Molecular cloning of cDNA for human prothymosin a - PNAS

Proc. Nati. Acad. Sci. USA Vol. 83j pp. 8926-8928, December 1986 Biochemistry

Molecular cloning of cDNA for human prothymosin a (thymosin/peptide secretion)

G. J. GOODALL*, FERNANDO DOMINGUEZtt, AND B. L. HORECKER* *Department of Biochemistry, Cornell University Medical College, 1300 York Avenue, New York, NY Roche Research Center, Nutley, NJ 07110

10021; and tRoche Institute of Molecular Biology,

Contributed by B. L. Horecker, August 19, 1986

ABSTRACT A cDNA library was constructed from human spleen mRNA and screened for clones containing eDNAs coding for prothymosin a. A clone containing a 503-base-pair insert including the entire coding sequence for the translated portion of the mRNA was isolated. The deduced amino acid sequence confirms and completes the partial sequence of human prothymosin a determined by protein sequencing methods. The presence of an initiator codon immediately preceding the codon for the NH2-terminal serine residue and of a terminator codon immediately following the codon for Asp-109, the COOHterminal residue, suggests that prothymosin a is synthesized without formation of a larger precursor polypeptide. Analysis of the 5' sequence preceding the initiator methionine codon excluded the presence of a signal peptide in the translated sequence.

EXPERIMENTAL PROCEDURES Preparation of a Human Spleen cDNA Library. Total RNA was prepared from a pathology laboratory waste specimen of human spleen by the guanidine thiocyanate method of Chirgwin et al. (10) and poly(A)+ RNA was separated as described by Aviv and Leder (11). cDNA was synthesized by using the RNase H method of Gubler and Hoffman (12). The plasmid vector was pMG5, a derivative of pBR322 from

which the BamHI site had been deleted and the sequence adjacent to the EcoRV site had been modified so as to generate two new BamHI sites after cleavage with EcoRV and addition of poly(dC) (Ueli Gubler, personal communication). This modification permits cDNA inserts to be retrieved from recombinant plasmids by BamHI digestion. The vector was linearized with EcoRV and prepared for annealing with cDNA as described (12), and the chimeric plasmids were used to transform Escherichia coli MC 1061 (13). Approximately 57,000 ampicillin-resistant transformants were obtained from 60 ng of double-stranded cDNA. Isolation of Prothymosin a cDNA Clones. Two mixedoligonucleotide probes were synthesized corresponding to regions of the rat prothymosin a sequence (Fig. 1). To screen for prothymosin clones, portions of the cDNA library were plated on nitrocellulose filters and replica filters were made. The colonies were lysed on the filters by the lysozyme/Triton X-100 method of Thayer (14). Hybridization with the oligonucleotide probes and washing of the filters was carried out under the conditions recommended by Wood et al. (15). The inclusion of tetramethylammonium chloride in the wash solutions was particularly important, since one of the probes (probe Ia) differed from a segment of the vector by only a single nucleotide. In an initial screening of 2500 colonies, a single clone was found that hybridized to both probe Ia and probe II. The plasmid from this clone contained an insert of approximately 240 base pairs (bp), and preliminary sequence analysis confirmed that it coded for a part of prothymosin a. An M13 phage subclone containing this cDNA was labeled by the method of Hu and Messing (16) and used as a hybridization probe to screen an additional 70,000 colonies from the cDNA library. Six clones that hybridized to the probe were isolated, and the sizes of their inserts were measured. The largest insert was approximately 565 bp long. This clone was named pHSpro565. Preparation of cDNA Inserts for Dideoxy Sequencing. The cDNA inserts containing the poly(dG-dC) tails were removed from recombinant plasmids by digestion with BamHI. It was necessary to remove the poly(dG-dC) tails before the insert could be sequenced by the M13-dideoxy method because the inserts released by BamHI subcloned directly in M13 yielded recombinant single-stranded DNAs containing the cDNA sequence flanked by a poly(dG) tract at one end and a poly(dC) tract at the other end. These formed an internal double-stranded region that, during sequencing, blocked the progress of the polymerase before it reached the cDNA sequence. To remove the poly(dG-dC) tails, we incubated 20 ,g of BamHI-digested plasmid for 10 min at 37°C in the presence of 33 mM Tris-acetate, pH 8.0/60 mM potassium acetate/10 mM magnesium acetate/0.5 mM dithiothreitol/ 0.1 mM dATP/0.1 mM dCTP/0.1 mM dTTP and 3 units of T4 DNA polymerase (Amersham) in a volume of 25 ,ul. After

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Abbreviation: bp, base pair(s). tPresent address: Department of Physiology, Faculty of Medicine, University of Santiago, Santiago de Compostela, Spain.

