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Prothymosin α and other thymic peptides related to thymosin α1
superoxide anion release from phagocytes by muramyl dipeptide or lipopolysaccharide. Infect. lmmun, 39:559-564. 9. hlurray, II. %%'.(1982). Cell-mediated immune response in experimental visceral leishmaniasis. I1. Oxygcn-dependent killing of intracellular Leishmania donovani amastigotes. J. lmmunol. 129:351-357. 10. Murray, ti. W., and D. hi. Cartelli. (1983). Killing of intracellular Leishmania donovani by human mononuclear phagocytes. Evidence for oxygen-dependent and independent teishmanicidal activity. J. Clin. Invest. 72:32-44. I 1. Murray, It. W., and Z. A. Cohn. (1980). Macrophage oxygen-dependent antimicrobial activity. I11. Enhanced oxidative metabolism as expression of macrophage activation. J. Exp. Med. 152:1596-1609. 12. Murray, H. W., C. F. Nathan, and Z. A. Cohn. (1980). Macrophage oxygen-dependent antimicrobial activity. IV. Role of endogenous scavengers and oxygen intermediates. J. Exp. Med. 152:1610-1624. 1"3. Murray, H. W., B. Y. Rubin, and C. D. Rothermel. (1983). Killing of , intracellular Leishmania donovani by lymphokine-stimulated human mononuclear phagocytes. Evidence that intcrfcron-~, is the activating [ymphokine. J. Clin. Invest. 72:15061510. 14. Nakagawara, A., et al. (1982). Lymphokines enhance the capacity of human monoeytes to secrete reactive oxygen intermediates. J. Clin. Invest. 70:1042-1048. 15. Nakagawara, A., C. F. Nathan, and
Prothymosin and Other Thymic Peptides Related to Thymosin czI B. L. Horecker, Ph.D. Roche Institute of Molecular Biology Roche Research Center Nutley, New Jersey A. A. Haritos, Ph.D. University of Athens Athens, Greece
The role of the thymus in the development and maintenance of cell-
Clinical ImmunologyNewsletter6:10.1985
A. Z. Cohn. (1981). Hydrogen peroxide metabolism in human monocytcs during differentiation in vitro. J. Clin. Invest. 68:1243-1252. 16. Nathan, C. F., et al. (1979). Activation of macrophages in vivo and in vitro. Correlation between hydrogen peroxide release and killing of trypanosoma cruzi. J. Exp. Med. 149:1056-1068. 17. Nathan, C. F., el al. (1983). Identification of interferon-~/as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J. Exp. Med. 158:670-689. 18. Nathan, C. F., el al. (1984). Activation of human macrophages. Comparison of other cytokines with interferon% J. Exp. Med. 160:600-605. 19. Pick, E., and Y. Bromberg. (1982). Quo vadis macrophage activation-Role of phospholipids in the elicitation of the oxidative burst in macrophages. Transpl. Proc. 14:570-575. 20. Pick, E., and Y. Bromberg. (1983). Regulation of maerophage function by lymphokines--Role of membrane phospholipids, pp. 243-249. hi J. W. Hadden et al. (eds.), Advances in immunopharmacology, vol. 2. Pergamon Press, Oxford. 21. Pick, E., Y. Bromberg, and M. Frcund. (1982). Extrinsic regulation of macrophage function by lymphokines--effect of lymphokines on the stimulated oxidative metabolism of macrophages. Adv. Exp. Med. Biol. 155:471-485. 22. Pick, E., and M. Freund. (1983). Biochemical mechanisms in macrophage activation by lymphokines: intracellular peroxide production by lyre-
mediated immunity is well established. There is, however, lack of agreement as to whether the effects of the thymus on undifferentiated lymphocytes require residence o f these lymphocytes in the thymus or are, at least in part, mediated by thymic hormones released into the circulation. Evidence to support both points of view has been reviewed (4, 15). A number of thymic peptides have been isolated and chemically characterized (for a review see ref. 16). Prominent among these is an acidic peptide containing 28 amino acid residues, named thymosin a I, isolated from a
© 1985ElsevierSciencePublishingCo.. Inc.
23.
24.
25.
26.
27.
28.
