by A. Felix of Hoffmann-La Roche. Trypsin (L-1-tosylamido-2-phenylmethyl chloromethyl ketone-treated) and Staphylococcus aureus V8 protease were from Worthington and Miles Laboratories respectively. Fluorescamine was a gift of W. E. Scott of Hoffmann-La Roche. Sephacryl S-200 (superfine) was purchased from Pharmacia. Other reagents and solvents were chromatography-grade commercial preparations; the solvents were redistilled as required.For preparation of the antibody, rabbits were in...

mentioning

confidence: 60%

“…The formation of these fragments may be related to its biological activity. Both thymosin a1 (7,18,19) and thymosin all (7) have been shown to protect sensitive strains of mice against opportunistic infections. Preliminary observations (unpublished data) indicate that prothymosin a is also active in these tests.…”

Section: Discussionmentioning

confidence: 99%

Prothymosin alpha: isolation and properties of the major immunoreactive form of thymosin alpha 1 in rat thymus.

Haritos¹,

Goodall²,

Horecker³

1984

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192

158

“…The library contained 2000 clones that hybridized. Of these, 2 clones, each about 900 bp in size, were chosen for further study because (i) when cleaved with Dde I they were found to contain a fragment apparently identical to the primer, a fact suggesting that the partial Dde I site at the 3' end of the antisense strand of the primer had been regenerated, and (it) when separated from the primer by treatment with Dde I and purified electrophoretically, the unique sequences generated by primer extension hybridized to an mRNA composed of =1300 nucleotides, the correct size for y-interferon mRNA. These characteristics were consistent with homology to y-interferon.…”

Section: Resultsmentioning

confidence: 99%

“…peptides: thymosin a1, thymosin a1l, and des-(25-28)-thymosin a1 (1,2). These hormones or hormone-like materials are derived in the rat from a polypeptide precursor containing 113 amino acids known as prothymosin a (3).…”

mentioning

confidence: 99%

The human prothymosin alpha gene is polymorphic and induced upon growth stimulation: evidence using a cloned cDNA.

Eschenfeldt¹,

Berger²

1986

184

Clones for human prothymosin a have been identified in cDNA libraries from staphylococcal enterotoxin A-stimulated normal human lymphocytes and from simian virus 40-transformed fibroblasts. The 1198-base-pair fibroblast clone has been sequenced. The encoded protein is highly acidic (54 residues out of 111) and shares >90% sequence homology with rat prothymosin a. The peptide "hormone" thymosin al appears at positions 2-29 of the prothymosin a amino acid sequence. There is no N-terminal signal peptide.

“…Both thymosin fraction 5 and thymosin a, protected immunodeficient mice against challenge with Candida albicans (13)(14)(15) and other opportunistic microorganisms (14). Two peptides closely related to thymosin a1 have recently been isolated from preparations of calf thymosin fraction 5 (16). They were found to be identical to a1 in amino acid sequence, except that one, des-(25-28)-thymosin a1, was four amino acid residues shorter and the other, thymosin a1l, contained seven additional amino acids at the COOH terminus.…”

mentioning

confidence: 99%

Primary structure of rat thymus prothymosin alpha.

Haritos¹,

Blacher²,

Stein³

et al. 1985

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The primary structure of prothymosin a from rat thymus, containing 113 amino acid residues, is reported as follows: AcSer-Asp-Ala-Ala-Val-Asp-Thr-Ser- Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys- Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Gly-Arg-Asp-Ala- 40 Pro-Ala-Asn-Gly-Asn-Ala-Gln-Asn-Glu-Glu- Gly-Glu-Gln-Glu-Ala-Asp- Glu-Glu-Glu-Glu-Gly-Gly-Gly-Glu-Glu (Asx,Gly,Gly, 70 Glx,Glx,Glx,Glx,Glx,Glx,Glx,Glx,Glx,Glx)Asn-Gly- 80Asp-Glu-Asp-Glu -Glu -Ala -Gl u -Ala-Pro -Th r-Gly -90 100Lys-Arg-Val-Ala-Glu-Asp-Asp-Glu-Asp-Asp-Asp- 110Val-Glu-Thr-Lys-Lys-Gln-Lys-Lys-Thr-Asp-GluAsp-AspOH. The sequence of the first 28 amino acids at the NH2 terminus is identical to that of calf thymosin al. The dicarboxylic amino acids, which account for nearly half of the total residues in prothymosin a, are largely clustered in the central portion of the polypeptide chain. The polypeptide contains no aromatic or sulfur-containing amino acids. A computer analysis of the three-dimensional structure based on the primary sequence suggests that the molecule is composed of at least five a-helical regions interrupted by one short extended chain and three short random coils.A mixture of peptides from calf thymus identified as thymosin fraction 5 (1-3) has been reported to restore immune function in thymectomized mice (1) and to yield positive results in other in vivo and in vitro assays for the induction and maintenance of immune function (ref. 4; for reviews of the earlier literature, see refs. 3, 5, and 6). Thymosin fraction 5 also has shown promise in clinical trials with children with immunodeficiency diseases (7) and with cancer patients (8).Thymosin fraction 5 was shown to be a mixture of peptides ranging in molecular weight from 1000 to 15,000 and in isoelectric point from 4.0 to 7.0 (9). The first component of thymosin fraction 5 to be isolated in pure form and characterized was thymosin a,, an acidic peptide containing 28 amino acid residues (9). This peptide was reported to show many of the biological activities of thymosin fraction 5 (refs. 9-12 (pH 5.5). To this buffer, 10-,/g aliquots of carboxypeptidase Y were added at 0, 4, and 8 hr. At timed intervals 5-10% of the total volume was removed, diluted to 100 ,Al with ice-cold buffer of 67 mM sodium citrate (pH 2.0),