Biosynthesis and immunobiochemical characterization of gp17/GCDFP-15. A glycoprotein from seminal vesicles and from breast tumors, in HeLa cells and in Pichia pastoris yeast.
The gp17 factor is a secretory product of human seminal vesicle cells which binds to CD4 and acts as a potent inhibitor of T lymphocyte apoptosis induced by CD4 crosslinking and subsequent T-cell receptor (TCR) activation. The protein is identical to gross cystic disease fluid protein-15 (GCDFP-15), a breast tumor secretory marker PIP (prolactin inducible protein), a prolactin-controlled and androgen-controlled protein; secretory actin binding protein (SABP), a seminal plasma actin binding protein and extra-parotid glycoprotein (EP-GP), a secretory protein from the salivary gland. The structure of this protein has not yet been elucidated and no biological function has been clearly attributed to date. Expression of recombinant gp17/GCDFP-15 cDNA in bacteria and insect cells leads to the production of a misfolded insoluble protein. In this study, we describe the production of gp17/GCDFP-15 in two different eukaryotic systems, namely HeLa cells and the Pichia pastoris yeast. Using constructs in which gp17/GCDFP-15 was tagged with enhanced green fluorescent protein (EGFP) in various combinations, we observed expression only when the fusion protein was directed to the secretory compartment by the correct signal peptide. The resulting fluorescent protein was inefficiently secreted, thus suggesting that gp17/GCDFP-15 is not appropriately post-translationally processed and/or transported in HeLa cells. The use of the P. pastoris secretory pathway allowed instead the accumulation in the culture medium of a GCDFP-15/gp17 species which retained the ability to bind to CD4 and also most of the biochemical and immunological properties of the native protein. The production of an active recombinant molecule opens the way to correlate the structural properties of this peculiar factor to its ability to bind several proteins, including CD4, and to block CD4-mediated T cell programmed death.
Caputo
E
E
International Institute of Genetics and Biophysics, Naples, Italy. caputo@iigbna.iigb.na.cnr.it
Carratore
V
V
Ciullo
M
M
Tiberio
C
C
Mani
J C
JC
Piatier-Tonneau
D
D
Guardiola
J
J
D001053:Apolipoproteins; D053399:Apolipoproteins D; D015704:CD4 Antigens / Q000737:chemistry*; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins; D008297:Male; D026901:Membrane Transport Proteins; D010843:Pichia; D011485:Protein Binding; D011994:Recombinant Proteins; D012669:Seminal Vesicles / Q000737:chemistry*; D014162:Transfection
C024328:APOD protein, human; D001053:Apolipoproteins; D053399:Apolipoproteins D; D015704:CD4 Antigens; D002352:Carrier Proteins; D006023:Glycoproteins; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; C488892:PIP protein, human; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins