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. 2010 Jul 2;285(27):20740-7.
doi: 10.1074/jbc.M110.120170. Epub 2010 May 3.

Alternative tasks of Drosophila tan in neurotransmitter recycling versus cuticle sclerotization disclosed by kinetic properties

Silvia Aust  1 , Florian Brüsselbach, Stefanie Pütz, Bernhard T Hovemann
Affiliations

Alternative tasks of Drosophila tan in neurotransmitter recycling versus cuticle sclerotization disclosed by kinetic properties

Silvia Aust et al. J Biol Chem. .

Abstract

Upon a stimulus of light, histamine is released from Drosophila photoreceptor axonal endings. It is taken up into glia where Ebony converts it into beta-alanyl-histamine (carcinine). Carcinine moves into photoreceptor cells and is there cleaved into beta-alanine and histamine by Tan activity. Tan thus provides a key function in the recycling pathway of the neurotransmitter histamine. It is also involved in the process of cuticle formation. There, it cleaves beta-alanyl-dopamine, a major component in cuticle sclerotization. Active Tan enzyme is generated by a self-processing proteolytic cleavage from a pre-protein at a conserved Gly-Cys sequence motif. We confirmed the dependence on the Gly-Cys motif by in vitro mutagenesis. Processing time delays the rise to full Tan activity up to 3 h behind its putative circadian RNA expression in head. To investigate its pleiotropic functions, we have expressed Tan as a His(6) fusion protein in Escherichia coli and have purified it to homogeneity. We found wild type and mutant His(6)-Tan protein co-migrating in size exclusion chromatography with a molecular weight compatible with homodimer formation. We conclude that dimer formation is preceding pre-protein processing. Drosophila tan(1) null mutant analysis revealed that amino acid Arg(217) is absolutely required for processing. Substitution of Met(256) in tan(5), on the contrary, does not affect processing extensively but renders it prone to degradation. This also leads to a strong tan phenotype although His(6)-Tan(5) retains activity. Kinetic parameters of Tan reveal characteristic differences in K(m) and k(cat) values of carcinine and beta-alanyl-dopamine cleavage, which conclusively illustrate the divergent tasks met by Tan.

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Figures

FIGURE 1.
FIGURE 1.
Tan and Ebony regulate histamine and dopamine concentrations in eye and cuticle, respectively. In the eye histamine neurotransmitter is inactivated by β-alanyl conjugation to yield β-alanyl histamine (carcinine). Later Tan can hydrolyze carcinine to again supply histamine for neurotransmission. In the cucticle the interplay between Tan and Ebony activity adjusts the concentration of free dopamine.
FIGURE 2.
FIGURE 2.
Self-processing activity of His6-Tan. Western blot analysis of E. coli expressed, affinity purified wild type, and mutant His6-Tan with the anti-Tan antiserum ap61/ap63 (5) directed against the amino- and carboxy-terminal ends. Tan mutant variants applied in each lane are indicated on the top using 1 letter amino acid abbreviations. Tan pre-protein (P), β-subunit (β) and α-subunit (α) are running at 45, 30, and 15 kDa, respectively.
FIGURE 3.
FIGURE 3.
Expression of the tan gene in wild type C-S, tan1, and tan5 heads. A–C, immunolabeling of Tan in wild type (A), tan1 (B), and tan5 (C) 10 μm Drosophila head freeze sections with anti-Tan antiserum ap61/ap63 (5). The Tan label is in blue. Ebony labeled in green is used as control. Re, La, and Me indicate retina, lamina, and medulla, respectively. D, Western blot analysis of wild type Canton-S (C-S) and mutants tan1 (t1) and tan5 (t5) head extracts with anti-Tan antiserum. E, RT-PCR amplification of a 456-nucleotide tan-RNA fragment (strong lower band) from head total RNA preparations of wild type Canton-S (C-S) and mutants tan1 (t1) and tan5 (t5). RT-PCR amplification of a 581-nucleotide ebony-RNA fragment (weak upper band) served as control.
FIGURE 4.
FIGURE 4.
Purification of His6-Tan proteins. A, Protino Ni-TED affinity purification of E. coli cell lysate. 1, whole cell lysate; 2–5, peak fractions containing His6-Tan and a 43-kDa contaminant. B, elution profile of a Source 15S column loaded with the peak fraction of the Ni-TED step. C, peak fraction of a Source 15S column purification of wild type Tan and TanC122A (C122A). Coomassie staining was used in A and B, silver staining in C.
FIGURE 5.
FIGURE 5.
Size evaluation of native wild type His6-Tan compared with mutant His6-TanC122A. Superdex 200 high resolution chromatography of wild type Tan (solid line) and TanC122A (broken line) as a ratio of elution volume (VE) to void volume (V0). Triangles indicate the elution ratio for size marker proteins alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa). From their linear regression indicated as a straight line, the molecular weights of Tan have been calculated.
FIGURE 6.
FIGURE 6.
His6-Tan pre-protein processing time. Western blot of His6-Tan pre-protein expressed in E. coli cell extracts after removal of IPTG and additional growth periods at 25 °C as indicated in minutes. Anti-Tan antiserum labels diminishing proportions of pre-protein.
FIGURE 7.
FIGURE 7.
Enzyme kinetics of carcinine and β-alanyl-dopamine hydrolysis. Representative Michaelis-Menten curves from each data set for each hydrolysis reaction are depicted. A, wild type His6-Tan with β-alanyl-dopamine as substrate. B, wild type His6-Tan and His6-TanM256I (broken line) with carcinine as substrate.
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