phokine-trcated macrophages, pp. 295-306. In Y. Yamamura and T. Tada (eds.), Progress in immunology, voI. V, Academic Press, New York. Pick, E., and Y. Keisari. (1980). A simple colorimetric method for the measurement of hydrogen peroxide produced by cells in culture. J. lmmunol. Meth. 38:161-170. Pick, E., and Y. Keisari. (1981). Superoxide and hydrogen peroxide production by chemically elicited peritoneal macrophages--induction by multiple nonphagocytic stimuli. Cell. Immunol. 59:30t-318. Sasada, M., hi. J. Pabst, and R. B. Johnston. (1983). Activation of mouse peritoneal macrophages by lipopolysaccharide alters the kinetic parameters of the superoxide-producing NADPH oxidase. J. Biol. Chem. 258:96319635. Tsunawaki, S., and C. F. Nathan. (1984). Enzymatic basis of macrophage activation. Kinetic analysis of superoxide production in lysates of resident and activated mouse peritoneal macrophages and granulocytes. J. Biol. Chem. 259:4305-4312. Weinberg, J. B., and M. A. Misukonis. (1983). Phorbol diester-induced H202 production by peritoneal macrophages. Different H202 production by macrophages from normal and BCGinfected mice despite comparable phorbol diester receptors. Cell. lmmunol. 80:405-415. Zwickel, J., S. Shpungin, and E. Pick. (1985). Arachidonic acid metabolism of lymphokine-activated macrophages. In C. Sorg and A. Schimpl (eds.), Proceedings of the 4th International Lymphokine Workshop. Academic Press, Orlando, FL, in press.
biologically active preparation from calf thymus called thymosin fraction 5. Thymosin a t has shown promise as an immune enhancer (5, 12). However, more recent studies from our laboratory indicate that calf thymosin fraction 5 contains at least two additional peptides structurally related to thymosin cq (1) and that these peptides may all represent fragments of a larger native polypeptide named prothymosin ¢x, which we have isolated from extracts of fresh rat thymus (7). A second peptide, similar in size and amino acid composition to prothymosin oL, was recovered from the same
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rat thymus extracts and named parathymosin a (10). The biochemical and immunological properties of these peptides are reviewed here. T h y m o s i n oh Sequence analysis of thymosin aj (5) showed it to contain 28 amino acid residues, with an acetylated NH2-terminus, for a calculated molecular weight of 3108 (Fig. 1). In several in vitro assays designed to measure the maturation and functional differentiation of T-lymphocytes, thymosin a I was reported to be 10-1000 times more active than thymosin fraction 5 (5, 12). T h y m o s i n oql a n d des-(25-28)t h y m o s i n cxl Two additional peptides closely related to thymosin cq have been isolated from preparations of calf thymus fraction 5 (1). One, designated des(25-28)-thymosin ¢xI, was identical to thym0sin al except that it lacked the last four amino acid residues at the COOH-terminus (Fig. 1). The other, named thymosin all, was found to contain seven additional residues (Fig. I). In the mouse-protection test employed by Salvin and his co-workers (13) thymosin all was as effective as thymosin a I (1). P r o t h y m o s i n ot The presence in calf thymosin fraction 5 of peptides having a common structure and differing only in length at the COOH-terminus suggested that they might all be proteolytic artifacts generated during the procedure employed for the preparation of the starting material, thymosin fraction 5.
Of particular significance was the fact that thymosin eq was not detected when proteolysis was avoided by extracting fresh thymus with guanidinium chloride (6). To isolate the native polypeptide containing the thymosin a I sequence, we employed a radioimmunoassay (RIA) based on an antibody raised against synthetic thymosin a I coupled to keyhole limpet hemocyanin (I 1). To avoid proteolysis during the extraction and isolation, the frozen tissue was pulverized and boiled in buffer before extraction. Extracts prepared in this manner were found to contain a single immunoreactive fraction, which, on purification, yielded a polypeptide containing 113 amino acids, corresponding to a molecular weight of 12,600 (7). This new polypeptide contained the sequence of thymosins a I and a H at its NH2-terminus (Fig. 1) and was named prothymosin a. It was evidently the source of the fragments present in thymosin fraction 5. Prothymosin a is a very acidic peptide, having an isoelectric point of 3.55 (7), consistent with an unusually high content of aspartic and glutamic acids, which together account for more than one-half of the total amino acid residues. These acidic amino acids are clustered in the middle region of the polypeptide chain (9), in contrast to the two arginyl residues and the nine lysyl residues that are located at either end of the molecule. Prothymosin a contains no cysteine or methionine and no aromatic amino acids. The amino acid sequence suggests that the secondary structure is predominantly a-helical. A computer search of nearly 2800 amino acid sequences
failed to reveal any that possessed significant structural homology with prothymosin a. Prothymosin a is present in highest concentration in thymus and spleen (8), but significant quantities could also be isolated from other rat tissues (Table 1). The origin of the peptide in nonlymphoid tissues remains to be determined, but its formation in the thymus is supported by the in vitro translation experiments (3) and by histochemical staining with an antibody directed against thymosin cq, which showed the immunoreactive material to be localized in thymic epithelial cells (2). In addition, hyperplastic thymus from patients with myasthenia gravis contained many strongly staining cells (2). The experiments using isolation procedures that avoid proteolysis (6, 7) suggest that the cross-reacting material detected by the immunofluorescent technique is likely to be prothymosin a. Prothymosin a appears to be a more potent enhancer of cellular immunity than either thymosin fraction 5 or thymosin cxI. It is 10-20 times more potent, on an equimolar basis, than thymosin a I in protecting immunodeficient murine strains against intravenous challenge with Candida albicans (13). For thymosin fraction 5, the optimum daily therapeutic dose was 5 t.tg for thymosin fraction 5 and 160 ng for thymosin al; higher and lower doses were relatively inactive. In contrast, the effective dose for prothymosin a ranged from 40 to 320 ng and, in addition, resulted in a more dramatic elimination of the infecting organisms (14). The development of cellmediated immunity as measured by resistance to infection is accompanied by
Figure 1, Sequences of prothymosht ~ and of the amino-terminal fragnwnts. The one-letter symbols for amino acids are as follows: A, alanh~e; R, arghdne; N, asparagbw; D, aspartic acid; B, asparagflTe or aspartic acid (not determhted); Q, glutamine; E, glutamic acid; Z, glutamine or ghttamic acid (not determhwd); G, gl)wine; 1, isoleucbTe; L, leuchte; K, lysbze; P, proline; S, serine; T, threonine; V, valine. Ac is N-acetyl. All four peptides have blocked NH2-termhfi; hz thymosin eq this has been identified as an acetyl group (5). 24 Des-(25-28)-thymosin ~'1 SDAAVDTSSEITTKDLKEKKEVVE
28
Thymosin C(I SDAAVDTSSE'rTTKDLKEKKEVVEEAEN
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150
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© 1985 Elsevier Science Publishing Co., Inc.
