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. 1997 Dec 23;94(26):14444-9.
doi: 10.1073/pnas.94.26.14444.

Generation of secretable and nonsecretable interleukin 15 isoforms through alternate usage of signal peptides

Y Tagaya  1 , G Kurys, T A Thies, J M Losi, N Azimi, J A Hanover, R N Bamford, T A Waldmann
Affiliations

Generation of secretable and nonsecretable interleukin 15 isoforms through alternate usage of signal peptides

Y Tagaya et al. Proc Natl Acad Sci U S A. .

Abstract

Two isoforms of human interleukin 15 (IL-15) exist. One isoform has a shorter putative signal peptide (21 amino acids) and its transcript shows a tissue distribution pattern that is distinct from that of the alternative IL-15 isoform with a 48-aa signal peptide. The 21-aa signal isoform is preferentially expressed in tissues such as testis and thymus. Experiments using different combinations of signal peptides and mature proteins (IL-2, IL-15, and green fluorescent protein) showed that the short signal peptide regulates the fate of the mature protein by controlling the intracellular trafficking to nonendoplasmic reticulum sites, whereas the long signal peptide both regulates the rate of protein translation and functions as a secretory signal peptide. As a consequence, the IL-15 associated with the short signal peptide is not secreted, but rather is stored intracellularly, appearing in the nucleus and cytoplasmic components. Such production of an intracellular lymphokine is not typical of other soluble interleukin systems, suggesting a biological function for IL-15 as an intracellular molecule.

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Figures

Figure 1
Figure 1
(A) Expression of a 1.2-kb IL-15 mRNA in testis. Total RNA from monkey tissues was electrophoresed, blotted onto Hybond N+ membrane, and probed by a DNA fragment of the human IL-15 entire coding region. Origin of RNA: a, kidney; b, testis; c, liver; d, pancreas; e, human HTLV-I(+) HuT 102 line; f, skeletal muscle; g, spleen; h, lung. HuT 102 expresses a chimeric IL-15 mRNA consisting of HTLV-I long terminal repeat and IL-15 (5) and this transcript migrates faster than the classical 1.5-kb IL-15 mRNA. Positions of 1.5-kb and 1.2-kb IL-15 transcripts are indicated. (B) Tissue expression of the mRNA encoding the SSP-IL-15. Human tissue blots (Northern Territory, Invitrogen) were probed by exon 4a fragment (see text for nomenclature). Origin of RNA: a, ovary; b, testis; c, prostate; d, gall bladder; e, lymphocytes; f, appendix; g, thymus; h, tonsil; i, skeletal muscle; j, spleen; k, pancreas; l, lung; m, liver; n, kidney; o, brain; p, heart.
Figure 2
Figure 2
The structure of the testis-derived IL-15 clone 9A1. Genomic architecture of the human IL-15 gene (Middle), structures of the classical IL-15 (Top) and testicular IL-15 (Bottom) are shown. Numbers over each drawing indicates the exon numbering (11).
Figure 3
Figure 3
(A) IL-15 activity from COS supernatants transfected with LSP-IL-15 or SSP-IL-15 expression constructs. COS cells were transfected with the LSP-IL-15 (○), or the SSP-IL-15 (•) construct by electroporation, and the supernatants were collected 48 hr after transfection. The supernatants were tested by the CTLL-2 bioassay to measure IL-15 activity. (B) Immunoblotting of COS lysates expressing LSP-IL-15 or SSP-IL-15. COS cell lysates were blotted onto poly(vinylidene fluoride) membrane (Immobilon-P, Millipore) and probed with an anti-human IL-15 polyclonal antibody. The lower arrow indicates the fully processed SSP-IL-15, and the upper arrow indicates the IL-15 with the full 21-aa SSP. In the LSP-IL-15 lane, the multiple bands were observed because of glycosylation and the cleavage of the SP. Position of the protein size marker is indicated: 46 kDa, 33 kDa, 21.5 kDa, and 14.3 kDa, from top to bottom.
Figure 5
Figure 5
(A) Fluorescence of the COS cells transfected with various GFP constructs. COS cells were transfected with GFP constructs with various SPs (IL-2Rα SP, IL-15 LSP, and IL-15 SSP). After 48 hr, GFP fluorescence was analyzed by the FACSort (Becton–Dickinson) analyzer. (B) Rabbit reticulocyte in vitro translation using IL-15 or IL-2 constructs. Proteins were labeled with [35S]methionine by using rabbit reticulocyte in vitro translation system (Ambion), immunoprecipitated, and electrophoresed. Because the IL-15 LSP is longer (48 amino acids) than the IL-15 SSP (21 amino acids) or the wt IL-2 SP (21 amino acids), fusion proteins associated with the IL-15 LSP migrate slower than other proteins.
Figure 4
Figure 4
(A) IL-2 activity detected in the supernatant of COS cells transfected with IL-15 LSP-IL-2, IL-15 SSP-IL-2, or wt IL-2. COS cells transfected the murine IL-2 (mIL-2) constructs with wt IL-2 SP (○), IL-15 LSP (□), or the IL-15 SSP (▪) were cultured for 48 hr. The supernatants of the transfected COS cells were serially diluted and assessed by using the murine CTLL-2 cell line for their secreted IL-2 activity. (B) Immunofluorescence of the COS cells expressing GFP proteins linked with various signal peptides. COS cells transfected with the GFP constructs with various SPs (a, no SP; b, IL-2Rα SP; c, IL-15 LSP; d, IL-15 SSP) were analyzed for their fluorescence by inverted immunofluorescence microscopy. (C) Confocal microscopic analysis of the COS cells expressing the IL-15 LSP-GFP or the IL-15 SSP-GFP protein. COS cells transfected the IL-15 LSP-GFP or the IL-15 SSP-GFP constructs were fixed and analyzed by scanning confocal immunofluorescence microscopy. (a) COS cells transfected with the IL-15 LSP-GFP construct; (b) COS cells transfected with the IL-15 SSP-GFP construct.
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