Prothymosin alpha gene in humans: organization of its promoter region and localization to chromosome 2.

A genomic clone encoding prothymosin alpha (gene symbol: PTMA), a nuclear-targeted protein associated with cell proliferation, was isolated and the 5'-regulatory region subcloned and sequenced. Because of previously reported discrepancies between several cDNA clones and a genomic clone for prothymosin alpha, we determined the sequence of the first exon and of a 1.7-kb region 5' to the first exon. The sequence of the genomic clone reported here corresponds to the published cDNA sequences, suggesting that the previously noted discrepancies may be due to genetic polymorphism in this region. In addition, our sequence data extend the known 5'-upstream sequence by an additional 1.5 kb allowing the identification of numerous, potential cis-acting regulatory sites. This 5'-flanking cloned probe permitted us to localize the prothymosin gene to chromosome 2 in humans.

Evidence for nuclear targeting of prothymosin and parathymosin synthesized in situ.

To test the hypothesis that prothymosin and parathymosin contain amino acid sequences that cause them to be targeted to the cell nucleus, expression vectors were constructed containing a simian virus 40 promoter and cDNAs that would code for chimeric proteins composed of truncated human growth hormone (hGH) linked to the NH2 terminus of prothymosin or parathymosin. The truncated hGH lacked the signal peptide sequence required for its secretion. After transfection of these constructs into HeLa S3 cells, which do not normally synthesize hGH, the use of indirect immunofluorescence staining to follow the localization of the hGH chimeras demonstrated that both prothymosin and parathymosin caused targeting to the cell nucleus. Controls with a construct coding for native hGH only, and one coding for the truncated hGH lacking the signal peptide, revealed secretion into culture medium and staining in the endoplasmic reticulum and Golgi apparatus in the first case, and diffuse staining throughout the cytoplasm in the second. The results provide direct evidence, with proteins synthesized in situ, for the presence of nuclear localization signals in both prothymosin and parathymosin.

Identification of two calpastatin forms in rat skeletal muscle and their susceptibility to digestion by homologous calpains.

Two forms of calpastatin, differing in their specificity for the homologous calpain isozymes I and II, have been separated from rat skeletal muscle extracts and purified to homogeneity. Calpastatin I, the first form to elute in chromatography on DE32, is more effective against calpain I, while calpastatin II is more effective as an inhibitor of calpain II. Based on their molecular mass (approximately 105 kDa) both calpastatin forms belong to the high molecular mass class found in muscles of other animal species (Murachi, T., 1989, Biochem. Int. 18, 263-294). For calpain I, which is active with low (mu-M) concentrations of Ca2+, maximum inhibition with either calpastatin form was observed over a wide range of Ca2+ concentrations. With calpain II, which requires high (mM) concentrations of Ca2+ for activity, maximum inhibition required Ca2+ concentrations above 1 mM. Both calpastatin forms were found to be highly sensitive to degradation by calpain II, but almost completely resistant to degradation by calpain I. Degradation of calpastatin by calpain II is competitively inhibited by the addition of a calpain substrate. Isovaleryl carnitine (IVC), an intermediate product of L-leucine catabolism, previously demonstrated to be a potent and specific activator of rat skeletal muscle calpain II (Pontremoli, S., Melloni, E., Viotti, P. L., Michetti, M., Di Lisa, F., and Siliprandi, N., 1990. Biochem. Biophys. Res. Commun. 167, 373-380) greatly enhances the rate of degradation of calpastatins by calpain II. IVC, which decreases the Ca2+ requirement for maximal calpain II activity, also decreases the concentration of Ca2+ required for digestion of the inhibitor. For calpain II, regulation by either calpastatins may occur only in the presence of high [Ca2+].

Identification of the proteolytically activated form of protein kinase C in stimulated human neutrophils.

The proteolytically activated form of protein kinase C has been identified in human neutrophils by using a monoclonal antibody that recognizes both the native kinase and the catalytically active proteolytic fragment (protein kinase M). Stimulation with fMet-Leu-Phe results in the conversion of approximately 30% of native protein kinase C to protein kinase M, with little evidence of further degradation. Stimulation with phorbol 12-myristate 13-acetate, on the other hand, causes only a transient formation of protein kinase M, with complete loss of total kinase activity. These differences are related to the differences in biochemical responses, reported earlier, in neutrophils exposed to these two activators.

