Thymosins and Fragments Products

Definition
Thymosins are small peptides, originally identified from the thymus, but now known to be more widely distributed in many tissues and cells ¹.

Related Peptides
Thymosins are divided into three main groups, alpha-, beta- and gamma-thymosins, based on their isoelectric points. Alpha-thymosins have nuclear localization and are involved in transcription and/or DNA replications; where as beta-thymosins have cytoplasmic localization and show high affinity to G-actin for cell mobility ¹.

Discovery
From a historical perspective, the studies that led to the isolation and characterization of the thymosins began in earnest in the early 1960s in the laboratory of Abraham White at the Albert Einstein College of Medicine in New York. In a 1966, Goldstein et al., first named these thymic-derived factors "Thymosins." By 1972, the thymosin team had moved to the University of Texas Medical Branch in Galveston (UTMB) where a group of scientists and students succeeded over the next 6 years in preparing and testing a highly active partially purified calf thymus preparation, termed thymosin fraction-5 (TF5), which was amenable for scale-up and suitable for clinical use. The immunorestorative and potentially life-saving properties of TF5 in clinical medicine were first documented in a landmark paper in 1975 by Drs. Arthur Ammann and Diane Wara in the New England Journal of Medicine. TF5 consists of a family of at least 40 mostly small acidic polypeptides, with molecular weights ranging from 1000 to 15 KDa ².

Structural Characteristics
Peptide hormones from the thymus gland are considered to play a role in the maturation and differentiation of T cells. A heat-stable fraction prepared from calf thymus extracts was designated thymosin fraction 5. TF-5 was found to contain a number of peptides, and several of them have been isolated in pure form and subjected to sequence determination. These include thymosin α_1, an acidic peptide (pI = 4.2) containing 28 amino acid residues, and thymosin β₄ (pI = 5.1) containing 44 amino acid residues. Each is modified by acetylation at the NH₂ terminus. Bovine fraction 5 has also been shown to contain significant quantities of ubiquitin, but this peptide is not considered to contribute to the biological activity of the preparation. However, ubiquitin may function as a modulator of the activities of other thymic peptides. The α-thymosins are a group of acidic peptides (pI 3.5–4.5) present in a calf thymus extract denominated TF-5, which shows immunoregulatory properties in several in vitro and in vivo assay systems. Thymosin α₁(Tα₁; 28 amino acids) is the most abundant α-thymosin in TF5 and was the first to be isolated and sequenced. A less abundant TF5 component named thymosin α₁₁ (Tα₁₁) has subsequently been characterized this peptide comprises the Tα₁ sequence plus an additional 7 residues at its C terminus (i.e. 35 residues in total) ³.

Two peptides, designated thymosin beta 8 and thymosin beta 9, respectively, have been isolated and their amino acid sequences established. Thymosin beta 8, isolated from calf thymus fraction 5, has a mass of 4.5 kDa and contains 39 amino acid residues, of which 31 are identical to the corresponding amino acid residues in thymosin beta 4 isolated from the same source. The NH₂ terminus of thymosin beta 8 is acetylalanine, compared with acetylserine in thymosin beta 4. Thymosin beta 9, isolated from fresh-frozen calf thymus by a procedure that minimizes proteolysis, is identical to thymosin beta 8 except for the presence of an additional dipeptide, -Ala-LysOH, at the COOH terminus. It has a mass of 4.7 kDa and 32 of its 41 amino acids are identical to those of thymosin beta 4. The similarity in structures of thymosin beta 4 and thymosin beta 9 suggests that they may have related functions ⁴.

Thymosin Fragments:
Thymosin α₁(Tα₁) and thymosin Tα₁₁(Tα₁₁) are polypeptides with immunoregulatory properties first isolated from thymic extracts, corresponding to the first 28 and 35 amino acid residues, respectively, of prothymosin α (ProTα), a protein involved in chromatin remodeling. Both Tα₁ and Tα₁₁ show immunoregulatory properties similar to those of TF5 ³.

