Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and a deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity. The disease is highly variable in its clinical expression, because of various mutations in the TNSALP gene. In approximately 14% of the patients tested in our laboratory, only one TNSALP gene mutation was found, despite exhaustive sequencing of the gene, suggesting that missing mutations are harbored in intron or regulatory sequences or that the disease is dominantly transmitted. The distinction between these two situations is of importance, especially in terms of genetic counseling, but dominance is sometimes difficult to conclusively determine by using familial analysis since expression of the disease may be highly variable, with parents of even severely affected children showing no or extremely mild symptoms of the disease. We report here the study of eight point mutations (G46 V, A99T, S164L, R167 W, R206 W, G232 V, N461I, I473F) found in patients with no other detectable mutation. Three of these mutations, G46 V, S164L, and I473F, have not previously been described. Pedigree and/or serum alkaline phosphatase data suggested possible dominant transmission in families with A99T, R167 W, and G232 V. By means of site-directed mutagenesis, transfections in COS-1 cells, and three-dimensional (3D) modeling, we evaluated the possible dominant effect of these eight mutations. The results showed that four of these mutations (G46 V, A99T, R167 W, and N461I) exhibited a negative dominant effect by inhibiting the enzymatic activity of the heterodimer, whereas the four others did not show such inhibition. Strong inhibition resulted in severe hypophosphatasia, whereas partial inhibition resulted in milder forms of the disease. Analysis of the 3D model of the enzyme showed that mutations exhibiting a dominant effect were clustered in two regions, viz., the active site and an area probably interacting with a region having a particular biological function such as dimerization, tetramerization, or membrane anchoring.

In addition to occupational exposures to acrylamide (AA), concerns about AA health risks for the general population have been recently raised due to the finding of AA in food. In this study, we evaluated the genotoxicity of AA and its metabolite glycidamide (GA) in L5178Y/ Tk +/− mouse lymphoma cells. The cells were treated with 2-18 mM of AA or 0.125-4 mM of GA for 4 h without metabolic activation. The DNA adducts, mutant frequencies and the types of mutations for the treated cells were examined. Within the dose range tested, GA induced DNA adducts of adenine and guanine [N3-(2-carbamoyl-2-hydroxyethyl)-adenine and N7-(2-carbamoyl-2-hydroxyethyl)-guanine] in a linear dose-dependent manner. The levels of guanine adducts were consistently about 60-fold higher across the dose range than those of adenine. In contrast, no GAderived DNA adducts were found in the cells treated with any concentrations of AA, consistent with a lack of metabolic conversion of AA to GA. However, the mutant frequency was significantly increased by AA at concentrations of 12 mM and higher. GA was mutagenic starting with the 2 mM dose, suggesting that GA is much more mutagenic than AA. The mutant frequencies were increased with increasing concentrations of AA and GA, mainly due to an increase of proportion of small colony mutants. To elucidate the underlying mutagenic mechanism, we examined the loss of heterozygosity (LOH) at four microsatellite loci spanning the entire chromosome 11 for mutants induced by AA or GA. Compared to GA induced mutations, AA induced more mutants whose LOH extended to D11Mit22 and D11Mit74, an alteration of DNA larger than half of the chromosome. Statistical analysis of the mutational spectra revealed a significant difference between the types of mutations induced by AA and GA treatments (P = 0.018). These results suggest that although both AA and GA generate mutations through a clastogenic mode of action in mouse lymphoma cells, GA induces mutations via a DNA adduct mechanism whereas AA induces mutations by a mechanism not involving the formation of GA adducts.

Host cells develop the OAS/RNase L [2′–5′–oligoadenylate synthetase (OAS)/ribonuclease L] system to degrade cellular and viral RNA, and/or the OASL/RIG-I (2′–5′–OAS like/retinoic acid inducible protein I) system to enhance RIG-I-mediated IFN induction, thus providing the first line of defense against viral infection. The 2′–5′–OAS-like (OASL) protein may activate the OAS/RNase L system using its typical OAS-like domain (OLD) or mimic the K63-linked pUb to enhance antiviral activity of the OASL/RIG-I system using its two tandem ubiquitin-like domains (UBLs). We first describe that divergent avian (duck and ostrich) OASL inhibit the replication of a broad range of RNA viruses by activating and magnifying the OAS/RNase L pathway in a UBL-dependent manner. This is in sharp contrast to mammalian enzymatic OASL, which activates and magnifies the OAS/RNase L pathway in a UBL-independent manner, similar to 2′–5′–oligoadenylate synthetase 1 (OAS1). We further show that both avian and mammalian OASL can reversibly exchange to activate and magnify the OAS/RNase L and OASL/RIG-I system by introducing only three key residues, suggesting that ancient OASL possess 2–5A [px5′A(2′p5′A)n; x = 1-3; n ≥ 2] activity and has functionally switched to the OASL/RIG-I pathway recently. Our findings indicate the molecular mechanisms involved in the switching of avian and mammalian OASL molecules to activate and enhance the OAS/RNase L and OASL/RIG-I pathways in response to infection by RNA viruses.

BackgroundOligoadenylate synthetases (OASs) are widely distributed in Metazoa including sponges, fish, reptiles, birds and mammals and show large variation, with one to twelve members in any given species. Upon double-stranded RNA (dsRNA) binding, avian and mammalian OASs generate the second messenger 2'-5'-linked oligoadenylate (2-5A), which activates ribonuclease L (RNaseL) and blocks viral replication. However, how Metazoa shape their OAS repertoires to keep evolutionary balance to virus infection is largely unknown. We performed comprehensive phylogenetic and functional analyses of OAS genes from evolutionarily lower to higher Metazoa to demonstrate how the OAS repertoires have developed anti-viral activity and diversified their functions.ResultsAncient Metazoa harbor OAS genes, but lack both upstream and downstream genes of the OAS-related pathways, indicating that ancient OASs are not interferon-induced genes involved in the innate immune system. Compared to OASs of ancient Metazoa (i.e. sponge), the corresponding ones of higher Metazoa present an increasing number of basic residues on the OAS/dsRNA interaction interface. Such an increase of basic residues might improve their binding affinity to dsRNA. Moreover, mutations of functional residues in the active pocket might lead to the fact that higher Metazoan OASs lose the ability to produce 3'-5'-linked oligoadenylate (3-5A) and turn into specific 2-5A synthetases. In addition, we found that multiple rounds of gene duplication and domain coupling events occurred in the OAS family and mutations at functionally critical sites were observed in most new OAS members.ConclusionsWe propose a model for the expansion of OAS members and provide comprehensive evidence of subsequent neo-functionalization and sub-functionalization. Our observations lay the foundation for interrogating the evolutionary transition of ancient OAS genes to host defense genes and provide important information for exploring the unknown function of the OAS gene family.Electronic supplementary materialThe online version of this article (10.1186/s12862-018-1315-x) contains supplementary material, which is available to authorized users.