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Role of Dopamine Chemistry in the Formation of Mechanically Strong Mandibles of Grasshoppers Kyueui Lee, Ekavianty Prajatelistia, Dong Soo Hwang, and Haeshin Lee Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.5b01680 • Publication Date (Web): 27 Jul 2015 Downloaded from http://pubs.acs.org on August 12, 2015
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Role of Dopamine Chemistry in the Formation of Mechanically Strong Mandibles of Grasshoppers Kyueui Lee†, Ekavianty Prajatelistia‡, Dong Soo Hwang‡, Haeshin Lee*,†,§ †
Department of Chemistry, KAIST, Daejeon 305-701, Korea School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang 790-784, Korea § Center for Nature-inspired Technology (CNiT) in KAIST Institute NanoCentury, KAIST, Daejeon 305-701, Korea ‡
ABSTRACT: Tooth hardness is an essential property for animal survival. In grasshoppers, chopping the tough cellulose of various plants is a critical survival function. Herein, we report the chemistry explaining the strong mechanical properties of mandibles that perform tooth functions in grasshoppers. Mandible hardness (~ 0.4 GPa in hardness vs. ~ 0.25 GPa for other insect cuticles) was solely achieved with organic components (no trace presence of any inorganic materials), which differs from the teeth of human and other animals. Mechanism studies revealed that dopamine plays an important role in the mechanical properties of grasshopper mandibles. N-acetyl-dopamine (known as NADA) and N-β-alanyl-dopamine (known as NBAD), in which the amine groups are chemically protected, were identified to act in tanning processes in insect cuticle formation. The use of chemically unprotected dopamine in tanning is reported for the first time and suggests that dopamine may be an effective molecule for producing hard surfaces via exposure to oxygen (i.e., air) environments and by creating gradient mechanical properties of the corresponding materials through the spatial regulation of dopamine oxidation.
Organs with high-stiffness such as jaws, shells, and teeth attribute survival of animals. These materials usually require bio-mineralization, which bestows strong mechanical properties. Human teeth, for example, consist of 95 wt.% minerals in the external enamel layers, which are primarily composed of hydroxyapatite and its related inorganic materials.1 Another example is the inner surface of a turtle shell, which contains various minerals such as calcium (15-20 wt.%) and magnesium.2 In contrast, mineralization is not the only way for creatures to build-up teeth-like hard materials. For example, the jaws of polychaete such as Nereis and Glycera contain only a few percent of minerals such as transition metals (Zn for Nereis and Cu for Glycera). Thus, organic components play an important role in exhibiting hardness of 0.4 - 0.9 GPa and elastic modulus of 7.0 - 13.0 GPa.3, 4 Interactions among proteins, catecholamine derivatives, and transition metals achieved the strong mechanical properties.5 Ali et al. recently reported on the mechanical properties of squid beaks, which exhibit remarkable hardness (~ 1 GPa) and elastic modulus (~ 5 GPa). Squid beaks achieve these properties in the absence of inorganic components, which demonstrates that inorganic components are not an essential element in naturally occurring hard materials.6 Similarly, the exoskeletons of insects exhibiting hard and resilient material properties (~ 0.3 GPa in hardness and ~ 7.0 GPa in Young’s modulus) are the results of purely organic components.7 However, the underlying chemical mechanisms in the stiffness of these organic materials substantially differ. For squid beak, two strategies co-exist in sclerotization. The catechol-containing molecule, 3,4-dihydroxy-L-phenylalanine (DOPA), is integrated into a polypeptide form, which are oxidatively crosslinked.6 Another catechol-containing molecule, 4-methyl catechol, is in fact not integrated into polypeptides, which are freely diffused and crosslinks polymers.8 For insect cuticles, the entire sclero-
