The Role of Specific Basic Amino Acids in SAA-Mediated Arterial Wall Lipid Retention: Cloning, site-directed mutagenesis, and the generation of SAA adenoviral vectors
Serum Amyloid A (SAA), a 102 amino acid protein expressed by the liver in response to inflammation, is associated with lipoproteins, specifically high-density lipoproteins (HDL). Recent studies have shown that SAA-containing HDLs are retained within atherosclerotic plaques. The mechanism of SAA-containing HDL retention in plaques may involve ionic interactions between positively-charged SAA amino acids and negatively-charged glycosaminoglycans (GAGs) of arterial wall proteoglycans (PGs). This project will focus on determining the specific amino acid residues on SAA that mediate the interaction with proteoglycan GAGs. One potential application of this project would be to design a drug that would interfere with this interaction, thereby decreasing lipoprotein retention in developing plaques. Goals for this project will include: 1) isolation of RNA from murine liver, 2) cloning of cDNAs for SAA1 and SAA2 from murine liver mRNA by reverse transcription/polymerase chain reaction (RT/PCR), 3) sequencing of the cDNA clones to confirm their identity and 4) PCR-based site-directed mutagenesis to generate a mutant SAA2 cDNA that encodes for neutral rather than basic amino acids at the 6 residues thought to mediate SAA2 binding to PGs, 5) generation of adenoviral vectors containing wild-type, 3-amino acid, and 6-amino acid mutant murine SAA2.