Clinical Immunology Newsletter 6:10.1985
Table 1 Content of Prothymosin o~ and Parathymosin o~ in Rat Tissues" Tissue
Prothymosin eL
Parathymosin e~
Sum
Thymus Spleen Lung Kidney Liver Brain
414 270 154 126 68 58
157 119 265 315 326 207
571 389 419 441 394 265
Data from (10). '~ Values given in Itg/g tissue.
an increase in the antigen-induced release of migration inhibitory factor (MIF) into the circulation in vivo. In addition, peritoneal exudate cells from animals treated with prothymosin were more active in ingesting cells of Candida krusei and also showed a greatly enhanced ability to kill the ingested cells (14). In low responder strains of mice, prothymosin a also stimulated the release of MIF into the circulation in ~ivo (14). Thus the development of cell-mediated immunity, as indicated by resistance to infection, is accompanied by a release of the lymphokine, MIF, into the circulation and by enhanced activity of peritoneal macrophages. P a r a t h y m o s i n o~ The procedure for the isolation of prothymosin a also yielded a related peptide, named parathymosin a (10). Parathymosin o~ is similar in size and
chromatographic behavior and shows significant sequence homology with prothymosin a (Fig. 2). The first 30 residues of parathymosin a have been sequenced, and the segment between residues 14 to 25 was found to be almost identical to the corresponding segment of prothymosin et. Lack of sequence homology at the NH2-terminus accounted for the absence of antigenic cross-reactivity with the antiserum raised against thymosin al. The content of parathymosin c~ in rat tissues shows a reciprocal relation to that of prothymosin a. The richest sources are rat liver and kidney, followed by lung, brain, spleen, and thymus. It may be significant that the total quantity of the two peptides is relatively constant in the five tissues analyzed (Table 1). In the mouseprotection test, parathymosin a did not exhibit the immunoenhancing properties of prothymosin c~. However, when parathymosin a was adminis-
tercd to susceptible strains of mice together with prothymosin a, it neutralized the immunoenhancing effect of the latter (10). Parathymosin a thus offers interesting possibilities as a suppressor of cellular immunity.
Conclusions All of the ~t-thymosins thus far characterized, thymosin Ctl, thymosin eql, and des-(25-28)-thymosin cq, appear to be proteolytie fragments of the native polypeptide, prothymosin ct. Isolation of the native form requires inactivation of endogenous proteinases before extraction of the tissue; we have found extraction of the frozen tissue with either guanidinium chloride or boiling buffer to be most effective. Similar procedures were employed earlier for the inactivation of ribonucleases in the preparation of mRNA. Prothymosin ~, which possesses an unusual amino acid composition and sequence, shows significant effects on parameters of cellular immunity when administered to immunodeficient mufine strains. Thus far an in vivo test for the biological activity of prothymosin ct has not been found, and its isolation was based on the quantitative RIA for thymosin cq. The fact that prothymosin ct is more potent on a weight basis than thymosin ct I in the mouse protection assay indicates that its biological activity is not due to its conversion in vivo to thymosin ct I or other peptide fragments.
Figure 2. Comparison of the amhzo-terminal sequences of parathymosin ~ and prothymosin ~. The identical positions are shown by the heavy bars.
5
IO
15
20
Parathymosin 0~ xSer-Lys-Ser-Glu-VaI-Glu-AIo-Ala-Ala-GluProthymosin c~ AcSer Asp Ala Ala VoI Asp Thr Ser Ser GluParathymosin o/ Prothymosin 0(.
