Mirna Mihelić

Use of bimanyl actin derivative (TMB-actin) for studying complexation of β-thymosins: Inhibition of actin polymerization by thymosin β9

By reacting trimethylammoniobromobimane bromide (TMB bromide) with rabbit muscle actin, a fluorescent reporter group was linked to cysteine at position 374. Fluorescence of TMB‐actin decreased significantly on addition of thymosin β4 (Tβ4), a peptide of 43 amino acid residues reported to bind to monomeric actin and to prevent filament formation. Based on this effect, we determined the K d value of the thymosin β4 complex as 0.8 μM, a value that is in agreement with previous determinations. In addition to the main compound thymosin β4, bovine tissue contains a related peptide, thymosin β9 (Tβ9), which has 41 amino acid residues and ca. 75% sequence homology. In the present study we show for the first time that Tβ9, similar to Tβ4, forms a 1:1 complex with monomeric actin, and hereby inhibits actin polymerization. With a K d value of 1.1 μM the affinity of Tβ9 is in the same range as that of Tβ4, suggesting that Tβ9, like Tβ4, contributes to maintaining the pool of monomeric actin in bovine non‐muscle cells. Further proof of the interaction of Tβ9 with actin was provided by native PAGE, where the complex showed the reported higher mobility, as well as by crosslinking experiments. Using different crosslinking reagents, like water‐soluble carbodiimide (EDC), m‐maleimidobenzoyl‐N‐hydroxysuccinimidate (MBS), and disuccinimidylsuberate (DSS), we were able to produce conjugates of 47 kDa. In one of these (from MBS) both actin and Tβ9 could be identified by immunoblotting. When, in the MBS crosslinking experiments, native actin was replaced with (374‐NEM)actin, the 47 kDa band was not seen, indicating that Cys‐374 takes part in the thiol‐specific crosslinking reaction. This suggests that part of the binding site of Tβ9 must be located close to the carboxy‐tenninus.

A thymosin β4 ELISA using an antibody against the N terminal fragment thymosin β4[1–14]

Abstract A thymosin β 4 ELISA was developed in which thymosin β 4 , absorbed on microwells, competed with thymosin β 4 in solution for the binding sites of an anti-thymosin β 4 antibody. The antibody molecules finally immobilized on the microwells were detected using a goat anti-rabbit immunoglobulin/horseradish peroxidase conjugate in combination with the substrate 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt, and measuring the relevant optical density values. Anti-thymosin β 4 antibodies were raised in rabbits against intact thymosin β 4 as well as against selected fragments of the peptide, i.e., the N terminal fragments thymosin β 4 [1–14] and thymosin β 4 [1–11]. The antibody against thymosin β 4 [1–14] was used in the thymosin β 4 ELISA, because it showed minimal cross-reactivity (0.1%) with the highly homologous peptide thymosin β 9 as well as exhibiting the highest titre. The ELISA procedure developed, apart from showing a minimal cross-reaction with thymosin β 9 , was fast, easy to perform and exhibited good assay characteristics.

MicroELISA method for the determination of thymosin β9 discriminating between thymosin β9 and the structurally closely related thymosin β4

In order to obtain specific antibodies against thymosin β9 showing minimal cross-reactivity with the highly homologous peptide thymosin β4, the N-terminal fragment 1–14 of thymosin β9 was used for immunization. These antibodies have been tested in a competitive ELISA and show less than 1% cross-reactivity with thymosin β4. On the other hand, antibodies raised against the native thymosin β9(1–14) cross-react 35% with thymosin β4. Specific antibodies against thymosin β9 are important for studying the concentration and localization of thymosin β9 in thymus and other bovine tissues because thymosin β9 is always accompanied by thymosin β4. Using N-terminal fragments of thymosin β4-like peptides may be a general approach for obtaining specific antibodies since this part of sequence is less conserved in thymosin β4-like peptides.

Polymerization of actin from the thymosin β4 complex initiated by the addition of actin nuclei, nuclei stabilizing agents or myosin S1

Thymosin β4 forms a 1:1 complex with actin and thereby prevents polymerization. Rapid formation of filaments from this complex was observed, however, when actin trimers were added. Polymerization can likewise be initiated by the addition of one equivalent of phalloidin or, less effectively, cytochalasin B. Since both toxins, which reportedly support nucleation, have similar effects as the covalently linked actin trimers, it appears that the formation of filaments from the actin—thymosin β4 complex depends on the availability of stable actin nuclei. Remarkably, rapid polymerization was also observed if small amounts of myosin S1 were added, suggesting that also myosin, a protein functionally connected with polymeric actin, can serve as a nucleation center. Considering the existence of thymosin β4 and related peptides in numerous mammalian tissues, our data suggest that spontaneous formation of microfilaments in non‐muscle cells may be regulated at the level of nucleation. Uncontrolled polymerization induced by the formation of phalloidin‐stabilized nuclei may explain the acute toxic effects of phalloidin in hepatocytes.

Theoretical and experimental epitope mapping of thymosin β4

Abstract Two rabbit polyclonal antisera, one directed against thymosin β 4 and the other one against the peptide fragment thymosin β 4 (1–14) were characterised by epitope mapping. Hexapeptides representing the whole sequence of the native peptide and overlapping by one amino acid were synthesised on polystyrene pins. The antigenic determinants were identified in microtitre plates with an ELISA procedure. The polyclonal serum against thymosin β 4 detected three epitopes (N-terminal, mid-region and C-terminal) whereas the polyclonal serum against the fragment contained only antibodies specific for the N-terminal epitope. These experimental results are consistent with theoretical predictions.

Immunohistochemical localization of thymosin β9 in bovine tissues

SummaryUsing an indirect fluorescent antibody technique with frozen sections, the localization of thymosin β9 was investigated for the first time in bovine thymus, spleen, lung, muscle and liver. The antibodies used have been raised against the N-terminal fragment 1–14 of thymosin β9 in order to minimize the cross-reactivity with thymosin β4 which was found to be also present in bovine tissues. The specific antibodies against thymosin β9 raised in our laboratory allowed us to localize this peptide in presence of the highly homologous and always accompanying thymosin β4 in different tissues. Although thymosin β9 was first isolated from calf thymus, it could be also detected in other bovine organs. The highest density of positive immunoreaction was found to be in spleen sections. In the muscle tissue a pronounced fluorescence intensity was present in the region of the sarcolemn.