Rongda Xu

Oxidative conjugation of catechols with proteins in insect skeletal systems

Abstract Cuticle sclerotization or tanning is a vital process that occurs during each stage of insect development to harden and stabilize the newly secreted exoskeleton. The structural polymers protein and chitin make up the bulk of the cuticle, and chemical interactions between these biopolymers with quinonoid tanning agents are largely responsible for the physical properties of the mature exoskeleton. The oxidative conjugation of catechols with cuticular proteins plays an important role in this metabolism. The main hypothesis for cuticle sclerotization involves the formation of adducts and cross-links between nucleophilic imidazole nitrogens of histidyl residues in the proteins and electrophilic ring or side-chain carbons of ortho-quinones and para-quinone methides derived from the catechols, N-acetyldopamine, N-beta-alanyldopamine, and 3,4-dihydroxyphenylethanol. C–N and C–O linkages between these quinone tanning agents and proteins in cuticles from a variety of insects from several orders have been elucidated. cDNAs for both the tyrosinase and laccase types of phenoloxidases that catalyze the cross-linking reactions have been isolated and sequenced. The sequences of laccase cDNAs from two insect species were more similar to fungal laccases than to those from plants. These results provide insights into how insects use structural proteins, catechols, and oxidative enzymes to form catechol–amino acid adducts during sclerotization.

Catecholamine and histidyl protein cross-linked structures in sclerotized insect cuticle

Catecholamines play an important role in cuticular sclerotization, an extracellular process used by insects at various stages of their life cycle to stabilize their exoskeletons. Analysis of products derived from the acid hydrolysis of insect cuticle provide for the first time direct structural evidence for the actual cross-links between catecholamines and histidyl residues of the cuticular proteins. Four adducts were purified by HPLC from a 6 M HCI hydrolysate of tobacco hornworm, Manduca sexta, sclerotized pupal cuticle. They were identified as 7-Nτ-, 6-Nτ-, 7-Nπ-, and 6-Nπ-histidyldopamine adducts using matrix-assisted laser desorption ionization mass spectrometry, 1D 1H NMR, and 2D homonuclear nuclear Overhauser effect NMR spectroscopy in combination with molecular modeling. The molar ratio of these adducts in the hydrolysate was approximately 6:3:2:1, respectively. These adducts apparently are formed from Michael 1,4- and 1,6-addition reactions of the putative quinonoid sclerotizing agents, N-β-alanyldopamine quinone and N-β-alanyldopamine quinone methide, with both imidazole nitrogens of histidyl residues of proteins.

Benzoquinone levels as a function of age and gender of the red flour beetle, Tribolium castaneum

Abstract Liquid chromatography with both photodiode array and electrochemical detection was used to analyze as a function of age and gender the levels of two ρ-quinones, methyl-1,4-benzoquinone (MBQ) and ethyl-1,4-benzoquinone (EBQ), which are found in defensive secretions of the red flour beetle, Tribolium castaneum. We developed a method to simultaneously analyze quinones and hydroquinones excreted from or in homogenates of individual beetles. The major components present in beetle extracts were the benzoquinones and not free or conjugated forms of the hydroquinones. Greater than 95% of the quinone/hydroquinone mixture in extracts was present in the oxidized form. Because of their lability, however, the quinones were quantified indirectly as their hydroquinone derivatives after extraction in dilute acid supplemented with ascorbic acid as a reducing agent. Comparisons of whole body rinses and homogenates revealed that rinses recovered only up to 60% of the total quinones that were extracted after homogenization. The levels recovered also depended on the age and sex of the individual beetles sampled. ρ-Benzoquinones in both male and female beetles increased after adult eclosion and cuticle sclerotization for 40–50 days and then remained at their highest levels (15–21 μg MBQ and 22–32 μg EBQ per beetle) through 80 days posteclosion. Virgin females that were collected 40–80 days after eclosion contained approximately 40% more of these compounds than males of the same age. The build-up of ρ-benzoquinones subsequent to cuticle sclerotization apparently reflects the need for an adequate cuticular barrier for self-protection from these defensive compounds.

Distinction of N-substituted histidines by electrospray ionization mass spectrometry.

