Phospholipid transfer protein is present in human tear fluid.
The human tear fluid film consists of a superficial lipid layer, an aqueous middle layer, and a hydrated mucin layer located next to the corneal epithelium. The superficial lipid layer protects the eye from drying and is composed of polar and neutral lipids provided by the meibomian glands. Excess accumulation of lipids in the tear film may lead to drying of the corneal epithelium. In the circulation, phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) mediate lipid transfers. To gain insight into the formation of tear film, we investigated whether PLTP and CETP are present in human tear fluid. Tear fluid samples were collected with microcapillaries. The presence of PLTP and CETP was studied in tear fluid by Western blotting, and the PLTP concentration was determined by ELISA. The activities of the enzymes were determined by specific lipid transfer assays. Size-exclusion and heparin-affinity chromatography assessed the molecular form of PLTP. PLTP is present in tear fluid, whereas CETP is not. Quantitative assessment of PLTP by ELISA indicated that the PLTP concentration in tear fluid, 10.9 +/- 2.4 microg/mL, is about 2-fold higher than that in human plasma. PLTP-facilitated phospholipid transfer activity in tears, 15.1 +/- 1.8 micromol mL(-)(1) h(-)(1), was also significantly higher than that measured in plasma. Inactivation of PLTP by heat treatment (+58 degrees C, 60 min) or immunoinhibition abolished the phospholipid transfer activity in tear fluid. Size-exclusion chromatography of tear fluid indicated that PLTP eluted in a position corresponding to a size of 160-170 kDa. Tear fluid PLTP was quantitatively bound to Heparin-Sepharose and could be eluted as a single peak by 0.5 M NaCl. These data indicate that human tear fluid contains catalytically active PLTP protein, which resembles the active form of PLTP present in plasma. The results suggest that PLTP may play a role in the formation of the tear film by supporting phospholipid transfer.
Jauhiainen
Matti
M
Department of Molecular Medicine, National Public Health Institute, Biomedicum, Helsinki, Finland.
Setälä
Niko L
NL
Ehnholm
Christian
C
Metso
Jari
J
Tervo
Timo M T
TM
Eriksson
Ove
O
Holopainen
Juha M
JM
D016632:Apolipoprotein A-I / Q000737:chemistry; D001057:Apolipoproteins E / Q000737:chemistry; D001693:Biological Transport, Active; D015153:Blotting, Western / Q000379:methods; D002352:Carrier Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D007106:Immune Sera / Q000737:chemistry; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism; D049294:Phospholipid Transfer Proteins / Q000037:antagonists & inhibitors / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives / Q000378:metabolism; D013666:Tears / Q000737:chemistry* / Q000201:enzymology / Q000378:metabolism
D016632:Apolipoprotein A-I; D001057:Apolipoproteins E; C506642:CETP protein, human; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters; D006023:Glycoproteins; D007106:Immune Sera; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; C037405:heparin-sepharose; D006493:Heparin; D012685:Sepharose