In Drosophila melanogaster, ebony and tan, two cuticle melanizing mutants, regulate the conjugation (ebony) of β-alanine to dopamine or hydrolysis (tan) of the β-alanyl conjugate to liberate dopamine. β-alanine biosynthesis is regulated by black. ebony and tan also exert unexplained reciprocal defects in the electroretinogram, at ON and OFF transients attributable to impaired transmission at photoreceptor synapses, which liberate histamine. Compatible with this impairment, both mutants have reduced histamine contents in the head, as measured by HPLC, and have correspondingly reduced numbers of synaptic vesicles in their photoreceptor terminals. Thus, the histamine phenotype is associated with sites of synaptic transmission at photoreceptors. When they receive microinjections into the head, wild-type Sarcophaga bullata (in whose larger head such injections are routinely possible) rapidly (<5 sec) convert exogenous [3H]histamine into its β-alanine conjugate, carcinine, a novel metabolite. Drosophila tan has an increased quantity of [3H]carcinine, the hydrolysis of which is blocked; ebony lacks [3H]carcinine, which it cannot synthesize. Confirming these actions, carcinine rescues the histamine phenotype of ebony, whereas β-alanine rescues the carcinine phenotype of black;tan double mutants. The equilibrium ratio between [3H]carcinine and [3H]histamine after microinjecting wild-type Sarcophaga favors carcinine hydrolysis, increasing to only 0.5 after 30 min. These findings help resolve a longstanding conundrum of the involvement of tan and ebony in photoreceptor function. It is suggested that reversible synthesis of carcinine occurs in surrounding glia, serving to trap histamine after its release at photoreceptor synapses; subsequent hydrolysis liberates histamine for reuptake (Borycz, 2002).
These findings indicate that a novel histamine phenotype underlies the reciprocal actions of ebony and tan. Unlike other aspects of their phenotypes, however, the mutants do not act reciprocally; both reduce the histamine contents of the head. Associated with reduced histamine in both mutants are parallel decreases in the number and packing of synaptic vesicles in the photoreceptor terminals, implying that the histamine phenotype is at least partly of photoreceptor origin. HPLC data are consistent with the conversion into [3H]carcinine of exogenous [3H]histamine, either taken up by or injected into the fly's head. Such uptake has been demonstrated previously in mutant hdc flies, which are unable to synthesize histamine and lack vision. The amount of [3H]carcinine converted in ebony and tan is consistent with the action of ebony in regulating β-alanyl conjugation of histamine and of tan in regulating the hydrolysis of carcinine back to histamine. The equilibrium between the actions of both enzymes evidently favors the hydrolysis of carcinine to liberate histamine, so that the wild-type carcinine content is normally low. Confirming this pathway, ebony flies fed carcinine have increased head histamine, as does the wild type. In addition, feeding β-alanine to double-mutant black;tan flies rescues their ability to synthesize carcinine by providing β-alanine as a substrate. Finally, evidence is provided for the existence of alternative metabolic pathways, with metabolites that convert back to histamine only slowly, if at all (Borycz, 2002).
The evidence for in vivo carcinine biosynthesis is novel for the visual system but has previously been reported biochemically for CNS extracts from the crab Carcinus maenas, which accumulates carcinine in the heart. Not all Carcinus tissues are able to metabolize carcinine, however, suggesting that the hydrolysis pathway represented by tan in Drosophila is either lacking or of low activity. The universal histamine immunoreactivity of, and likely prevalence of histaminergic transmission at, arthropod photoreceptors suggests that a carcinine biosynthesis pathway could be widely used at this site. Carcinine was not sought in a previous study of insect histamine metabolites but is certainly not restricted to arthropods. It is also a minor metabolite in mammals, in which it exerts a positive inotropic action at the heart (Borycz, 2002).
