Silvestro Duprè

Pantetheinase activity of membrane‐bound Vanin‐1: lack of free cysteamine in tissues of Vanin‐1 deficient mice

Pantetheinase (EC 3.5.1.‐) is an ubiquitous enzyme which in vitro has been shown to recycle pantothenic acid (vitamin B5) and to produce cysteamine, a potent anti‐oxidant. We show that the Vanin‐1 gene encodes pantetheinase widely expressed in mouse tissues: (1) a pantetheinase activity is specifically expressed by Vanin‐1 transfectants and is immunodepleted by specific antibodies; (2) Vanin‐1 is a GPI‐anchored pantetheinase, and consequently an ectoenzyme; (3) Vanin‐1 null mice are deficient in membrane‐bound pantetheinase activity in kidney and liver; (4) in these organs, a major metabolic consequence is the absence of detectable free cysteamine; this demonstrates that membrane‐bound pantetheinase is the main source of cysteamine in tissues under physiological conditions. Since the Vanin‐1 molecule was previously shown to be involved in the control of thymus reconstitution following sublethal irradiation in vivo, this raises the possibility that Vanin/pantetheinase might be involved in the regulation of some immune functions maybe in the context of the response to oxidative stress.

Vanin-1-/- mice exhibit a glutathione-mediated tissue resistance to oxidative stress

ABSTRACT Vanin-1 is an epithelial ectoenzyme with pantetheinase activity and generating the amino-thiol cysteamine through the metabolism of pantothenic acid (vitamin B5). Here we show that Vanin-1−/− mice, which lack cysteamine in tissues, exhibit resistance to oxidative injury induced by whole-body γ-irradiation or paraquat. This protection is correlated with reduced apoptosis and inflammation and is reversed by treating mutant animals with cystamine. The better tolerance of the Vanin-1−/− mice is associated with an enhanced gamma-glutamylcysteine synthetase activity in liver, probably due to the absence of cysteamine and leading to elevated stores of glutathione (GSH), the most potent cellular antioxidant. Consequently, Vanin-1−/− mice maintain a more reducing environment in tissue after exposure to irradiation. In normal mice, we found a stress-induced biphasic expression of Vanin-1 regulated via antioxidant response elements in its promoter region. This process should finely tune the redox environment and thus change an early inflammatory process into a late tissue repair process. We propose Vanin-1 as a key molecule to regulate the GSH-dependent response to oxidative injury in tissue at the epithelial level. Therefore, Vanin/pantetheinase inhibitors could be useful for treatment of damage due to irradiation and pro-oxidant inducers.

The copper catalyzed oxidation of cysteine to cystine

Abstract The addition of cysteine to an alkaline solution of CuII produces the immediate appearance of a yellow color. The spectrum of this color is characterized by a broad peak at 330 mμ and a shoulder at 400 mμ. By chemical analysis and EPR spectrometry, it has been demonstrated that this yellow compound is to be identified with a cysteine-copperII complex, in which the cysteine to copper ratio is 2:1. This complex accounts for nearly all the copper present in solution; remains unchanged during the cysteine oxidation by molecular oxygen, and quickly disappears at the end of the reaction. At this time no more cysteine is present in solution in its reduced form and all the copper may be recovered as CuI if immediately trapped by neocuproine. The yellow cysteine-CuII complex is also immediately produced in the absence of oxygen, however, under this condition, it disappears slowly, CuII being reduced by excess of cysteine. Some kinetic approaches indicate that both the formation of the yellow complex, and its anaerobic reduction, are very complex reactions. The results obtained indicate that the cysteine-CuII complex represents the real intermediate catalyst in the copper-catalyzed oxidation of cysteine.

Is pantetheinase the actual identity of mouse and human vanin-1 proteins?

Pantetheinase is an amidohydrolase involved in the dissimilative pathway of CoA, allowing the turnover of the pantothenate moiety. We have determined the N‐terminal sequence as well as the sequences of a number of tryptic and chymotryptic peptides of the protein isolated from pig kidney. These sequence stretches were used as probes to search in the SwissProt database and significant similarities were found with a GPI‐anchored protein (mouse vanin‐1, with a suggested role in lymphocyte migration), with two putative proteins encoded by human cDNAs (VNN1 and VNN2) and with human biotinidase. On the basis of sequence similarity, we propose that vanin‐1 and VNN1 should be identified as pantetheinase.

Luminol chemiluminescence studies of the oxidation of cysteine and other thiols to disulfides.