A peptide containing 28 amino acid residues, named thymosin a, (1), was originally isolated from calf thymosin fraction 5 (2) and shown to restore various aspects of immune function in several in vivo and in vitro test systems (ref. 3; for reviews see refs. 4 and 5). Thymosin a1 was subsequently isolated from a similar fraction from human thymus and reported to have the same amino acid sequence as bovine thymosin a1 (cited in ref. 7). However, thymosin a1 could not be isolated from fresh thymus (6, 7), suggesting that it might be a proteolytic fragment of a larger native polypeptide. We have recently reported the isolation from fresh rat thymus of a larger polypeptide, named prothymosin a, which contains the thymosin a1 sequence at its NH2 terminus (8). Prothymosin a has also been isolated from human thymus, and the identity of the thymosin a, portion of the amino acid sequence has been confimed (9). However, a number of amino acid substitutions and deletions were detected in other portions of human prothymosin a, particularly in the COOHterminal region. The human polypeptide was also found to be significantly less active than the rat polypeptide in restoring immune function in immunodeficient strains of mice (9). We undertook the cloning of the cDNA for human prothymosin a to complete and confirm the information on its primary sequence and also to determine whether or not the mRNA coded for a signal peptide that would support a role for this polypeptide as a secretory thymic hormone.

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Proc. Natl. Acad. Sci. USA 83 (1986)

Biochemistry: Goodall et al. Amino Acid Sequence

Asp

Asp

Giu

Asp

Asp

ASD

Val

GAA GG ~GACU

GACU

GAAG

GACU

GACU

GACU

GUN

3'

Probe Ia

3'

T CT C

CTAGG CTG CTGA

CA

5'

Probe lb

3'

CTT

CTA

CTA

CTG

CA

5'

c G

CTTC

crGA

CTGA

ciGA~

5'

Giu

Predicted mRNA Sequence S

Probe II

3'

CTTC

CTGA

A~

8927

FIG. 1. Oligonucleotides synthesized for use as probes. Note that the probe sequences are shown 3'-*5'. Probe I was made in two pools, with a single base difference, at position 6, counting from the 5' end. Duplicate filters were hybridized with each pool separately.

incubation the DNA was isolated by extraction with phenol and precipitation with ethanol, the latter carried out in the presence of 2 M ammonium acetate to minimize precipitation of dNTPs (17). The DNA was redissolved and treated with 1 unit of S1 nuclease for 15 min at 370C in 50 1ul of a solution containing 200 mM NaCl, 50 mM sodium acetate at pH 4.5, 1 mM ZnSO4, and 0.5% glycerol. The mixture was then treated with 2 ,ul of 0.5 M EDTA (pH 8) and 2.5 ,ul of 2 M Tris base, and the aqueous solution was extracted with phenol and then three times with diethyl ether, and the DNA was precipitated with ethanol. The DNA was made blunt-ended by incubation at 25°C for 30 min in 30 Al of 50 mM Tris HCl, pH 7.5/5 mM MgCl2/0.1 mM dithiothreitol/0.08 mM each of dATP, dCTP, dGTP, and dTTP, and 1 unit of the Klenow fragment of DNA polymerase. The cDNA was then separated from vector DNA by electrophoresis in 1% low-melting point agarose and the DNA was isolated by using an Elutip (Schleicher & Schuell) according to the manufacturer's recommendations. The DNA was ligated to Sma I-cut M13mp19 for sequencing by the dideoxy method of Sanger et al. (18). RESULTS Sequence of the cDNA Insert. The insert was found to contain 503 bp and to include the entire coding sequence for prothymosin a (Fig. 2). The deduced amino acid sequence completed the gap from residues 60 through 74 that could not be determined by conventional amino acid sequencing (9). This sequence contains 11 glutamic residues and is part of a glutamic acid-rich region extending from residue 53 through residue 82, accounting for 20 of the 34 glutamic residues in the polypeptide sequence. The amino acid sequence deduced from the cDNA sequence also confirms the previously reported differences in primary structure between rat prothymosin a and the human polypeptide, differences that appear to affect the biological activities of these peptides (9). The AUG codon immediately preceding the NH2-terminal serine residue must function as the initiator codon, because no other AUG sequence is present between the codon for the NH2-terminal serine and an in-frame terminator at position 79-81, 54 bases upstream from the initiator AUG codon. A terminator codon was also found to be present at positions 461 to 463, immediately following the codon for the COOHterminal aspartic acid. Thus, the only processing required to produce prothymosin a from the primary gene product would be removal of the NH2-terminal methionine residue and acetylation of the exposed NH2-terminal serine residue. The identification of the GAT codon for aspartic acid at the positions coding for residue 74 in the amino acid sequence was unexpected. In our previous analyses of the amino acid sequences of both rat and human prothymosin a (5, 9), we assigned asparagine to this position, based on the cleavage of

this site by hydroxylamine, a reagent reported to be specific for the Asn-Gly bond (19). The presence of a codon for aspartic acid in the cDNA clone studied may reflect allelic polymorphism at this site. Size of the mRNA for Prothymosin a. The size was established by blot hybridization analysis of the total RNAs isolated from the human fibroblast cell line WISH (supplied by Sidney Pestka) (Fig. 3). A single RNA species 1.4 kilobases in size was detected, much larger than the 503-bp cDNA insert in the clone pHSproS65. Much of the nontranslated region is lacking in this clone, including the AATAAA polyadenylylation signal (22).