Cellular levels of thymosin immunoreactive peptides are linked to proliferative events: evidence for a nuclear site of action.

Thymosin alpha 1 (T alpha 1), the N-terminal 28-amino acid fragment of prothymosin alpha (ProT alpha), and ProT alpha, although originally isolated from whole thymus extracts, are also present in nonthymic cells and tissues. We used an ELISA with an antibody raised against T alpha 1 to investigate the relationship between intracellular levels of thymosin immunoreactive peptide(s) (TIP) and cell proliferation in a rat small intestinal IEC-6 cell line. Increasing TIP levels were observed during cell proliferation, which decreased when proliferation was halted by cellular contact inhibition. Serum feeding of cells previously rendered quiescent by serum starvation resulted in a significant increase in TIP within 1 hr. Conversely, serum starvation decreased TIP levels within 1 hr. Peak TIP levels appeared after 3 hr of serum incubation, while maximum [3H]thymidine incorporation was noted after 9 hr, suggesting maximum TIP concentrations in the G1 phase of the proliferative cycle. Immunoelectron microscopy demonstrated an association of TIP with condensed nuclear chromatin. These results support a relation of intracellular TIP levels to IEC-6 cell proliferation and also a nuclear site of action. HPLC analysis of cellular homogenates from proliferating IEC-6 cells revealed a peak of immune reactivity that elutes in the position of T alpha 1.

Sequencing of peptides and proteins with blocked N-terminal amino acids: N-acetylserine or N-acetylthreonine.

Many proteins cannot be directly sequenced by Edman degradation because they have a blocked N-terminal residue. A method is presented for deblocking such proteins when the N-terminal residue is N-acetylserine (which occurs frequently in eukaryotic proteins) or N-acetylthreonine. The method has been applied successfully to the determination of the N-terminal amino acid sequence of human, bovine, and rat parathymosins. Prothymosin alpha and other blocked proteins and peptides were also readily deblocked and sequenced by this procedure. It is proposed that the mechanism of the deblocking reaction involves an acid-catalyzed N----O shift of the acetyl group followed by a beta-elimination.

Isozymes of protein kinase C in human neutrophils and their modification by two endogenous proteinases.

Two major protein kinase C (PKC) isozymes, accounting for approximately 95% of the total activity in human neutrophils, were separated by hydroxyapatite chromatography and were identified as beta-PKC (60% of the total) and alpha-PKC (35% of the total). No gamma-PKC was detected. A minor Ca2+/phospholipid requiring kinase that eluted from hydroxyapatite after alpha-PKC did not react significantly with any of the specific antisera employed for identification. Modification of beta-PKC or the minor PKC isozyme by calpain yielded Ca2+/phospholipid-independent forms (PKM) that retained only 50% of the original activities. In contrast, PKM formed from alpha-PKC retained full catalytic activity. For each native isozyme the rate of conversion by calpain was accelerated in the presence of Ca2+ and the lipid effectors, and the PKM form generated in each case was resistant to further digestion by calpain. All three PKC isozymes were also modified by a neutral serine proteinase isolated from human neutrophils, with this proteinase the major effect being loss of kinase activity, via a transient production of a Ca2+/phospholipid-independent form. This neutral serine proteinase appears to be localized at sites of interaction of cytoskeletal proteins with the cell membrane. Following stimulation of intact neutrophils with phorbol 12-myristate 13-acetate complete loss of native cytosolic kinase activity was observed, with recovery of approximately 30% of the original activity as a cytosolic Ca+/phospholipid independent form, presumably PKM. Loss of native PKC activity was greatest for the beta-isozyme. In cells stimulated by fMet-Leu-Phe approximately 60% of the original PKC activity was recovered as native cytosolic PKC and 30% as cytosolic PKM. Inhibitors of calpain reduced the extent of down-regulation of PKC, increased the proportion of PKC that remained associated with the plasma membrane and significantly reduced the proteolytically generated fully active PKM. Taken together, the in vitro and in vivo results suggest that calpain is involved primarily in the conversion of the PKC isozymes to the irreversibly activated PKM forms, and that the neutral serine proteinase may be the enzyme responsible for down-regulation, possibly via PKM as an intermediate.

Activation of neutrophil calpain following its translocation to the plasma membrane induced by phorbol ester or fMet-Leu-Phe.

