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The ARH family of ADP-ribose-acceptor hydrolases consists of three 39-kDa members (ARH1-3), with similarities in amino acid sequence. ARH1 was identified based on its ability to cleave ADP-ribosyl-arginine synthesized by cholera toxin. Mammalian ADP-ribosyltransferases (ARTCs) mimicked the toxin reaction, with ARTC1 catalyzing the synthesis of ADP-ribosyl-arginine. ADP-ribosylation of arginine was stereospecific, with β-NAD+ as substrate and, α-anomeric ADP-ribose-arginine the reaction product. ARH1 hydrolyzed α-ADP-ribose-arginine, in addition to α-NAD+ and O-acetyl-ADP-ribose. Thus, ADP-ribose attached to oxygen-containing or nitrogen-containing functional groups was a substrate. Arh1 heterozygous and knockout (KO) mice developed tumors. Arh1-KO mice showed decreased cardiac contractility and developed myocardial fibrosis. In addition to Arh1-KO mice showed increased ADP-ribosylation of tripartite motif-containing protein 72 (TRIM72), a membrane-repair protein. ARH3 cleaved ADP-ribose from ends of the poly(ADP-ribose) (PAR) chain and released the terminal ADP-ribose attached to (serine)protein. ARH3 also hydrolyzed α-NAD+ and O-acetyl-ADP-ribose. Incubation of Arh3-KO cells with H2O2 resulted in activation of poly-ADP-ribose polymerase (PARP)-1, followed by increased nuclear PAR, increased cytoplasmic PAR, leading to release of Apoptosis Inducing Factor (AIF) from mitochondria. AIF, following nuclear translocation, stimulated endonucleases, resulting in cell death by Parthanatos. Human ARH3-deficiency is autosomal recessive, rare, and characterized by neurodegeneration and early death. Arh3-KO mice developed increased brain infarction following ischemia-reperfusion injury, which was reduced by PARP inhibitors. Similarly, PARP inhibitors improved survival of Arh3-KO cells treated with H2O2. ARH2 protein did not show activity in the in vitro assays described above for ARH1 and ARH3. ARH2 has a restricted tissue distribution, with primary involvement of cardiac and skeletal muscle. Overall, the ARH family has unique functions in biological processes and different enzymatic activities.

Poly(ADP-ribosyl)ation refers to the covalent attachment of ADP-ribose to protein, generating branched, long chains of ADP-ribose moieties, known as poly(ADP-ribose) (PAR). Poly(ADP-ribose) polymerase 1 (PARP1) is the main polymerase and acceptor of PAR in response to DNA damage. Excessive intracellular PAR accumulation due to PARP1 activation leads cell death in a pathway known as parthanatos. PAR degradation is mainly controlled by poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose-acceptor hydrolase 3 (ARH3). Our previous results demonstrated that ARH3 confers protection against hydrogen peroxide (H(2)O(2)) exposure, by lowering cytosolic and nuclear PAR levels and preventing apoptosis-inducing factor (AIF) nuclear translocation. We identified a family with an ARH3 gene mutation that resulted in a truncated, inactive protein. The 8-year-old proband exhibited a progressive neurodegeneration phenotype. In addition, parthanatos was observed in neurons of the patient’s deceased sibling, and an older sibling exhibited a mild behavioral phenotype. Consistent with the previous findings, the patient’s fibroblasts and ARH3-deficient mice were more sensitive, respectively, to H(2)O(2) stress and cerebral ischemia/reperfusion-induced PAR accumulation and cell death. Further, PARP1 inhibition alleviated cell death and injury resulting from oxidative stress and ischemia/reperfusion. PARP1 inhibitors may attenuate the progression of neurodegeneration in affected patients with ARH3 deficiency.

Anosmin is an extracellular matrix protein, and genetic defects in anosmin result in human Kallmann syndrome. It functions in neural crest formation, cell adhesion, and neuronal migration. Anosmin consists of multiple domains, and it has been reported to bind heparan sulfate, FGF receptor, and UPA. In this study, we establish cell adhesion/spreading assays for anosmin and use them for antibody inhibition analyses to search for an integrin adhesion receptor. We find that α5β1, α4β1, and α9β1 integrins are needed for effective adhesive receptor function in cell adhesion and cell spreading on anosmin; adhesion is inhibited by both RGD and α4β1 CS1-based peptides. This identification of anosmin-integrin adhesion receptors should facilitate studies of anosmin function in cell and developmental biology.

Key roles for fibronectin and its integrin receptors have been postulated in the multiple cell-matrix interactions essential for chick embryo morphogenesis. However, mechanistic studies of these processes have been hampered by the current absence of sequence data and chicken cDNA clones for the major fibronectin receptor subunit, integrin α5 (ITGA5). We report here the sequence, endogenous expression pattern, and transfection of full-length chicken integrin α5. During early chicken embryonic development, α5 is highly expressed in cranial neural folds and migrating neural crest cells, suggesting potential roles in neural crest formation and migration. In fact, over-expression of this integrin in early neural tube selectively induces BMP5, a growth factor recently implicated in neural crest formation. Availability of these α5 integrin tools should facilitate studies of its functions in early embryonic development.

Cholera toxin, an 84-kDa multimeric protein and a major virulence factor of Vibrio cholerae, uses the ADP-ribosyltransferase activity of its A subunit to intoxicate host cells. ADP-ribosylation is a posttranslational modification of proteins, in which the ADP-ribose moiety of NAD+ is transferred to an acceptor. In mammalian cells, ADP-ribosylation of acceptors appears to be reversible. ADP-ribosyltransferases (ARTs) catalyze the modification of acceptor proteins, and ADP-ribose-acceptor hydrolases (ARHs) cleave the ADP-ribose-acceptor bond. ARH1 specifically cleaves the ADP-ribose-arginine bond. We previously demonstrated a role for endogenous ARH1 in regulating the extent of cholera toxin-mediated fluid and electrolyte abnormalities in a mouse model of intoxication. Murine ARH1-knockout (KO) cells and ARH1-KO mice exhibited increased sensitivity to cholera toxin compared to their wild-type (WT) counterparts. In the current report, we examined the sensitivity to cholera toxin of male and female ARH1-KO and WT mice. Intestinal loops derived from female ARH1-KO mice when injected with cholera toxin showed increased fluid accumulation compared to male ARH1-KO mice. WT mice did not show gender differences in fluid accumulation, ADP-ribosylarginine content, and ADP-ribosyl Gαs levels. Injection of 8-Bromo-cAMP into the intestinal loops also increased fluid accumulation, however, there was no significant difference between female and male mice or in WT and KO mice. Female ARH1-KO mice showed greater amounts of ADP-ribosylated Gαs protein and increased ADP-ribosylarginine content both in whole intestine and in epithelial cells than did male ARH1-KO mice. These results demonstrate that female ARH1-KO mice are more sensitive to cholera toxin than male mice. Loss of ARH1 confers gender sensitivity to the effects of cholera toxin but not of cyclic AMP. These observations may in part explain the finding noted in some clinical reports of enhanced symptoms of cholera and/or diarrhea in women than men.