Leu-Ser-AIo-Lys-Asp-Leu-Lys-Glu-Lys-LysTie -Thr -Th r- Lys-Asp-Le u-Lys-G Iu- Lys- Lys-
Perathymosin O(. Prothymosin 06
As p-I_ys-VaI-GIu-G lu -L_ys-A Io-Gly-Arg-LysGlu-VoI-VeI-Glu-Glu-AIo-Glu-Asn-Gly-Arg-
Clinical Immunology Newsletter 6:10ol985
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The reciprocal relation in the quantities of prothymosin et and parathymosin a and the indication that parathymosin a may neutralize the immunoenhancing effects of prothymosin et raise interesting possibilities for the function of these peptides as regulators of cell-mediated immunity.
11. Ilarilos, A. A., et al. (1985). A radioimmunoassay for thymosin otI that detects the native polypeptide, prothymosin et. J. Immunol. Methods, in press. 12. Low, T. L. K., et al. (1979). The chemistry and biology of thymosin. I. Isolation, characterization and biological activities of thymosin cq and polypeptide 131 from calf thymus. J. Biol. Chem. 254:981-986. 13. Salvin, S. B. (1984). In vivo effects of thymosin on cellular immunity. Clin. Immunol. Newsletter 5:129133. 14. Salvin, S. B., et al. (1985). Immunoenhancing activities of the thymic polypeptide prothymosin et, (submitted for publication). 15. Stutman, O. (1983). Role of thymic hormones in T cell differentiation, pp. 9-81. h~ Clinics in immunology and allergy, vol. 3, no. 1, W. B. Saunders Company, Ltd. 16. White, A. (1980). Chemistry and biological actions of products with thymic hormone-like activity, pp. 1-46. h~ G. Litwack ted.), Biochemical actions of hormones, vol. VII. Academic Press, Inc., New York.
tions" appearing after some 15-20 years in all forms of the disease and leading to blindness, renal failure, coronary artery disease, and amputations. The daily challenge for the diabetologist is that such complications are not genetically determined (22, 25) but mostly proceed from chronic exposure of tissues to the array of metabolic abnormalities characteristic of the diabetic milieu (8). Thus we strive for more effective means of treating and, ultimately, for ways of preventing the onset of diabetes. Over the past ten years it has become increasingly clear that the immune system is heavily implicated in multiple pathogenetic aspects of the disease, and I shall summarize the available information below.
Type I is insulin-dependent diabetes (IDDM), ketosis prone, most often with an early onset (previously called juvenile-onset diabetes), and it is associated with increased frequency of certain HLA antigens and with islet-cell antibodies, suggestive of an autoimmune contribution to the destruction of pancreatic islet [3-cells and consequent insulin deficiency. Type II is non insulin-dependent diabetes (NIDDM), ketosis-resistant, and in western societies 6 0 - 9 0 % of this form of diabetes occurs in obese individuals (29) in whom the pathophysiology of the disease is based on resistance to hzsulin action. No characteristic aggregation of HLA types and islet-cell antibodies have been described in this form of diabetes, and the insulin resistance is probably initiated by the overeating and obesity leading to decreased number and responsiveness of the insulin receptors on target tissues. Among the cases of diabetes associated with certain conditions and syndromes, a very small number are caused by autoantibodies directed
5.
6.
7.
References I. Caldarella, J., et al. (1983). Thymosin etu: a peptide related to thymosin eq isolated from calf thymosin fraction 5. Proc. Natl. Aead. Sci. USA 80:7414-7427. 2. Dalakas, M. C., et al. (1981). Immunocytochemical localization of thymosin eq in thymic epithelial cells of normal and myasthenia gravis patients and in thymic cultures. J. Neurol. Sci. 50:239-247. 3. Freire, M., et al. (1981). Purification of thymus mRNA coding for a 16,000dalton polypeptide containing the thymosin cq sequence. Proe. Natl. Aead. Sci. USA 78:192-195. 4. Frledman, tt. (1979). Subcellular fac-
Immunological Aspects of Diabetes Mellitus Mara Lorenzi, M.D. Diabetes Clhlic U.C.S.D. Medical Center University of California San Diego San Diego, California
The abnormality that establishes the diagnosis of diabetes mellitus is elevated plasma glucose in the fasting state or after a standard glucose challenge (29). The hyperglycemia is in turn mediated by insufficient production of insulin by the 13-cells of the endocrine pancreas (insulin deficiency) or by decreased biologic action of the circulating hormone (insulin resistance). Diabetes is one of the most common chronic diseases of the western world, affecting at least 5% of the United States population. The extraordinary social impact of diabetes is a consequence of its late vascular "complica-
152
tors in immunity. Ann. New York Acad. Sci., vol. 332, New York, NY. Goldstein, A. L., et al. (1977). Thymosin at: isolation and sequence analysis of an immunologically active thymic polypeptide. Proc. Natl. Acad. Sci. USA 74:725-729. Ilannappel, E., et al. (1982). Isolation of peptides from calf thymus. Biochem. Biophys. Res. Commun. 104:266-271. Haritos, A. A., et al. (1984). Prothymosin or: isolation and properties of the major immunoreactive form of thymosin eq in rat thymus. Proc. Natl. Aead. Sci. USA 81:1008-1011. llarilos, A. A., et al. (1984). Distribution of prothymosin ot in rat tissues. Proe. Natl. Acad. Sci. USA 81:13911393. Haritos, A. A., et al. (1985). Primary structure of rat thymus prothymosin ct. Proc. Natl. Acad. Sci. USA 82:343346. Haritos, A. A., et al. (1985). Parathymosin ct--a peptide from rat tissues with structural homology to prothymosin et. Proc. Natl. Acad. Sci. USA 82:1050-1053.