tization process solely relies on catechol derivatives that are not integrated into polymers; instead, N-acetyl-dopamine (known as NADA) and N-β-alanyl-dopamine (known as NBAD) oxidatively crosslinks cuticular macromolecules.9 The benefits of small-molecule catecholamine crosslinkers include a high diffusion rate (which resulted in fast inter-molecular crosslinking) and the availability of oxygen at the air/body interface where the cuticles are formed. Notably, the oxygen at an air/water interface largely accelerates polymeric crosslinking.10-12 A low diffusion rate is one representative benefit of catechol integration into polymers. Low diffusion inhibits dilution due to seawater in the case of beak formation in squids. In 1940’s, Pryor reported the role of small catecholamine derivatives in insect cuticle sclerotization.13, 14 Sugumuran described the mechanisms of insect sclerotization: quinone tanning, β-sclerotization, and quinone methide sclerotization.15 In these mechanisms, the amine group in the catecholamines is chemically protected by acylation as characterized in NADA and NBAD. Ex vivo experiments on sclerotization processes have been performed with chemically unprotected version catecholamines. For example, unprotected isotopic-labeled dopamine, a representative catecholamine, was directly injected into Manduca sexta pupae16-18 and ecdysed cockroaches19. The injected labeled dopamine was integrated into the newly formed cuticles during sclerotization. However, the role of chemically unprotected catecholamines in insect cuticle for intermolecular crosslinkings to increase mechanical strength has not been reported in nature. Herein, we report for the first time that dopamine, a widely known neurotransmitter, crosslinks proteins in the mandibles of grasshoppers and thus strengthens their mechanical properties. Unlike the conventional catecholamines such as NADA and NBAD found in tanned cuticles, the mandibles of grass-
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hoppers utilize un-protected dopamine for tanning. This was identified using high performance liquid chromatography connected with mass spectrometry (HPLC-MS) of the acid hydrolyzed grasshopper mandibles. Furthermore, we found that the mandibles consist of purely organic compounds (similar to squid beaks). In addition, three distinct highly, moderately, and sparsely tanned areas were observed. Measuring the mechanical properties of the grasshopper mandibles revealed that the hardness value was approximately 0.4 GPa, and Young’s modulus was approximately 6.0 GPa. In the tanned area, the dopamine was chemically reacted with the secondary amine of histidine, which is consistent with the typical sclerotization observed in animals.8, 20, 21 Considering rapid, air-sensitive oxidation of dopamine10 compared with the previously identified N-protected catecholamines (NADA and NBAD), our study suggests that rapid hardening of mandibles is critical to obtain nutrients, in which dopamine plays an important role via rapid oxidative tanning processes. This might help for grasshoppers to eat plant cellulose immediately after birth.
Figure 1. Mandible extraction and composition analysis of Shirakiacris shirakii. (a) A photograph of Shirakiacris shirakii. (b) The mandibles of Shirakiacris shirakii shown by lifting the labrum. (c) A photograph of the extracted mandible with the tanning gradient from the black tip to the white base. (d) EDS analysis of the mandibles demonstrating carbon (white), oxygen (light gray), and nitrogen (dark gray) (nor inorganic components). (e) Gravimetric measurements: chitin (cross-hatch), tanned pigments (dark gray), proteins (light gray), and water (white).
After lifting the labrum of the grasshopper (Shirakiacris shirakii) (Figure 1a and b), two distinct pigmentation areas (tanned and untanned) were observed (Figure 1c). As previously hypothesized, we used energy dispersive X-ray spectroscopy (EDS) for the elemental analysis of mandibles to demonstrate the absence of inorganic elements. All components were organics (carbon, nitrogen, and oxygen; no indication of calcium or silicon) (Figure 1d, S1). From untanned to tanned areas, the carbon content decreased: approximately 72 % for the untanned regions, 62-69 % for the moderately tanned areas, and approximately 56 % for the fully tanned areas. Conversely, the nitrogen content increased from the untanned to tanned regions: approximately 8 % for the untanned, approximately 8-15 % for the moderately tanned, and approximately 18 % for the fully tanned regions (Figure 1d).
