Abstract
Purpose:
Single nucleotide polymorphisms (SNPs) within a transcript’s coding region that do not change the amino acid sequence of the protein product are intuitively assumed to have no effect on protein properties and function. The synonymous SNPs rs1049331 and rs2293870 within the exon 1 of HTRA1 are associated with increased risk of neovascular age-related macular degeneration (NvAMD), and convert the common codons for Ala34 and Gly36 to least frequently used codons. The purpose of this project was to assess the impact of these SNPs on HtraA1’s properties and functions.
Methods:
To evaluate the effect of optimum-to-rare codon conversion on the rate of protein synthesis, synthetic mRNA templates encoding HtrA1+SNPs or WT HtrA1 were used in in vitro translation. We evaluated the effect of the synonymous SNPs on protein structure by partial proteolysis and immunoprecipitation with a conformation-sensitive antibody. The expression level of HtrA1 in neovascular AMD (NvAMD) patients homozygotic for rs1049331 and rs2293870 were compared to age-matched unaffected controls. Given that rs1049331 and rs2293870 lie within HtrA1’s Mac25 domain, we evaluated the ability of HtrA1 to influence IGF-1 signaling in endothelial cells.
Results:
We report that this common-to-rare codon conversion reduced mRNA translation rate, increased susceptibility to partial proteolysis, and reduced recognition by an antibody raised against the region of HtrA1 that includes exon 1. Exon 1 encodes a Mac25 domain, which is structurally very similar to proteins that bind insulin-like growth factor-1 (IGF-1). The NvAMD-associated SNPs reduced HtrA1’s ability to antagonize IGF-1-stimulated signaling events, and to suppress IGF-1-dependent tube formation of primary human choroidal endothelial cells.
Conclusions:
These observations demonstrate the potential of synonymous SNPs within HTRA1 to impact its previously unappreciated ability to titrate IGF-1, and suggest that the loss of this capacity contributes to pathogenesis of NvAMD.
Keywords: 412 age-related macular degeneration •
539 genetics •
714 signal transduction