Molecular cloning and functional expression of cDNA encoding the pig plasma phospholipid transfer protein.
Humans and the pig show marked similarities in lipoprotein metabolism; therefore, the pig has been used as a model in numerous nutritional studies. Pig plasma displays no activity of cholesteryl ester transfer protein (CETP), which is known to be responsible for half of the phospholipid mass transfer in human plasma, the other half being accounted for by the plasma phospholipid transfer protein (PLTP). This makes the pig an ideal subject for the study of PLTP structure and function. Here we report the molecular cloning of pig PLTP and the eukaryotic cell expression of its complementary DNA. Pig PLTP was found to share 93% amino acid sequence identity with human PLTP and 81% with mouse PLTP. Tissue expression of PLTP mRNA was examined by a method based on reverse transcription-polymerase chain reaction (RT-PCR) and solid-phase minisequencing in nine pig tissues. The highest PLTP mRNA levels were found in the pancreas, brain, lung, and liver. Medium from COS-1 cells expressing PLTP possessed phospholipid transfer activity, and the secreted recombinant PLTP was detectable by Western blotting in the culture supernatant. A mutant protein with a substitution of Cys at position 22 by Arg was found to display impaired secretion into growth medium indicating a role for cysteines in the correct folding of PLTP. This study forms the basis for future work on the structure-function relationships in pig PLTP.
Pussinen
P J
PJ
Department of Biochemistry, National Public Health Institute, Helsinki, Finland.
Olkkonen
V M
VM
Jauhiainen
M
M
Ehnholm
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D053480:Cholesterol Ester Transfer Proteins; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression; D006023:Glycoproteins; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013552:Swine / Q000235:genetics*; D014162:Transfection
C506642:CETP protein, human; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D018076:DNA, Complementary; D006023:Glycoproteins; D011994:Recombinant Proteins