Plasma phospholipid transfer protein fused with green fluorescent protein is secreted by HepG2 cells and displays phosphatidylcholine transfer activity.
Phospholipid transfer protein (PLTP) is a serum glycoprotein with a central role in high-density lipoprotein metabolism. We created a fusion protein in which enhanced green fluorescent protein (EGFP) was fused to the carboxyl-terminus of PLTP. Stably transfected HepG2 cells, which overexpress this fusion protein, were generated. PLTP-EGFP was translocated into the ER and fluoresced within the biosynthetic pathway, showing a marked concentration in the Golgi complex. The transfected cells secreted into the growth medium phospholipid transfer activity 7-fold higher than that of the mock-transfected controls. The medium of the PLTP-EGFP - expressing cells displayed EGFP fluorescence, demonstrating that both the PLTP and the EGFP moieties had attained a biologically active conformation. However, the specific activity of PLTP-EGFP in the medium was markedly reduced as compared with that of endogenous PLTP. This suggests that the EGFP attached to the carboxyl-terminal tail of PLTP interferes with the interaction of PLTP with its substrates or with the lipid transfer process itself. Fluorescently tagged PLTP is a useful tool for elucidating the intracellular functions of PLTP and the interaction of exogenously added PLTP with cells, and will provide a means of monitoring the distribution of exogenously added PLTP between serum lipoprotein subspecies.
Siggins
Sarah
S
Department of Molecular Medicine, National Public HealthInstitute, Biomedicum, Helsinki, Finland.
Ehnholm
Christian
C
Jauhiainen
Matti
M
Olkkonen
Vesa M
VM
D002460:Cell Line; D003470:Culture Media; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008856:Microscopy, Fluorescence; D010713:Phosphatidylcholines / Q000378:metabolism; D049294:Phospholipid Transfer Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D003470:Culture Media; C493430:PLTP protein, human; D010713:Phosphatidylcholines; D049294:Phospholipid Transfer Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins