ABSTRACT
Transmembrane protein CD36 binds multiple ligands, including oxidized low-density lipoproteins (oxLDLs) and long-chain fatty acids (LCFAs). Our aim was to determine whether LCFAs compete with oxLDLs for binding to CD36. We addressed this issue by examining the inhibitory effect of LCFAs against the binding of Alexa-fluor-labeled oxLDLs (AFL-oxLDL) to a synthetic peptide representing the oxLDL-binding site on CD36 (3S-CD36150â168). All of the unsaturated LCFAs tested, inhibited the binding of AFL-oxLDL to 3S-CD36150â168, albeit to varying degrees. For instance, the concentrations required for 50% inhibition of binding for oleic, linoleic, and α-linolenic acids were 0.25, 0.97, and 1.2 mM, respectively. None of the saturated LCFAs tested (e.g. stearic acid) exhibited inhibitory effects. These results suggest that at least unsaturated LCFAs can compete with oxLDLs for binding to CD36. The study also provides information on the structural requirements of LCFAs for inhibition of oxLDLs-CD36 binding.
Subject(s)
CD36 Antigens/metabolism , Fatty Acids, Unsaturated/chemistry , Fatty Acids, Unsaturated/pharmacology , Lipoproteins, LDL/metabolism , Amino Acid Sequence , CD36 Antigens/chemistry , Glycerophospholipids/metabolism , HEK293 Cells , Humans , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein BindingABSTRACT
CD36 binds oxidized low-density lipoprotein (oxLDL). A synthetic peptide comprising amino-acid residues 149-168 of mouse CD36 was recently found to bind fluorescence-labeled oxLDL particles. Based on our oxLDL-binding analysis of various synthetic CD36 peptides, we suggest that not only hydrophilic residues (e.g., Lys164 and Lys166) but also hydrophobic ones (e.g., Phe153, Leu158, and Leu161) are critical to binding.
Subject(s)
CD36 Antigens/chemistry , CD36 Antigens/metabolism , Lipoproteins, LDL/metabolism , Amino Acid Sequence , Animals , Biotin/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Ligands , Mice , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Binding , Substrate SpecificityABSTRACT
CD36 is an integral membrane protein that mediates the cellular uptake of oxidized low-density lipoprotein (oxLDL) through recognition of the oxidized glycerophospholipids (oxPLs) formed during LDL oxidation. We aimed to devise an assay system to detect binding between CD36 and oxLDL/oxPL without using recombinant proteins. A peptide corresponding to amino-acid residues 149-168 of mouse CD36 with biotin at its N-terminus (named biotin-CD36(149-168)) and variants of it were synthesized and immobilized onto streptavidin-coated plates. oxLDL labeled with Alexa-Fluor-488 bound specifically and saturably to immobilized biotin-CD36(149-168), but poorly or not at all to the variants, such as that with a scrambled amino-acid sequence. The binding of fluorescence-labeled oxLDL to biotin-CD36(149-168) was inhibited efficiently by an oxPL species, but not by a nonoxidized glycerophospholipid. This assay system using biotin-CD36(149-168) provides a convenient means not only of characterizing binding profiles between CD36 and oxLDL/oxPL but also of finding competitors for the binding.
Subject(s)
Biological Assay , Biotin/chemistry , CD36 Antigens/chemistry , Fluorescent Dyes/chemistry , Lipoproteins, LDL/analysis , Lipoproteins, LDL/chemistry , Peptides/chemical synthesis , Amino Acid Sequence , Animals , Binding Sites , Binding, Competitive , Mice , Molecular Sequence Data , Peptides/chemistry , Protein BindingABSTRACT
Reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR were used to detect 14 (6.6%) influenza C virus (InfC) among 213 clinical samples collected from children with respiratory symptoms in Mie Prefecture, Japan, between January 2012 and December 2012. Virus isolation using Madin-Darby canine kidney cells and/or embryonated chicken eggs was also successful for 3 of the 14 PCR-positive samples. Eleven patients (78.6%) were aged <3 years. Phylogenetic analysis of the hemagglutinin-esterase gene showed that the InfC detected in Mie Prefecture belonged to the C/Sao Paulo/82-related lineage. To determine the seroprevalence of InfC, a total of 575 serum samples from patients aged 1 month to 69 years in Mie Prefecture were screened by hemagglutination inhibition test using the C/Mie/199/2012 (C/Sao Paulo/82-related lineage) strain as the antigen. The samples with an antibody titer of ≥1:16 were designated as antibody-positive. The results showed that 53.7% of the 296 serum samples collected in 2011 and 85.3% of the 279 samples collected in 2012 were positive for antibodies against InfC, suggesting that an outbreak of InfC infection occurred in Mie Prefecture in 2012. Therefore, continuous and proactive monitoring is important to determine the number of InfC-infections and to better understand the epidemiology.