Site Directed Mutagenesis on Serum Amyloid A Type II
Serum Amyloid A (SAA), a 102 amino acid protein expressed by the liver in response to inflammation, long has been known to associate with lipoproteins, specifically high-density lipoproteins (HDL). Recent studies have shown that SAA-containing HDLs are retained in atherosclerotic plaques. It is believed that the mechanism of SAA-containing HDL retention in plaques is through ionic interactions between positively-charged SAA amino acids and negatively-charged glycosaminoglycans (GAGs) of arterial wall proteoglycans. Research will focus on determining the specific amino acid residues on SAA that mediate this interaction with proteoglycan GAGs. One potential application of this project would be to design a drug that interferes with this interaction, thereby decreasing lipoprotein retention in developing atherosclerotic plaques. In this project, the student will perform the following: 1) isolate RNA from murine liver, 2) clone cDNAs for SAA1 and SAA2 from murine liver mRNA by reverse transcription/polymerase chain reaction (RT/PCR), 3) sequence the cDNA clones to confirm their identity and 4) perform PCR-based site-directed mutagenesis to generate a mutant SAA2 cDNA that encodes for alanines rather than basic amino acids at the 6 residues thought to mediate SAA2 binding to proteoglycans.