Six common amino acid sequence fragments of thymosins β4,β8 and β9,H-Lys-Leu-Lys-Lys-Thr-Glu-Thr-Gln-Glu-Lys-Asn-OH (positions 16-26), H-Lys-Glu-Thr-Ile-Glu-Gln-Glu-Lys-Gln-OH (positions 31-39), H-Asp-Lys-Pro-Asp-OH (positions 2-5), H-Phe-Asp-Lys-OH (positions 12-14), H-Leu-Pro-OH (positions 28-29) and H-Glu-Ile-OH (positions 8-9), were synthesized by the solution method and were tested to determine their effects on the low E-rosette forming cells of lupus nephritis patients. Two of the fragments, H-Lys-Leu-Lys-Lys-Thr-Glu-Thr-Gln-Glu-Lys-Asn-OH and H-Lys-Glu-Thr-Ile-Glu-Gln-Glu-Lys-Gln-OH, increased E-rosette forming capacity when incubated in vitro with patient's blood though they were less effective than thymosin β9,but the other four peptide fragments, H-Asp-Lys-Pro-Asp-OH, H-Phe-Asp-Lys-OH, H-Leu-Pro-OH and H-Glu-Ile-OH, had no effect ⁵.

Mode of Action
Peptides of fraction V of thymosin induce expression of T cell surface antigens (Thy, T1, Ly) 4-6 and differentiation of precursor cells into T₁, and then T₂ immunologically competent cells. Data from several laboratories indicate that interaction of a target cell population with thymic peptides is mediated by cyclic AMP ⁶. Tα₁ induces functional maturation and interleukin-12 production by fungus-pulsed DCs through the p38 mitogen-activated protein kinase/nuclear factor (NF)–κB-dependent pathway. This occurs by signaling through the myeloid differentiation pathway, involving distinct Toll-like receptors ⁷.

Functions
Both alpha- and beta-thymosins play important roles in modulating immune response, vascular biology, and cancer pathogenesis. More importantly, thymosins may have significant clinical applications. They may serve as molecular markers for the diagnosis and prognosis of certain diseases. In addition, they could be molecular targets of certain diseases or be used as therapeutic agents to treat certain diseases ¹.

References

1. Chen C, Li M, Yang H, Chai H, Fisher W, Yao Q (2005). Roles of thymosins in cancers and other organ systems. World J Surg., 29(3):264-270.
2. Goldstein AL (2007). History of the discovery of the thymosins. Ann N Y Acad Sci., 1112:1-13.
3. Sarandeses CS, Covelo G, Díaz-Jullien C, Freire M (2003). Prothymosin α Is Processed to Thymosin α₁ and Thymosin α₁₁ by a Lysosomal Asparaginyl Endopeptidase. Journal of Biological Chemistry, 278(15):13286-13293.
4. E Hannappel, S Davoust, and B L Horecker. 1982. Thymosins beta 8 and beta 9: two new peptides isolated from calf thymus homologous to thymosin beta 4.  PNAS.,79(6): 1708-1711.
5. Abiko T, Sekino H (1984). Synthesis of six common amino acid sequence fragments of thymosins beta 4, beta 8 and beta 9 and determination of their effects on the low E-rosette forming cells of lupus nephritis patients. Chem Pharm Bull.,  32(1):228-236.
6. Brelinska R, Warchoł JB (1982). Receptors for thymosin fraction V on rat thymic lymphocytes. Experientia., 38(5)618-619.
7. Romani L, Bistoni F, Gaziano R, Bozza S, Montagnoli C, Perruccio K, Pitzurra L, Bellocchio S, Velardi A, Rasi G, Di Francesco P, Garaci E (2004). Thymosin α1 activates dendritic cells for antifungal Th1 resistance through Toll-like receptor signaling. Blood, 103(11):4232-4239.