The simultaneous action of both Ebony and Tan proteins in wild-type flies indicates that carcinine forms rapidly. Within 5 sec of injecting Sarcophaga, [3H]histamine gains access to Ebony and is already converted to carcinine. The independent regulation of synthase and hydrolase activity means that the rates for carcinine biosynthesis and hydrolysis can be independently regulated by differential transcription under different physiological conditions. For example, ebony transcription exhibits a circadian modulation (Claridge-Chang, 2001). The significance of carcinine as a metabolite may in fact lie not as much in the identity of the metabolite itself as in the rates of its biosynthesis and reversible hydrolysis, which the [3H]histamine evidence indicates are normally adjusted to a 2:1 equilibrium ratio in favor of hydrolysis. Although it is clear that Ebony acts rapidly, the methods used do not allow assessment of whether it contributes to the termination of histamine action at the cleft. Resolution of this question is crucial to understand how insects, especially fast-flying diurnal flies, are able to use the high-temporal resolution of their photoreceptors. The latter depends on the rapid clearance of released histamine at photoreceptor terminals (Borycz, 2002).
Exact sites of histamine metabolism in the lamina and distal medulla are still not clear. The photoreceptor terminals R1-R6 that surround the axons of L1 and L2 within a cartridge are wrapped in turn by three epithelial glial cells. These are well placed to metabolize histamine from the synaptic cleft and thereby regulate its postsynaptic action at sites on L1 and L2. Moreover, the epithelial glia do indeed express Ebony strongly (Richardt, 2002). Carcinine biosynthesis by Ebony in the epithelial glia would remove histamine released into the lamina, presumably from the synaptic cleft, but would store histamine in a form that can then rapidly liberate it by hydrolysis. The site of that hydrolysis is unknown in detail, but mosaic studies indicate that tan acts in or close to the eye, compatible with its action in the photoreceptors (Borycz, 2002).
The accumulation of [3H]carcinine in tan, but its lack in ebony, can be explained by the reciprocal regulation of β-alanyl conjugation of histamine in the two mutants. However, this still fails to explain how a reduction in head histamine results from the reciprocal action of the two genes. It is proposed that histamine content is reduced in tan because of the failure to liberate histamine from accumulated carcinine, a function that is autonomous to the mutant eye, but is reduced in ebony because carcinine fails to trap histamine after it is released, leaving the histamine free to diffuse away from the compound eye. The fate of histamine after diffusion is unclear but could finally be loss, to the thorax, thence by excretion. It is proposed that this loss is the primary reason for the reduced head content of histamine in ebony. In the absence of functional Ebony protein, mutant flies also fail to trap exogenous [3H]histamine, much of which is likewise lost by excretion. As a result, not only is total head histamine reduced but also the amount of 3H incorporation. In tan, a reciprocal effect occurs, with [3H]histamine incorporation increasing with respect to wild type. It is believed that this may signify increased efficiency in the histamine uptake mechanisms in response to the greater reduction in the head histamine of tan. In Drosophila gynandromorphs with a single mutant ebony eye, the defect in the ERG transients is nonautonomous (R. Hodgetts, personal communication to Borycz, 2002). One interpretation of this difference from tan is that a mutant ebony lamina is unable to convert released histamine to carcinine, so that the histamine remains extracellular and may be free to diffuse to other sites, including the other eye, where it is converted to carcinine by functional Ebony. The fact that such sites can rescue the ERG defect in the mutant eye suggests that the lack of transients when both eyes are mutant for ebony could reflect the presence in the synaptic cleft of residual histamine, even that released in the dark. It is proposed that carcinine that accumulates by the action of functional Ebony in a mutant tan eye is localized initially to the epithelial glia and is not free to diffuse. Therefore, it sequesters much of the histamine pool. In that case, the ERG defect in the mutant tan eye may be attributed to insufficient release of histamine. Thess findings thus help shed light on the involvement in lamina function of tan and ebony and offer a possible explanation for why both, albeit for different reasons, result in the loss of the ON transients of the ERG. An alternative interpretation, offered without reference to histamine metabolism and possibly an independent effect, is that the loss of the lamina transients of ERG in tan could result from the decreased availability of dopamine during larval development (Neckameyer, 2001), with ebony showing reciprocal defects to those shown by tan. Still left to be resolved is whether the carcinine pathway operates at other sites. These include (1) terminals of head mechanoreceptors, which also contain histamine and in which function is both lost and rescued by exogenous histamine in flies mutant for hdc; (2) wide-field histamine-like immunoreactive neurons in the central brain; and (3) dopaminergic neurons in the brain (Borycz, 2002).