0197-1859/85/S0.00 + 02.20
8.
9.
I0.
Classification o f D i a b e t e s We should think of diabetes as a syndrome (because of its heterogenicity), which is currently subclassified as 1) type I diabetes; 2) type II diabetes; and 3) diabetes associated with certain conditions and syndromes (29).
© 1985 Elsevier Science Publishing Co., Inc.
Clinical Immunology Newsletter 6:10.1985
Prothymosin and Other Thymic Peptides Related to Thymosin czI B. L. Horecker, Ph.D. Roche Institute of Molecular Biology Roche Research Center Nutley, New Jersey A. A. Haritos, Ph.D. University of Athens Athens, Greece
The role of the thymus in the development and maintenance of cell-
Clinical ImmunologyNewsletter6:10.1985
A. Z. Cohn. (1981). Hydrogen peroxide metabolism in human monocytcs during differentiation in vitro. J. Clin. Invest. 68:1243-1252. 16. Nathan, C. F., et al. (1979). Activation of macrophages in vivo and in vitro. Correlation between hydrogen peroxide release and killing of trypanosoma cruzi. J. Exp. Med. 149:1056-1068. 17. Nathan, C. F., el al. (1983). Identification of interferon-~/as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J. Exp. Med. 158:670-689. 18. Nathan, C. F., el al. (1984). Activation of human macrophages. Comparison of other cytokines with interferon% J. Exp. Med. 160:600-605. 19. Pick, E., and Y. Bromberg. (1982). Quo vadis macrophage activation-Role of phospholipids in the elicitation of the oxidative burst in macrophages. Transpl. Proc. 14:570-575. 20. Pick, E., and Y. Bromberg. (1983). Regulation of maerophage function by lymphokines--Role of membrane phospholipids, pp. 243-249. hi J. W. Hadden et al. (eds.), Advances in immunopharmacology, vol. 2. Pergamon Press, Oxford. 21. Pick, E., Y. Bromberg, and M. Frcund. (1982). Extrinsic regulation of macrophage function by lymphokines--effect of lymphokines on the stimulated oxidative metabolism of macrophages. Adv. Exp. Med. Biol. 155:471-485. 22. Pick, E., and M. Freund. (1983). Biochemical mechanisms in macrophage activation by lymphokines: intracellular peroxide production by lyre-
mediated immunity is well established. There is, however, lack of agreement as to whether the effects of the thymus on undifferentiated lymphocytes require residence o f these lymphocytes in the thymus or are, at least in part, mediated by thymic hormones released into the circulation. Evidence to support both points of view has been reviewed (4, 15). A number of thymic peptides have been isolated and chemically characterized (for a review see ref. 16). Prominent among these is an acidic peptide containing 28 amino acid residues, named thymosin a I, isolated from a
© 1985ElsevierSciencePublishingCo.. Inc.
23.
24.
25.
26.
27.
28.
phokine-trcated macrophages, pp. 295-306. In Y. Yamamura and T. Tada (eds.), Progress in immunology, voI. V, Academic Press, New York. Pick, E., and Y. Keisari. (1980). A simple colorimetric method for the measurement of hydrogen peroxide produced by cells in culture. J. lmmunol. Meth. 38:161-170. Pick, E., and Y. Keisari. (1981). Superoxide and hydrogen peroxide production by chemically elicited peritoneal macrophages--induction by multiple nonphagocytic stimuli. Cell. Immunol. 59:30t-318. Sasada, M., hi. J. Pabst, and R. B. Johnston. (1983). Activation of mouse peritoneal macrophages by lipopolysaccharide alters the kinetic parameters of the superoxide-producing NADPH oxidase. J. Biol. Chem. 258:96319635. Tsunawaki, S., and C. F. Nathan. (1984). Enzymatic basis of macrophage activation. Kinetic analysis of superoxide production in lysates of resident and activated mouse peritoneal macrophages and granulocytes. J. Biol. Chem. 259:4305-4312. Weinberg, J. B., and M. A. Misukonis. (1983). Phorbol diester-induced H202 production by peritoneal macrophages. Different H202 production by macrophages from normal and BCGinfected mice despite comparable phorbol diester receptors. Cell. lmmunol. 80:405-415. Zwickel, J., S. Shpungin, and E. Pick. (1985). Arachidonic acid metabolism of lymphokine-activated macrophages. In C. Sorg and A. Schimpl (eds.), Proceedings of the 4th International Lymphokine Workshop. Academic Press, Orlando, FL, in press.