The amino acid histidine is one of the primary sites of protein conjugation with catecholamines that occurs during the process of insect cuticle sclerotization.1,2 Oxidative conjugation of the model compounds N-acetylhistidine (NAcHis) with N-acetyldopamine (NADA) via an o-quinone intermediate (NADA quinone) has been studied previously and the site of NAcHis attachment at the aromatic ring of NADA has been elucidated by NMR spectroscopy.3 Analysis of products derived from the acid hydrolysis of insect cuticle provided further evidence for the cross-links between catecholamines and histidyl residues of the cuticular proteins.4,5 A more difficult task is to establish the site of catecholamine attachment at the histidine moiety, for which two di†erent nucleophilic groups, Nq and Nn, can react with N-acetyldopaquinone (Scheme 1). Because of the substitution pattern in the imidazole ring, conjugates formed via nucleophilic attack by the non-equivalent imidazole nitrogen atoms (Nq and Nn) are difficult to distinguish by 2D-NMR without the assistance of molecular modeling and with small quantities of adducts obtained from the reaction mixture by reversed-phase liquid chromatography.4 Here, we report a facile distinction of N-1 (Nq) and N-3 (Nn) substituted histidines using electrospray ionization tandem mass spectrometry (ESI-MS/MS). A mechanistic rationale for the distinct dissociations of the gasphase ions is also discussed. As model compounds, we used N-1 (Nq) and N-3 (Nn) methylated histidines 1 and 2 (purchased from Sigma and Aldrich, respectively, and used as received). The compounds were infused in methanol or aqueous methanol solutions containing 0.5% acetic acid or ammonium acetate. ESI-MS of 1 and 2 resulted in efficient protonation to yield ions 1H` and 2H`, respectively, which appeared at m/z 170 (spectra not shown). Collisionally activated dissociations (CAD) of 1H` and 2H` were investigated in a radiofrequency-only quadrupole collision cell of a Sciex API-III triple-quadrupole tandem mass spectrometer (argon as collision gas, 26 eV laboratory collision energy) and in a quadrupole ion trap (Finnigan LCQ, helium at 10~3 Torr (1 Torr \ 133.3 Pa) as bu†er gas). The N-1 methylated isomer 1H` underwent dominant loss of to form a fragment ion at m/z 124. This dissoH2O] CO ciation had a sharp energy threshold ; the m/z 124 peak was weak at low collision energies, e.g., D1% relative to m/z 170 at D0.5 eV, corresponding to 5% relative collision energy (RCE) in the ion trap instrument. However, at 10% RCE (D1 eV), CAD was almost complete and the m/z 124 ion was by far the predominant species in the spectrum [Fig. 1(a)]. Other primary fragments of 1H` were weak, e.g., m/z 153 (loss of ammonia), 152 (loss of water) and 126 (loss of A second2 ary fragment appeared at m/z 109, which was due to sequential elimination of and To deduce the elemental 2 3 composition of the m/z 109 ion, the [13C,15N]-isotopomer of 1H` at m/z 171 was selected and collisionally dissociated. The relative abundances of the m/z 110 and 109 ions (82.9 and 17.1%, respectively, data not shown) were close to those calculated for loss of (83.3 and 16.7%, respectively), 2 ] 3 but di†ered from those calculated for loss of CO] 2 (75.0 and 25.0%, respectively). By comparison, the ] 3 measured relative abundances of the m/z 125 and 124 ions (87.0 and 13.0%, respectively) agreed well with the calculated values for loss of (87.5 and 12.5%, respectively). H2O] CO The CAD spectrum of the N-3 methylated isomer 2H` differed substantially from that of 1H` [Fig. 1(b)]. Ion 2H` showed loss of ammonia (m/z 153), (m/z 126) and COOH 2 (m/z 125) as important primary dissociations. The m/z 126 ion underwent further dissociations by loss of ammonia (m/z 109) and (m/z 97). The di†erences in primary dissociations 2 observed in the CAD spectra thus allowed unequivocal distinction of the methylation site in the imidazole ring, since the N-1 methylated isomer 1H` lost but the N-3 H2O] CO, isomer did not. CAD spectra obtained on the triple-quadrupole tandem mass spectrometer showed more extensive dissociations, including side-chain cleavages (m/z 95È97) and losses of substituents (m/z 81È83, 68) (Fig. 2). However, the characteristic loss of was dominant in the CAD spectrum of H2O] CO 1H` and virtually absent for 2H`. Similar results were obtained for several isomeric Nacetyldopamine-N-acetylhistidine conjugates and deacetylated derivatives, the latter isolated from acid hydrolyzates of insect

Catechols Involved in Sclerotization of Cuticle and Egg Pods of the Grasshopper, Melanoplus sanguinipes, and Their Interactions With Cuticular Proteins

N-Acetyldopamine (NADA) is the major catechol in the hemolymph of nymphal and adult grasshoppers, Melanoplus sanguinipes (F.), and mainly occurs as an acid-labile conjugate indicated to be a sulfate ester. Its concentration increases in last instar nymphs and peaks during adult cuticle sclerotization. Dopamine (DA), the precursor of NADA and melanic pigments, is about 10 times lower in concentration than NADA, but shows a similar pattern of accumulation. NADA also predominates in cuticle, but its concentration is lowest during the active period of sclerotization, reflecting its role as a precursor for quinonoid tanning agents. Two other catechols, 3,4dihydroxybenzoic acid (DOBA) and 3,4-dihydroxyphenylethanol (DOPET), also occur in hemolymph and cuticle, and their profiles suggest a role in cuticle stabilization. Solid-state NMR analysis of sclerotized grasshopper cuticle (fifth instar exuviae) estimated the relative abundances of organic components to be 59% protein, 33% chitin, 6% catechols, and 2% lipid. About 99% of the catechols are covalently bound in the cuticle, and therefore are involved in sclerotization of the protein-chitin matrix. To determine the types of catechol covalent interactions in the exocuticle, samples of powdered exuviae were heated in HCl under different hydrolytic conditions to release adducts and cross-linked products. 3,4-Dihydroxyphenylketoethanol (DOPKET) and 3,4-dihydroxyphenylketoethylamine (arterenone) are the major hydrolysis products in weak and strong acid, respectively, and primarily represent NADA oligomers that apparently serve as cross-links and filler material in sclerotized cuticle. Intermediate amounts of norepinephrine (NE) are released, which represent N-acetylnorepinephrine