Stimulation of human neutrophils with phorbol myristate acetate or fMet-Leu-Phe results in translocation to the plasma membrane of approximately 25-40% of the cellular calpain activity. In the membrane-bound form the Ca2+-requirement for proteolytic activity is substantially reduced. An anti-calpain monoclonal antibody that is internalized by stimulated neutrophils is recovered in the same subcellular fraction that contains the membrane-bound calpain, apparently in the form of pinocytotic vesicles. When both monoclonal antibody and calpain were present in these vesicles, a pronounced inhibition of the membrane bound proteinase activity was observed. These results provide an explanation for the previously observed inhibitory effect of the monoclonal antibody on intracellular calpain activity and on the concomitant inhibition of granule exocytosis. The activated calpain associated with the plasma membrane compartment is therefore identified as the form specifically involved in mediating the physiological responses.

The sequence of human parathymosin deduced from a cloned human kidney cDNA.

The amino acid sequence of human parathymosin has been deduced from the cDNA sequence of a clone isolated from a human kidney cDNA library. Screening of the cDNA library with a probe containing a partial rat cDNA sequence yielded two clones containing inserts of 1200 and 1100 base pairs respectively, each including the complete open reading frame for human parathymosin. The open reading frame contains 306 nucleotides, including the codon for the initiator methionine. Analysis of the 5' flanking sequence excluded the presence of a hydrophobic signal peptide in the translated sequence. It may therefore be concluded that parathymosin, like prothymosin alpha, is synthesized without formation of a large precursor polypeptide. Comparison of the deduced amino acid sequence with the known primary structure of rat and bovine parathymosins shows that the primary structure of parathymosin is highly conserved among these species.

Prothymosin alpha and parathymosin: mRNA and polypeptide levels in rodent tissues.

Blot hybridization analyses have established the presence of mRNAs for prothymosin alpha (ProT alpha) and for parathymosin (ParaT) in rat and mouse lung, liver, kidney, and brain, confirming the biosynthesis of these peptides in nonlymphoid tissues. In these tissues the levels of mRNAs paralleled the content of the polypeptides, determined with specific radioimmunoassays. The mRNA levels also confirmed the reciprocal relation between the two polypeptides; ProT alpha and its mRNA were found in highest concentrations in spleen and thymus, followed by lung, kidney, and brain, with lowest concentrations in liver. On the other hand, liver contained highest concentrations of ParaT and the mRNA for ParaT, with lowest levels present in spleen and thymus. In comparison to tissues from young (6-8 week) mice, older (18 month) mice contained lower concentrations (20-40%) of both polypeptides, with qualitatively similar decreases in mRNA content.

Localization of the gene coding for parathymosin to chromosome 17 in humans.

Parathymosin is a 101-amino acid polypeptide involved in the regulation of cellular immunity. In situ hybridization of rat parathymosin cDNA to human metaphase chromosomes localized the gene for human parathymosin to the q12----q22 region of chromosome 17.

The amino acid sequence of bovine thymus prothymosin alpha.

Prothymosin alpha has been purified from calf thymus and its amino acid sequence determined. It contains 109 amino acid residues and closely resembles human prothymosin alpha, with only two substitutions, glutamic acid for aspartic acid at position 31 and alanine for serine at position 83. This is in contrast to six differences between rat and bovine prothymosins, including four substitutions and two deletions. The structural similarity of the bovine and human polypeptides makes the former a good candidate for studies on the evaluation of the biological activities of prothymosin alpha in human systems.

Bovine parathymosin: amino acid sequence and comparison with rat parathymosin.

Parathymosin has been purified from calf liver and its primary sequence established, except for a segment containing approximately 11 amino acid residues in the central part of the polypeptide chain. Bovine parathymosin contains approximately 101 amino acid residues and shows 90% identity with rat parathymosin, with substitution of Glu for Asp at positions 21, 57, and 58, Asp for Glu at positions 60 and 63, and Ala for Val at position 77. Three non-conservative substitutions were Ala for Thr at position 81, Leu for Arg at position 78, and Val for Lys at position 79. The replacement at the last two positions of a pair of basic by hydrophobic amino acid residues may account for differences in chromatographic behavior observed for the bovine and rat polypeptides. Analysis of the NH2-terminus employed a new deblocking procedure which was also employed to analyze rat parathymosin, requiring correction of the previously published NH2-terminal sequence for that polypeptide.

