Three distinct human thymopoietins are derived from alternatively spliced mRNAs.

Abstract

Thymopoietin (T7P) was orignlly sated as a 5-kDa 49-aa protein from bovine thymus in studies of the effects of thymic extracts on neuromuscular tAsi n and was subsequently observed to affect T-cell differentiation and function. We now report the Isolation of cDNA clones for three alternatively spliced mRNAs that encode three distinct human T-cell TPs. Proteins encoded by these mRNAs, whih we have Thymopoietin (TP) was originally isolated as a 5-kDa polypeptide from bovine thymus on the basis ofits ability to affect neuromuscular transmission when injected into mice (1). Subsequently, purified 5-kDa TP was found to affect certain immunological functions, inducing differentiation of prothymocytes to thymocytes, as measured by changes in expression of Thy-1 and other cell surface phenotypic markers (2), and enhancing allogeneic responses of peripheral T cells (3). The 5-kDa TP and a synthetic peptide corresponding to aa 32-36 of TP, Arg-Lys-Asp-Val-Tyr (thymopentin) gave similar results in both neurophysiological and immunological assays, suggesting that thymopentin may act as a TP mimetic (4, 5). Furthermore, in clinical studies with thymopentin being used as a putative immunoregulatory drug, promising findings have been obtained in double-blind, placebocontrolled studies of subjects with rheumatoid arthritis (6), recurrent herpes simplex (7), atopic dermatitis (8), and asymptomatic human immunodeficiency virus infection (9).To better define the cellular function(s) of TP and to provide a molecular basis for the mechanism of action of thymopentin, we isolated and characterized cDNAs encoding this protein. previously isolated a bovine cDNA that encodes the originally determined 49-aa bovine TP sequence (11, 12) at the 5' end of a larger open reading frame. Here we report the isolation, using a probe derived from the bovine sequence of Zevin-Sonkin et al. (10), of human TP cDNA clones that encode three distinct human TP proteins, derived from three alternatively spliced mRNAs, each ofwhich contains a sequence at the N terminus that is homologous to the originally determined bovine 49-aa TP sequence. TPa (75 kDa), TP(3 (51 kDa), and TPy(39 kDa), contain some domains in common and some unique domains,

The characterization and localization of the mouse thymopoietin/lamina-associated polypeptide 2 gene and its alternatively spliced products.

“…Mouse Tmpo cx, [3, 8, e, % and ~ share an identical aminoterminal 186-amino-acid domain. Like the hum a n and the bovine Tmpo isoforms, amino acids 1-49 are highly homologous to the originally purified 49-amino-acid bovine Tmpo (Schlesinger and Goldstein 1975 are 78%, 90%, and 91% identical to the previously published h u m a n TMPO u, [3, and y sequences (Harris et al 1994), respectively, suggesting interspecies conserved functions.…”

“…Nucleotides of the intronic sequences are in lowercase. The numbers that flank the exonic sequences correspond to the numbered base pairs of the human TMPOI3 gene (Harris et al 1994).…”

“…The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the recombination frequencies between the loci are shown in Figure 7. Tmpo ~, [3, and ~/mRNAs appear to vary in different tissues and cell lines (Harris et al 1994;Berger et al 1995), suggesting that both expression and alternative splicing of Tmpo may be regulated in a tissue-specific manner. One possible mechanism for control of the formation of Tmpo c~ and Identical sequences between the murine and the human 3'-splice sites are in boldface type and underlined.…”

“…The following human TMPO~ oligonucleotide primers were synthesized: 5'-AGCTATTCTCAAGCTGGAA-3', sense nucleotides 661-680 and 5'-TTCAGCTCTTGTCAATGG-3', antisense nucleotides 987-970 (Harris et al 1994). A 50-~1 reaction mixture containing 500-1000 ng of placental genomic human DNA in 50 mM KC1, 10 mM Tris-HC1 (pH 8.3), 1.5 mM MgC12, 0.001% gelatin, 250 mM (each) dNTPs: dATP, dCTP, dGTP, dTTP, 20 pmoles of each of the primers, and I unit of Taq DNA polymerase was subjected to 35 cycles of amplification.…”

“…Isolation of a bovine thymopoietin cDNA (Zevin-Sonkin et al 1992) and subsequently hum a n cDNAs, encoding three related but distinct TMPOs (TMPO c~, 13, and ~/) (Harris et al 1994), expanded our knowledge about Tmpo. Human TMPO c~, ~, and ~/ share an identical aminoterminal domain of 187 amino acids, which is followed in TMPO~ by a specific domain (506 amino acids).…”