Age-Related Macular Degeneration-Associated Silent Polymorphisms in HtrA1 Impair Its Ability To Antagonize Insulin-Like Growth Factor 1

Abstract

Synonymous single nucleotide polymorphisms (SNPs) within a transcript's coding region produce no change in the amino acid sequence of the protein product and are therefore intuitively assumed to have a neutral effect on protein function. We report that two common variants of high-temperature requirement A1 (HTRA1) that increase the inherited risk of neovascular agerelated macular degeneration (NvAMD) harbor synonymous SNPs within exon 1 of HTRA1 that convert common codons for Ala34 and Gly36 to less frequently used codons. The frequent-to-rare codon conversion reduced the mRNA translation rate and appeared to compromise HtrA1's conformation and function. The protein product generated from the SNP-containing cDNA displayed enhanced susceptibility to proteolysis and a reduced affinity for an anti-HtrA1 antibody. The NvAMD-associated synonymous polymorphisms lie within HtrA1's putative insulin-like growth factor 1 (IGF-1) binding domain. They reduced HtrA1's abilities to associate with IGF-1 and to ameliorate IGF-1-stimulated signaling events and cellular responses. These observations highlight the relevance of synonymous codon usage to protein function and implicate homeostatic protein quality control mechanisms that may go awry in NvAMD. Single nucleotide polymorphisms (SNPs) that fall within the coding region have the potential to alter the amino acid sequence of a gene's product and therefore can serve as a Rosetta stone for understanding the pathogenesis of human disorders (1, 2). A curiosity that emerged from human genome-wide association studies is that exonic SNPs that do not alter the amino acid sequence of the protein product (i.e., SNPs that are synonymous) are as common as SNPs that do (i.e., SNPs that are nonsynonymous) (3). Furthermore, some of the disease-associated synonymous SNPs constitute the molecular underpinnings of pathology, meaning that they are enriched in affected human subjects and alter the gene product. For the majority (95%) of disease-associated synonymous SNPs, aberrant gene products were attributed to unstable mRNA transcripts with a reduced half-life or mutations in splice sites that resulted in exon skipping (4, 5). In other cases, synonymous SNPs caused translational defects independently of mRNA splicing errors (6-9).A parsimonious mechanism by which synonymous SNPs impact the integrity of protein products involves the alteration of codon usage. In vivo, folding of nascent proteins proceeds cotranslationally (10, 11) and is influenced by the rate of ribosome transit through the mRNA template. What distinguishes synonymous codons that encode a given degenerate amino acid is the abundance of their corresponding tRNA, which is lower for infrequently used codons than for frequently used codons (12, 13). Consequently, codon frequency can influence the rate of translation. Of note, codon bias has been widely described in prokaryotes and single-celled eukaryotes but less extensively in complex eukaryotes.In humans, correlations between optimum codon usage and either protein expression...

Rare codons capacitate Kras-driven de novo tumorigenesis

“…There is also emerging evidence that rare codons within mammalian genes can have biochemical or cellular consequences (4,(32)(33)(34)(35). We now show that altering rare codons.…”

“…Growth arrest of primary human cells. As previously described (23), IMR90 cells (pd [35][36][37] were infected with retroviruses derived from pBabePuro with no insert or the indicated HRAS or Kras oncogenes, selected with 2 μg/ml puromycin, and plated at 4,000 cells per well in 24-well dishes. On the indicated days, cells were fixed with 10% formalin and stained with 0.1% crystal violet (Sigma-Aldrich).…”

Insulin-like Growth Factor 1 (IGF-1) Stabilizes Nascent Blood Vessels

“…IGF-1 Stabilized Them-To study remodeling and destabilization of nascent capillaries in vitro, tubes were generated uniformly from primary HMRECs treated with VEGF-A in a modified version of the collagen gel sandwich assay described previously (19,20). When such retinal endothelial tubes formed, they were stable and did not significantly remodel in terms of total length or branch points for at least 4 h in serum-and growth factor-free medium (Fig.…”

“…Bovine microvascular retinal endothelial cells were isolated from 3-month-old bovine eyes (Research 87, Inc., Boylston, MA) and used at P4-P10. Cells were plated on tissue culture dishes coated with 0.2% gelatin (Sigma) and maintained in M199 medium that contained 20% bovine calf serum (Hyclone, Logan, UT), 100 g/ml heparin, 12 g/ml bovine pituitary extract (Hammond Cell Tech, Windsor, CA), and 100 units/ml penicillin G and 100 g/ml streptomycin C (Gemini Bioproducts, West Sacramento, CA) as described previously (19,20). Cell culture medium was replaced every 2 days unless cultures needed to be passaged for in vitro tube assays.…”

“…For regression studies, endothelial tubes were generated as described previously (19,20) with a few modifications. 48 h prior to the tube regression assay, cells were collected in trypsin (0.5% in EDTA, 2 min at 37°C).…”