The production of carcinine is not the sole metabolic pathway for photoreceptor histamine. In the horseshoe crab Limulus, histamine is also a putative photoreceptor transmitter, and an additional or alternative metabolic pathway involves gamma-glutamyl histamine, a means of histamine inactivation reported previously in the opisthobranch Aplysia. It is not clear what additional metabolites might also exist in Drosophila, but the presence of 3H peaks with HPLC retention times shorter than that for carcinine allows a number of candidates, possibly up to three. It was not possible to detect acetyl-histamine or imidazol-4-acetic acid, but a 3H peak with the same retention time as γ-glutamyl histamine exists, so this metabolite could be present. Other metabolites probably exist as well. For example, the separate actions of pargyline and deprenyl and of clorgyline could indicate a role for monoamine oxidases. Therefore, it is surprising that the monoamine oxidase gene appears to have been lost from the Drosophila genome, making the identity of these metabolites a topic for future clarification as well. Moreover, insensitivity to semicarbazide and hydroxylamine could indicate the lack of SSAO action (Borycz, 2002).
In addition to their activities at one time and in one genetic background, the relative activities of the metabolic pathway for carcinine (regulated by ebony and tan) and for alternative metabolites indicate that each pathway can be differentially regulated. Regulation is seen in black;tan double mutants, which have a large early retention peak suggesting that, in the congenital absence of a capacity to synthesize carcinine, histamine metabolism switches into another pathway, possibly for γ-glutamyl histamine. That pathway is not increased in the single mutant tan, possibly because tan is able to store released histamine as carcinine. Such shifts can also apparently occur in the short term, as for example in Sarcophaga injected with pargyline and deprenyl. Under the influence of these drugs, the early HPLC retention peaks are diminished, and the histamine peak is larger, suggesting that histamine metabolism via carcinine is upregulated (Borycz, 2002).
Using Ebony protein either expressed in Escherichia coli or in Schneider S2 cells, evidence is provided for its substrate specificity and reaction mechanism. Ebony activates beta-alanine to aminoacyladenylate by an adenylation domain and covalently attaches it as a thioester to a thiolation domain in a nonribosomal peptide synthetase (NRPS) related mechanism. In a second reaction, biogenic amines act as external nucleophiles on beta-alanyl-S-pantetheine-Ebony, thereby releasing in a fast reaction the dipeptide (peptidoamine) in a process that is novel in higher eucaryotes. Therefore, Ebony is defined as a beta-alanyl-biogenic amine synthetase. Insight into the reaction mechanism stems from mutational analysis of an invariant serine that disclosed Ebony as a multienzyme with functional analogy to the starting modules of NRPSs. In light of a putative biogenic amine-deactivating capacity, Ebony function in the nervous system must be reconsidered. It is proposed that in the Drosophila eye Ebony is involved in the transmission process by inactivation of histamine through beta-alanyl conjugation (Richardt, 2003). Ebony similar to NRPSs belongs to the large family of aminoacyladenylate-forming enzymes. A relation to the thioesterification process, however, is in addition to NRPSs only present in the group of acyl carrier proteins including polyketide synthases and fatty acid synthases. The homology between polyketide and fatty acid synthases and Ebony is limited to the core sequence element of the thiolation domain, which contains the invariant serine, the P-pant cofactor-mediated acyl carrier. Acyl carrier proteins, NRPSs, and Ebony need to be activated by P-pant cofactor transfer, which requires a corresponding transferase activity. Searching for two conserved amino acid sequence motifs detected in previously sequenced P-pant transferases, a reading frame was indeed identified in the Drosophila data base that showed a considerable homology to this conserved region. Expression of the corresponding putative P-pant transferase cDNA in E. coli gave rise to a protein that in vitro enhanced low level phosphantetheinylated S2 cell-derived Ebony activity depending on the presence of CoA comparable with the phosphopantetheinyltransferase (Richardt, 2003).