biologically active preparation from calf thymus called thymosin fraction 5. Thymosin a t has shown promise as an immune enhancer (5, 12). However, more recent studies from our laboratory indicate that calf thymosin fraction 5 contains at least two additional peptides structurally related to thymosin cq (1) and that these peptides may all represent fragments of a larger native polypeptide named prothymosin ¢x, which we have isolated from extracts of fresh rat thymus (7). A second peptide, similar in size and amino acid composition to prothymosin oL, was recovered from the same
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rat thymus extracts and named parathymosin a (10). The biochemical and immunological properties of these peptides are reviewed here. T h y m o s i n oh Sequence analysis of thymosin aj (5) showed it to contain 28 amino acid residues, with an acetylated NH2-terminus, for a calculated molecular weight of 3108 (Fig. 1). In several in vitro assays designed to measure the maturation and functional differentiation of T-lymphocytes, thymosin a I was reported to be 10-1000 times more active than thymosin fraction 5 (5, 12). T h y m o s i n oql a n d des-(25-28)t h y m o s i n cxl Two additional peptides closely related to thymosin cq have been isolated from preparations of calf thymus fraction 5 (1). One, designated des(25-28)-thymosin ¢xI, was identical to thym0sin al except that it lacked the last four amino acid residues at the COOH-terminus (Fig. 1). The other, named thymosin all, was found to contain seven additional residues (Fig. I). In the mouse-protection test employed by Salvin and his co-workers (13) thymosin all was as effective as thymosin a I (1). P r o t h y m o s i n ot The presence in calf thymosin fraction 5 of peptides having a common structure and differing only in length at the COOH-terminus suggested that they might all be proteolytic artifacts generated during the procedure employed for the preparation of the starting material, thymosin fraction 5.
Of particular significance was the fact that thymosin eq was not detected when proteolysis was avoided by extracting fresh thymus with guanidinium chloride (6). To isolate the native polypeptide containing the thymosin a I sequence, we employed a radioimmunoassay (RIA) based on an antibody raised against synthetic thymosin a I coupled to keyhole limpet hemocyanin (I 1). To avoid proteolysis during the extraction and isolation, the frozen tissue was pulverized and boiled in buffer before extraction. Extracts prepared in this manner were found to contain a single immunoreactive fraction, which, on purification, yielded a polypeptide containing 113 amino acids, corresponding to a molecular weight of 12,600 (7). This new polypeptide contained the sequence of thymosins a I and a H at its NH2-terminus (Fig. 1) and was named prothymosin a. It was evidently the source of the fragments present in thymosin fraction 5. Prothymosin a is a very acidic peptide, having an isoelectric point of 3.55 (7), consistent with an unusually high content of aspartic and glutamic acids, which together account for more than one-half of the total amino acid residues. These acidic amino acids are clustered in the middle region of the polypeptide chain (9), in contrast to the two arginyl residues and the nine lysyl residues that are located at either end of the molecule. Prothymosin a contains no cysteine or methionine and no aromatic amino acids. The amino acid sequence suggests that the secondary structure is predominantly a-helical. A computer search of nearly 2800 amino acid sequences
failed to reveal any that possessed significant structural homology with prothymosin a. Prothymosin a is present in highest concentration in thymus and spleen (8), but significant quantities could also be isolated from other rat tissues (Table 1). The origin of the peptide in nonlymphoid tissues remains to be determined, but its formation in the thymus is supported by the in vitro translation experiments (3) and by histochemical staining with an antibody directed against thymosin cq, which showed the immunoreactive material to be localized in thymic epithelial cells (2). In addition, hyperplastic thymus from patients with myasthenia gravis contained many strongly staining cells (2). The experiments using isolation procedures that avoid proteolysis (6, 7) suggest that the cross-reacting material detected by the immunofluorescent technique is likely to be prothymosin a. Prothymosin a appears to be a more potent enhancer of cellular immunity than either thymosin fraction 5 or thymosin cxI. It is 10-20 times more potent, on an equimolar basis, than thymosin a I in protecting immunodeficient murine strains against intravenous challenge with Candida albicans (13). For thymosin fraction 5, the optimum daily therapeutic dose was 5 t.tg for thymosin fraction 5 and 160 ng for thymosin al; higher and lower doses were relatively inactive. In contrast, the effective dose for prothymosin a ranged from 40 to 320 ng and, in addition, resulted in a more dramatic elimination of the infecting organisms (14). The development of cellmediated immunity as measured by resistance to infection is accompanied by
Figure 1, Sequences of prothymosht ~ and of the amino-terminal fragnwnts. The one-letter symbols for amino acids are as follows: A, alanh~e; R, arghdne; N, asparagbw; D, aspartic acid; B, asparagflTe or aspartic acid (not determhted); Q, glutamine; E, glutamic acid; Z, glutamine or ghttamic acid (not determhwd); G, gl)wine; 1, isoleucbTe; L, leuchte; K, lysbze; P, proline; S, serine; T, threonine; V, valine. Ac is N-acetyl. All four peptides have blocked NH2-termhfi; hz thymosin eq this has been identified as an acetyl group (5). 24 Des-(25-28)-thymosin ~'1 SDAAVDTSSEITTKDLKEKKEVVE
28
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Table 1 Content of Prothymosin o~ and Parathymosin o~ in Rat Tissues" Tissue
Prothymosin eL
Parathymosin e~
Sum
Thymus Spleen Lung Kidney Liver Brain
414 270 154 126 68 58
157 119 265 315 326 207
571 389 419 441 394 265
Data from (10). '~ Values given in Itg/g tissue.
