Accelerated evolution of Fetuin-A (FETUA, also AHSG) is driven by positive Darwinian selection, not GC-biased gene...

Human Fetuin-A (FETUA, also termed AHSG) is a serum protein composed of two cystatin-like domains D1 and D2 of together 235 amino acids (aa) and an unrelated domain D3 of 114 aa. Though the protein plays a role in diverse physiological and pathological processes, comparably little is known about sequence evolution of FETUA. We therefore analyzed its molecular evolution on the basis of coding sequences of 16 primate species. Ratios of non-synonymous to synonymous substitution rates (d(n)/d(s)=omega) suggest that a previously reported acceleration of sequence evolution of exon 7, which encodes domain D3, is driven by positive selection instead of neutral evolution. Irrespective of the maximum likelihood model used, a remarkable 40% of codon sites is estimated to be positively selected in domain D3, with an average omega of 4.3. Analyses of GC/AT- and AT/GC-substitutions reveal that GC-biased gene conversion (gBGC) cannot explain the observed pattern. We thus reject the extended null model of molecular evolution (neutral evolution and gBGC) and conclude that positive Darwinian selection contributes to the sequence evolution of FETUA domain D3. Remarkably, domain D3 of human FETUA is characterized by a significant accumulation of proteolytic cleavage sites. Though speculative at present, the accelerated evolution of domain D3 might be due to the evolution of differential cleavage site patterns across primates reflecting different paths to achieve one goal, the decomposition of FETUA under pathological conditions. Present analyses use a PERL-script ("Substitution Bias Indicator", SBI) for the extraction of the numbers of GC/AT- and AT/GC-substitutions from supplemental results files of PAML analyses. The script is available upon request and the presented approach of testing the extended null hypothesis of molecular evolution can be transferred to every other protein coding locus.