Abstract The luminol chemiluminescence excited by autooxidizing thiols has been investigated. When the copper-catalyzed thiol oxidation in alkaline medium is allowed to take place in the presence of luminol, a sharp light flash is observed at the end of the reaction. It has been demonstrated that the luminol chemiluminescence is due to the sudden degradation of the H2O2 accumulated during the reaction. By a chemical method, H2O2 production during thiol oxidation has been demonstrated. H2O2 is accumulated during the reaction and is immediately destroyed when all the thiol is oxidized to the disulfide. A comparison of the degradation of H2O2 and the excitement of luminol chemiluminescence shows that both reactions are strongly dependent upon pH. At high pH values H2O2 is suddenly destroyed by cupric ions, and chemiluminescence appears. At neutral pH values it is more stable and cannot excite luminol chemiluminescence. It has been shown that, under the experimental conditions described, the degradation of H2O2 requires the presence of a free metal, and only in these conditions may it excite the chemiluminescence of luminol. The results obtained indicate that during thiol oxidation the metal is strongly complexed by the thiol itself. At the end of the reaction, when thiol is no longer present in solution, the metal is again free and immediately decomposes the accumulated H2O2, thereby exciting the luminol chemiluminescence.

Identification of pantethinase in horse kidney extract

Purification of an oxygenase oxidizing cysteamine to hypotaurine has been recently reported from this laboratory [l] . Cysteamine, however, is not a common metabolite and we have sought a possible mechanism capable of producing the cysteamine used as substrate by this enzyme. Although a direct decarboxylation of cysteine has never been reported, it is known that cysteine is decarboxylated to cysteamine when it is bound to pantothenic acid in the course of the biosynthesis of coenzyme A [2]. Moreover pantetheine and phosphopantetheine are present in considerable amounts in the free or bound form in a number of tissues [3,4] . The enzymic cleavage of these compounds could represent, therefore, a source of free cysteamine. We have used pantethine as a representative substrate for checking the occurrence of a cysteamine (cystamine) producing enzyme in horse kidney. For convenience this enzyme will be referred to as pantethinase.

Antioxidant properties of sulfinates: protective effect of hypotaurine on peroxynitrite-dependent damage.

Fig. 3. Prevention of ONOO-mediated a1AP inactivation by hypotaurine and cysteine sulfinic acid (CSA). Sulfinates, at the indicated concentrations, were incubated with a1AP in 0.5 M K-phosphate buffer, pH 7.4 before ONOO ) addition (0.5 mM). The a1AP inactivation was measured by its ability to inhibit elastase activity as described in experimental procedure. Results are the mean ± SEM of three experiments.

Cystamine restores GSTA3 levels in Vanin-1 null mice.

Free cysteamine levels in mouse tissues have been strictly correlated to the presence of membrane-bound pantetheinase activity encoded by Vanin-1. Vanin-1 is involved in many biological processes in mouse, from thymus homing to sexual development. Vanin-1 -/- mice are fertile and grow and develop normally; they better control inflammation and most of the knockout effects were rescued by cystamine treatment. Gene structure analysis showed the presence of an oxidative stimuli-responsive ARE-like sequence in the promoter. In this paper we investigate antioxidant-detoxifying enzymatic activities at the tissue level, comparing Vanin-1 -/- and wild-type mice. In Vanin-1 null animals we pointed out a decrease in the Se-independent glutathione peroxidase activity. The decrease in enzymatic activity appeared to be correlated to an impairment of GST isoenzyme levels. In particular a significant drop in GSTA3 together with a minor decrement in GSTM1 and an increase in GSTP1 levels was detected in Vanin-1 -/- livers. Cystamine administration to Vanin-1 -/- mice restored specifically GSTA3 levels and the corresponding enzymatic activity without influencing protein expression. A possible role of cystamine on protein stability/folding can be postulated.

Enzymatic synthesis of S-aminoethyl-L-cysteine from pantetheine.

The recently characterized compound S-aminoethylcysteine ketimine can be synthesized from purified S-aminoethylcysteine by enzymatic systems (transaminases or L-amino acid oxidase) present in mammalian tissues. S-Aminoethylcysteine, which could be considered as the natural precursor of the ketimine, is produced from L-serine and cysteamine by the action of the enzyme cystathionine-beta-synthase. We demonstrate in this paper that pantetheine, a normal cellular component, is an efficient cysteamine donor for the synthesis of S-aminoethylcysteine and of S-aminoethylcysteine ketimine in the place of free cysteamine, and we describe the enzymatic system, composed of partially purified enzymes, for the in vitro synthesis of S-aminoethylcysteine ketimine from pantetheine. This seems to indicate a new biological role for pantetheine.

Biosynthesis of lipoic acid in the rat: Incorporation of 35S- and 14C-labeled precursors

Abstract After injection of various 35 S- and 14 C-containing compounds, the incorporation of the label into the lipoic acid present in the liver of growing rats has been determined. The best precursor for sulfur atoms, after 24 h, is cysteine; methionine and cystamine are scarcely incorporated and thiosulfate not at all. Good precursors of the carbon moiety are acetate and octanoate, whereas the incorporation of butyrate and cysteine is very low. It is concluded that lipoic acid is biosynthesized in the rat liver, and that sulfur atoms probably originate from cysteine.