an increase in the antigen-induced release of migration inhibitory factor (MIF) into the circulation in vivo. In addition, peritoneal exudate cells from animals treated with prothymosin were more active in ingesting cells of Candida krusei and also showed a greatly enhanced ability to kill the ingested cells (14). In low responder strains of mice, prothymosin a also stimulated the release of MIF into the circulation in ~ivo (14). Thus the development of cell-mediated immunity, as indicated by resistance to infection, is accompanied by a release of the lymphokine, MIF, into the circulation and by enhanced activity of peritoneal macrophages. P a r a t h y m o s i n o~ The procedure for the isolation of prothymosin a also yielded a related peptide, named parathymosin a (10). Parathymosin o~ is similar in size and
chromatographic behavior and shows significant sequence homology with prothymosin a (Fig. 2). The first 30 residues of parathymosin a have been sequenced, and the segment between residues 14 to 25 was found to be almost identical to the corresponding segment of prothymosin et. Lack of sequence homology at the NH2-terminus accounted for the absence of antigenic cross-reactivity with the antiserum raised against thymosin al. The content of parathymosin c~ in rat tissues shows a reciprocal relation to that of prothymosin a. The richest sources are rat liver and kidney, followed by lung, brain, spleen, and thymus. It may be significant that the total quantity of the two peptides is relatively constant in the five tissues analyzed (Table 1). In the mouseprotection test, parathymosin a did not exhibit the immunoenhancing properties of prothymosin c~. However, when parathymosin a was adminis-
tercd to susceptible strains of mice together with prothymosin a, it neutralized the immunoenhancing effect of the latter (10). Parathymosin a thus offers interesting possibilities as a suppressor of cellular immunity.
Conclusions All of the ~t-thymosins thus far characterized, thymosin Ctl, thymosin eql, and des-(25-28)-thymosin cq, appear to be proteolytie fragments of the native polypeptide, prothymosin ct. Isolation of the native form requires inactivation of endogenous proteinases before extraction of the tissue; we have found extraction of the frozen tissue with either guanidinium chloride or boiling buffer to be most effective. Similar procedures were employed earlier for the inactivation of ribonucleases in the preparation of mRNA. Prothymosin ~, which possesses an unusual amino acid composition and sequence, shows significant effects on parameters of cellular immunity when administered to immunodeficient mufine strains. Thus far an in vivo test for the biological activity of prothymosin ct has not been found, and its isolation was based on the quantitative RIA for thymosin cq. The fact that prothymosin ct is more potent on a weight basis than thymosin ct I in the mouse protection assay indicates that its biological activity is not due to its conversion in vivo to thymosin ct I or other peptide fragments.
Figure 2. Comparison of the amhzo-terminal sequences of parathymosin ~ and prothymosin ~. The identical positions are shown by the heavy bars.
5
IO
15
20
Parathymosin 0~ xSer-Lys-Ser-Glu-VaI-Glu-AIo-Ala-Ala-GluProthymosin c~ AcSer Asp Ala Ala VoI Asp Thr Ser Ser GluParathymosin o/ Prothymosin 0(.
Leu-Ser-AIo-Lys-Asp-Leu-Lys-Glu-Lys-LysTie -Thr -Th r- Lys-Asp-Le u-Lys-G Iu- Lys- Lys-
Perathymosin O(. Prothymosin 06
As p-I_ys-VaI-GIu-G lu -L_ys-A Io-Gly-Arg-LysGlu-VoI-VeI-Glu-Glu-AIo-Glu-Asn-Gly-Arg-
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The reciprocal relation in the quantities of prothymosin et and parathymosin a and the indication that parathymosin a may neutralize the immunoenhancing effects of prothymosin et raise interesting possibilities for the function of these peptides as regulators of cell-mediated immunity.