Isolation and partial characterization of prothymosin alpha from porcine tissues.

Prothymosin alpha, an immunoactive polypeptide of 12 kDa, has been isolated from porcine thymus, spleen, lung and kidney. It lacks aromatic and sulfur-containing amino acids and has a high content of glutamic and aspartic acids. Tryptic digestion of porcine thymus prothymosin alpha yielded peptides which on separation, amino acid analysis and alignment with the known sequence of prothymosin alpha from rat and man showed that the amino terminal portion of the molecule is conserved and the few differences present are confined to the carboxy terminal.

Prothymosin alpha and parathymosin: amino acid sequences deduced from the cloned rat spleen cDNAs.

A rat spleen cDNA library was screened for clones carrying the cDNAs for prothymosin alpha and parathymosin. Sequence analysis of a clone carrying the entire coding region for prothymosin alpha confirmed and completed the amino acid sequence for this polypeptide and established the number of amino acid residues as 111. Rat prothymosin alpha differs from human prothymosin alpha at six positions, including four substitutions and two insertions. The nucleotide sequences of the cDNAs for the rat and human polypeptides are more than 90% identical in the open reading frames, with significant homology extending into the 5' and 3' flanking regions. From the same library, we also isolated a clone carrying 80% of the coding region for rat parathymosin. The number of amino acid residues in rat parathymosin is 101, based on the sequence deduced from the cDNA insert and earlier information on the sequence in the amino-terminal portion of this polypeptide. Despite their similarity in size and amino acid composition, rat prothymosin alpha and rat parathymosin show only limited sequence homology, primarily in the segment including residues 14 through 25, where 10 of 12 positions are identical in the two polypeptides. this is also the region of significant sequence similarity to a 12-amino-acid segment in the p17 protein of the human immunodeficiency disease associated virus (HTLV-IIIB).

An endogenous activator of the Ca2+-dependent proteinase of human neutrophils that increases its affinity for Ca2+.

An endogenous activator of the Ca2+-dependent proteinase (calpain) has been identified in human neutrophils. In the presence of the activator, the affinity of calpain for Ca2+ is increased by greater than 100-fold and maximum catalytic activity is observed with Ca2+ concentration below 1 microM. The activator is a heat-stable protein having an apparent molecular mass of approximately equal to 40 kDa. It appears to be associated with the cytoskeletal fraction of human neutrophils. Neutrophils also contain an endogenous cytosolic calpain inhibitor (calpastatin), which is readily separated from the activator by size-exclusion chromatography. The effects of the activator and inhibitor appear to be antagonistic and may constitute a physiological mechanism for modulating intracellular calpain activity.

Effects of a monoclonal anti-calpain antibody on responses of stimulated human neutrophils. Evidence for a role for proteolytically modified protein kinase C.

A monoclonal antibody directed against the Ca2+-requiring proteinase (calpain) of human neutrophils was employed to assess the role of this proteinase in mediating the responses to stimuli such as phorbol 12-myristate 13-acetate or fMet-Leu-Phe. In the presence of either phorbol 12-myristate 13-acetate or fMet-Leu-Phe the antibody is taken up by the neutrophils, and a marked inhibition of intracellular calpain is observed. The decreased calpain activity is accompanied by (a) a significant decrease in the proteolytic conversion of native protein kinase C (Ca2+/phospholipid-dependent enzyme) to the soluble form that does not require Ca2+ or phospholipids for activity; (b) a marked increase in the production of superoxide anion; and (c) a decrease in the exocytosis of granule contents. The increase in superoxide production can be attributed to a more prolonged association of native protein kinase C with the plasma membrane, thus enhancing the phosphorylation of membrane proteins that precedes O(2-) production (Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Michetti, M., Sacco, O., and Horecker, B. L. (1986), Biochem. Biophys. Res. Commun. 140, 1121-1126). The decreased exocytosis can be attributed to a decreased phosphorylation of certain cytoskeletal proteins, catalyzed by the soluble form of protein kinase C (Pontremoli, S., Melloni, E., Michetti, M., Sparatore, B., Salamino, F., Sacco, O., and Horecker, B. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3604-3608); the subsequent reorganization of the cytoskeleton appears to be related to degranulation. These effects of the monoclonal anti-calpain provide direct evidence for an essential role for calpain in the activation of human neutrophils.