Development of a new high-throughput screening model for human high density lipoprotein receptor (CLA-1) agonists.
OBJECTIVE
To develop a new high-throughput screening model for human high-density lipoprotein (HDL) receptor (CD36 and LIMPII analogous-1, CLA-1) agonists using CLA-1-expressing insect cells.


METHODS
With the total RNA of human hepatoma cells BEL-7402 as template, the complementary DNA (cDNA) of CLA-1 was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Bac-to-Bac baculovirus expression system was used to express CLA-1 in insect cells. CLA-1 cDNA was cloned downstream of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV) into donor vector pFastBac1 and recombinant pFastBac1-CLA-1 was transformed into E. coli DH10Bac to transpose CLA-1 cDNA to bacmid DNA. Recombinant bacmid-CLA-1 was transfected into Spodoptera frugiperda Sf9 insect cells to produce recombinant baculovirus particles. Recombinant CLA-1 was expressed on the membrane of Sf9 cells infected with the recombinant baculoviruses. A series of parameters of DiI-lipoprotein binding assays of CLA-1-expressing Sf9 cells in 96-well plates were optimized.


RESULTS
Western blot analysis and DiI-lipoprotein binding assays confirmed that CLA-1 expressed in insect cells had similar immunoreactivity and ligand binding activity as its native counterpart. A reliable and sensitive in vitro cell-based assay was established to assess the activity of CLA-1 and used to screen agonists from different sample libraries.


CONCLUSIONS
Human HDL receptor CLA-1 was successfully expressed in Sf9 insect cells and a novel high-throughput screening model for CLA-1 agonists was developed. Utilization of this model allows us to identify potent and selective CLA-1 agonists which might possibly be used as therapeutics for atherosclerosis.
Tian
De-Feng
DF
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Hong
Bin
B
Si
Shu-Yi
SY
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D001681:Biological Assay; D002232:Carbocyanines / Q000378:metabolism; D045744:Cell Line, Tumor; D008076:Cholesterol, HDL / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008075:Lipoproteins, HDL / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000378:metabolism; D018110:Receptors, Lipoprotein / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051122:Scavenger Receptors, Class B / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000235:genetics* / Q000378:metabolism
D002232:Carbocyanines; D008076:Cholesterol, HDL; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D018110:Receptors, Lipoprotein; D011994:Recombinant Proteins; C495124:SCARB1 protein, human; D051122:Scavenger Receptors, Class B; C032629:acetyl-LDL; C031616:high density lipoprotein receptors; C024286:3,3'-dioctadecylindocarbocyanine