11. Ilarilos, A. A., et al. (1985). A radioimmunoassay for thymosin otI that detects the native polypeptide, prothymosin et. J. Immunol. Methods, in press. 12. Low, T. L. K., et al. (1979). The chemistry and biology of thymosin. I. Isolation, characterization and biological activities of thymosin cq and polypeptide 131 from calf thymus. J. Biol. Chem. 254:981-986. 13. Salvin, S. B. (1984). In vivo effects of thymosin on cellular immunity. Clin. Immunol. Newsletter 5:129133. 14. Salvin, S. B., et al. (1985). Immunoenhancing activities of the thymic polypeptide prothymosin et, (submitted for publication). 15. Stutman, O. (1983). Role of thymic hormones in T cell differentiation, pp. 9-81. h~ Clinics in immunology and allergy, vol. 3, no. 1, W. B. Saunders Company, Ltd. 16. White, A. (1980). Chemistry and biological actions of products with thymic hormone-like activity, pp. 1-46. h~ G. Litwack ted.), Biochemical actions of hormones, vol. VII. Academic Press, Inc., New York.
tions" appearing after some 15-20 years in all forms of the disease and leading to blindness, renal failure, coronary artery disease, and amputations. The daily challenge for the diabetologist is that such complications are not genetically determined (22, 25) but mostly proceed from chronic exposure of tissues to the array of metabolic abnormalities characteristic of the diabetic milieu (8). Thus we strive for more effective means of treating and, ultimately, for ways of preventing the onset of diabetes. Over the past ten years it has become increasingly clear that the immune system is heavily implicated in multiple pathogenetic aspects of the disease, and I shall summarize the available information below.
Type I is insulin-dependent diabetes (IDDM), ketosis prone, most often with an early onset (previously called juvenile-onset diabetes), and it is associated with increased frequency of certain HLA antigens and with islet-cell antibodies, suggestive of an autoimmune contribution to the destruction of pancreatic islet [3-cells and consequent insulin deficiency. Type II is non insulin-dependent diabetes (NIDDM), ketosis-resistant, and in western societies 6 0 - 9 0 % of this form of diabetes occurs in obese individuals (29) in whom the pathophysiology of the disease is based on resistance to hzsulin action. No characteristic aggregation of HLA types and islet-cell antibodies have been described in this form of diabetes, and the insulin resistance is probably initiated by the overeating and obesity leading to decreased number and responsiveness of the insulin receptors on target tissues. Among the cases of diabetes associated with certain conditions and syndromes, a very small number are caused by autoantibodies directed
5.
6.
7.
References I. Caldarella, J., et al. (1983). Thymosin etu: a peptide related to thymosin eq isolated from calf thymosin fraction 5. Proc. Natl. Aead. Sci. USA 80:7414-7427. 2. Dalakas, M. C., et al. (1981). Immunocytochemical localization of thymosin eq in thymic epithelial cells of normal and myasthenia gravis patients and in thymic cultures. J. Neurol. Sci. 50:239-247. 3. Freire, M., et al. (1981). Purification of thymus mRNA coding for a 16,000dalton polypeptide containing the thymosin cq sequence. Proe. Natl. Aead. Sci. USA 78:192-195. 4. Frledman, tt. (1979). Subcellular fac-
Immunological Aspects of Diabetes Mellitus Mara Lorenzi, M.D. Diabetes Clhlic U.C.S.D. Medical Center University of California San Diego San Diego, California
The abnormality that establishes the diagnosis of diabetes mellitus is elevated plasma glucose in the fasting state or after a standard glucose challenge (29). The hyperglycemia is in turn mediated by insufficient production of insulin by the 13-cells of the endocrine pancreas (insulin deficiency) or by decreased biologic action of the circulating hormone (insulin resistance). Diabetes is one of the most common chronic diseases of the western world, affecting at least 5% of the United States population. The extraordinary social impact of diabetes is a consequence of its late vascular "complica-
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tors in immunity. Ann. New York Acad. Sci., vol. 332, New York, NY. Goldstein, A. L., et al. (1977). Thymosin at: isolation and sequence analysis of an immunologically active thymic polypeptide. Proc. Natl. Acad. Sci. USA 74:725-729. Ilannappel, E., et al. (1982). Isolation of peptides from calf thymus. Biochem. Biophys. Res. Commun. 104:266-271. Haritos, A. A., et al. (1984). Prothymosin or: isolation and properties of the major immunoreactive form of thymosin eq in rat thymus. Proc. Natl. Aead. Sci. USA 81:1008-1011. llarilos, A. A., et al. (1984). Distribution of prothymosin ot in rat tissues. Proe. Natl. Acad. Sci. USA 81:13911393. Haritos, A. A., et al. (1985). Primary structure of rat thymus prothymosin ct. Proc. Natl. Acad. Sci. USA 82:343346. Haritos, A. A., et al. (1985). Parathymosin ct--a peptide from rat tissues with structural homology to prothymosin et. Proc. Natl. Acad. Sci. USA 82:1050-1053.
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Classification o f D i a b e t e s We should think of diabetes as a syndrome (because of its heterogenicity), which is currently subclassified as 1) type I diabetes; 2) type II diabetes; and 3) diabetes associated with certain conditions and syndromes (29).
© 1985 Elsevier Science Publishing Co., Inc.
Clinical Immunology Newsletter 6:10.1985