Drosophila has preserved an amino acid activation mechanism that until now was considered to be specific for microbial NRPSs. Ebony combines this unique feature with a functional domain that allows peptide bond formation with a structurally constrained group of amines. However, a connection between two or more NRPS-like modules that enable the activation of amino acids and the formation of dipeptides has not yet been detected in higher eucaryotes even though genuine dipeptides such as β-alanyl-histidine (carnosine) have been shown to exist in vertebrates. Given the existence of a single NRPS-like activation domain in Ebony, nonribosomal synthesis of dipeptides in higher eucaryotes cannot generally be excluded. However, it would require two amino acid activation modules in addition to a functional domain for condensation of the two activated amino acids as well as a thioesterase activity for peptide release. Evidence that this complex structure of multimodular NRPS activity has been preserved through evolution to higher eucaryotes is still lacking (Richardt, 2003).
Ebony uses a novel two-step reaction mechanism including amino acid activation and binding followed by peptide bond formation. The procedure of amino acid activation and binding resembles that of NRPSs. Peptide bond formation and product release require a nucleophilic attack of an incoming primary amine that must meet the observed structural prerequisites. This is different in multimodular NRPSs in two ways. 1) The nature of peptide bond forming amino acid is predetermined by the specificity of a second adenylation domain within the multimodular enzyme, and 2) in NRPSs, a condensation domain located between the modules is essential for peptide bond catalysis. Such a condensation domain is missing in Ebony. Instead, a C-terminal domain with a yet unknown function seems to be responsible for catalyzing the nucleophilic attack of the primary amines (with relaxed substrate specificity) on the activated carboxyl thioester group of β-alanine. The mechanism of this reaction and that of dipeptide product release are still unknown (Richardt, 2003).
Ebony is expressed in diverse tissues at different times during development. Nervous system activity has been predicted from the behavioral and visual phenotype of the mutant but was only recently confirmed by activity staining of an ebony-lacZ fusion gene transformant and by immunocytochemistry. The puzzling fact that evidence for dopaminergic neurons in the lamina was lacking led to experiments that revealed that Ebony is involved in beta-alanyl-histamine formation in the eye. The capacity of capturing the biogenic amines histamine, dopamine, tyramine, octopamine (see Tyramine β hydroxylase), and serotonin that clearly fulfill different functions in Drosophila to β-alanine might reflect a key function of Ebony at specific sites of the body (Richardt, 2003).
This study provides evidence that Ebony is indeed capable of binding biogenic amines including histamine to β-alanine. Therefore, it is plausible to assign to Ebony a function in histamine neurotransmitter metabolism at the photoreceptor synapse of the eye. Because histamine synthesis as well as metabolic degradation in the eye is relatively slow, the almost infinite transmitter supply must be maintained by a fast re-uptake system. Therefore, at the synapse where transmitter removal excites the postsynaptic cell by disinhibition, a mechanism of fast retraction of histamine from the synaptic cleft is essential. Interestingly, in illuminated barnacle photoreceptor preparations, [3H]histamine was concentrated over the photoreceptor terminals, whereas after incubation in the dark, the label was found at the glia. This observation lends support to the concept that at darkening a fast clearance of transmitter out of the synaptic cleft would be achieved by transport of histamine into the surrounding glia where it could be trapped by Ebony via β-alanine binding. The model requires that β-alanine is sufficiently loaded in the glia to prime Ebony for histamine capture and a biochemical pathway that allows the subsequent reuse of the withdrawn histamine in the photoreceptor. Although both histamine transport into photoreceptor as well as into glia has been reported previously, it remains to be investigated whether a mechanism exists that darkening and concomitant reduction of histamine release shifts uptake toward glia followed by immediate inactivation by β-alanine binding (Richardt, 2003).
Fast histamine removal from the synaptic cleft is essential for the function of arthropod photoreceptor synapses that operate with tonic release of histamine. In vitro product formation from [β-alanine-loaded Ebony with histamine or any of the other biogenic amine substrates was already completed within 10 s, the shortest time point that could be determined under standard assay conditions. This time point is still far away from the reaction velocity expected for a function in neurotransmitter inactivation. Beyond this point, reaction velocity may differ among the biogenic amines serving as substrate. Determination of Vmax and Km values of individual biogenic amines requires specific analytical methods operating in the millisecond range. They will disclose whether Ebony can fulfill the kinetic prerequisites for neurotransmitter inactivation (Richardt, 2003).
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