Thymosin ␣ 1 (T␣ 1 ) and thymosin T␣ 11 (T␣ 11 ) are polypeptides with immunoregulatory properties first isolated from thymic extracts, corresponding to the first 28 and 35 amino acid residues, respectively, of prothymosin ␣ (ProT␣), a protein involved in chromatin remodeling. It has been widely supposed that these polypeptides are not natural products of the in vivo processing of ProT␣, since neither was found in extracts in which proteolysis was prevented. Here we show that a lysosomal asparaginyl endopeptidase is able to process ProT␣ to generate T␣ 1 and T␣ 11 . In view of its catalytic properties and structural and immunological analyses, this protease was identified as mammalian legumain. It selectively cleaves some of the asparaginylglycine residues in the ProT␣ sequence; specifically, Asn 28 -Gly 29 and Asn 35 -Gly 36 residues are cleaved with similar efficiency in vitro to generate T␣ 1 and T␣ 11 , respectively. By contrast T␣ 1 is the main product detected in vivo, free in the cytosol, at concentrations similar to that of ProT␣. The data here reported demonstrate that T␣ 1 is not an artifact but rather is naturally present in diverse mammalian tissues and raise the possibility that it has a functional role.

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'251-Labeled prothymosin a (ProTa) was used to study the presence and characteristics of receptors for ProTa on human peripheral blood mononuclear cells (PBMC). The kinetics of '251-ProTa binding to PBMC was fast at 37"C, whilst it required 50 min to reach equilibrium at 4°C and room temperature. Analysis of steady state binding data by the method of Scatchard and by unlabeled ProTa competition experiments identified two binding sites with an apparent equilibrium dissociation constant of 216321 pM for the high-affinity receptor and of 11.421.1 nM for the low-affinity one; the sites per cell ranged from 1,479 to 1,519 and from 47,547 to 56,169, respectively. The kinetically derived equilibrium dissociation constant agreed with these data and showed no interaction between receptors.

Recent results indicate that prothymosin alpha (ProT alpha) may be useful in designing future therapeutic interventions in cancer patients and in potentiating the immune system. We described recently the presence and characteristics of two binding sites for ProT alpha on human peripheral blood mononuclear cells (PBMC). In search of a receptor upregulation, we decided to corroborate this finding on two lymphocytic populations, (PHA-activated) lymphoblasts and YT cells. The kinetics of [125I]ProT alpha binding to lymphoblasts were fast at room temperature but with YT cells were slower. Analysis of steady-state binding data identified two binding sites in lymphoblasts with an apparent equilibrium Kd of 44-75 pM and 4228-9143 sites per cell for the high-affinity receptor and 1.7-2.9 nM and 20,534-35,044 sites per cell for the low-affinity receptor. However, it identified only one site with a Kd of 265-435 pM and 8318-27,237 sites per cell in YT cells. We conclude that exists a ProT alpha receptor in the CD3+ T cell population, and this presence is regulated. After binding to cell surface, [125I]ProT alpha is internalized in a short period of time and then degraded; therefore, we conclude that the dynamics of ProT alpha receptor turnover in part determines the concentration of ProT alpha available to induce its enhancing effects.

The acidic protein prothymosin alpha (ProTalpha), with a broad presence in mammalian cells, has been widely considered to have a role in cell division, through an unrevealed mechanism in which histones may be involved in view of their ability to interact with ProTalpha in vitro. Results of co-immunoprecipitation experiments presented here demonstrate that ProTalpha interacts in vivo with core histones in proliferating B-lymphocytes (NC-37 cells). This interaction occurs with histones H3, H2A, H2B and H4 located free in the nucleoplasm, whereas no interaction was detected with histone H1, mono-nucleosome particles or chromatin. Moreover, the core histones form part of a nuclear multiprotein complex of about 700 kDa separated by ProTalpha-Sepharose affinity, with components including H3 and H4 acetyltranferases, H3 methyltransferases, hnRNP isotypes A3, A2/B1 and R, ATP-dependent and independent DNA helicases II, beta-actin and vimentin, all co-purifying by gel filtration. This indicates that the interaction of ProTalpha with core histones in the nucleus may be related to the structural modification of histones H3 and H4, and hence to chromatin activity, raising the possibility that the other proteins in the nuclear complex may play a role in this process.