DISCUSSION McClure et al. (23), using a radioimmunoassay, have reported the presence of thymosin al (or an immunologically related peptide) in human plasma. The results reported have TCC TTG CCC GCC GCA GTC GCC TCC GCC GCG CGC CTC CTC CGC CGC CGC 48

GGA CTC CGG CAG CTT TAT CGC CAG AGT CCC TGA ACT CTC GCT TTC TTT 96

TTA ATC CCC TGC ATC GGA TCA CCG GCG TGC CCC ACC ATG TCA GAC GCA 144 Met Ser Asp Ala

GCC GTA GAC ACC AGC TCC GM ATC ACC ACC MG GAC TTA AAG GAG AAG 192 Ala Val Asp Thr Ser Ser Glu Ile Thr Thr Lys Asp Leu Lys Glu Lys 10 MG GAA GTT GTG GAA GAG GCA GAA MT GGA AGA GAC GCC CCT GCT MC 240 Lys Glu Val Val Glu Glu Ala Glu Asn Gly Arg Asp Ala Pro Ala Asn 20 30 GGG AAT GCT AAT GAG GAA MC GGG GAG CAG GAG GCT GAC MT GAG GTA 288 Gly Asn Ala Asn Glu Glu Asn Gly Glu GIn Glu Ala Asp Asn Glu Val 40 50 GAC GAA GAA GAG GAA GAA GGT GGG GAG GAA GAG GAG GAG GAA GAA GM 336 Asp Glu Glu Glu Glu Glu Gly Gly Glu Glu Glu Glu Glu Glu Glu Glu 60 GGT GAT GGT GAG GAA GAG GAT GGA GAT GAA GAT GAG GAA GCT GAG TCA 384 Gly Asp Gly Glu Glu Glu Asp Gly Asp Glu Asp Glu Glu Ala Glu Ser 70 80 GCT ACG GGC AAG CGG GCA GCT GM GAT GAT GAG GAT GAC GAT GTC GAT 432 Ala Thr Gly Lys Arg Ala Ala Glu Asp Asp Glu Asp Asp Asp Val Asp 90

ACC AAG AAG CAG MG ACC GAC GAG GAT GAC TAG ACA GCA AAA MG GAA 480 Thr Lys Lys Gin Lys Thr Asp Glu Asp Asp --109 100 MG TTA AAC TAA MA AM AM AA 503

FIG. 2. Sequence of the prothymosin a cDNA and of the predicted polypeptide. The numbering of the residues of the polypeptide corresponds to that of mature prothymosin a, which has acetylserine at the NH2 terminus (9).

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Biochemistry: Goodall et al.

kb -9.5 7.4 4.4

2.4

-1.4

-

0.3

FIG. 3. Blot hybridization of RNA from the human fibroblast cell line WISH, probed with prothymosin a cDNA. Total RNA (2 lag) from WISH cells was electrophoresed on a 1% agarose gel containing formaldehyde (20). The RNA was transferred to a nylon membrane and hybridized with prothymosin

a

cDNA, labeled by random-

primed synthesis (21). The hybridized mRNA band was visualized by autoradiography. The mRNA markers (from Bethesda Research Laboratories) were visualized by staining with ethidium bromide. kb, Kilobases.

raised interesting questions about the origin of this material, since it is apparent that prothymosin a, the native intracellular form of thymosin a,, lacks the biosynthetic features associated with most secretory peptides. Not only the lack of a signal peptide sequence in the primary translation product but also the extremely hydrophilic nature of the native polypeptide and the absence of any significant hydrophobic regions make it unlikely that prothymosin a would be secreted by the usual mechanism evoked for secretory peptides. This absence of a hydrophobic signal peptide sequence is shared by at least two secreted peptidesnamely, interleukin 1 (24-26) and endothelial cell growth factor (27). Interleukin 1 has been reported to be membrane associated (28), and its release from activated monocytes is promoted by extracellular proteinases such as trypsin or plasmin (29). However, prothymosin a is a hydrophilic peptide (8), and analysis by the algorithm of Hopp and Woods (30) did not reveal the presence of any hydrophobic regions, rendering unlikely its association with the cell membrane. Its release from cells may be due to leakage from damaged or injured cells, as suggested for interleukin 1 (25). We are indebted to Dr. Ueli Gubler (Department of Molecular Genetics, Roche Research Center) for helpful discussions. This work was supported in part by Grant NP-534 from the American Cancer Society and by the Biomedical Research Support Grant to the Cornell University Medical College from the National Institutes of Health.

Proc. Natl. Acad. Sci. USA 83

(1986)

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