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A similar increase in nitrogen content was observed using Xray photoelectron spectroscopy (XPS). The survey scans showed that the nitrogen content of ~ 2.4 % for untanned areas and it was increased to ~ 8.4 % for tanned ones (Figure S2). The tanning process typically involves oxidative crosslinking between the nitrogen-containing catecholamines and basic cuticle proteins.9 The atomic percentage of nitrogen in chitin is about 7 %, which is relatively low compared with catecholamine-histidine adducts (~ 18 %) or basic cuticular proteins (~ 20 %). Thus, an increase in nitrogen content obtained in XPS and EDS both explains that the tanned areas are rich in catecholamine crosslinked cuticular proteins. We additionally performed an assay aiming for quantitative determination of catecholamine crosslinkers which has often been used for DOPA detection.22 For purification, each hydrolysate (untanned and tanned) was applied to a phenylboronate resin, which selectively binds to catechol-containing compounds based on cis-diol interactions.23, 24 After elution via acetic acid, the eluent was continuously reacted with hydrochloric acid, nitrite, and sodium hydroxide, which develops quinone derivatives showing reddish color (Figure S3a). Comparing the ultraviolet–visible (UV-Vis) absorbance at 500 nm, we determined that the amount of catechols in tanned area is approximately four times greater than that of untanned area (Figure S3b). Considering the role of catecholamines in insect cuticle as crosslinking agents13, 14 and the results shown above, the increased content in nitrogen determined spectroscopic methods might attribute to the presence of catecholamines. The primary biological function of tanning chemistry is to control the mechanical strength of cuticles for protection. Other function of tanning includes strengthening of ant mandibles25 and spider fangs26. However, the presence of dopamine in these hardened tissues has not been reported. Previously, the tanning reaction of dopamine predominantly and rapidly occurs only at liquid/air interfaces.10 These results show that oxidation of dopamine is extremely air-sensitive compared to NADA and NBAD in which the amines are chemically protected. The air-sensitive oxidation may explain the rapid build-up of hard teeth (i.e. mandibles) in an airsensitive way, which might be essential to tooth functions of newborn insects from caterpillars. Gravimetric measurements were performed to quantitatively assess chitin, tanned pigments, proteins, and water (see the supporting information for experimental procedures).6 This analysis has been widely used in determining the components of biological hard tissues.27-29 Notably, the weight percentage of water largely decreased from the untanned to tanned regions: ~ 76 to ~ 10 %) (white bars, Figure 1e). However, the proportions of proteins and tanned pigments increased from the untanned to tanned areas: ~ 10 to ~ 59 % for proteins and ~ 4 to 22 % for pigments. The positive correlation between the tanned pigments and proteins could indicate oxidative crosslinking (i.e., tanning) between proteins and catecholamine molecules (pigments). Previous reports found that the crosslinking of catechol groups in biopolymers resulted in dehydration,27, 30 which reduces water content (i.e., solvation) in local environments.31, 32 These results support the observed decrease in water weight percentage in the more thoroughly tanned pigments. The formation of water-insoluble films spontaneously formed by tanning-mimicking reactions represents the recent findings in dehydration.10-12
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Figure 2. Result of mass spectrometry (MS) from directly injected acid-hydrolyzed grasshopper mandibles.
The chemical identification of catecholamine pigments is necessary to assess tanning in mandibles. The catecholamine pigments of mandibles were isolated from acid hydrolysates using a phenylbornate affinity column. After elution process, the purified eluent was analyzed using mass spectrometry (MS). The MS analysis primarily revealed dopamine (m/z = 154) and histidine-dopamine conjugates (m/z = 307). Additionally, arternone (m/z = 168, the adduct product of dopamine in acid hydrolysis33) as well as O-tyrosino-3,4-dihydroxyacetophenone (m/z = 332, originating from tyrosine34) were obtained (Figure 2). Multiple components were identified in the previous MS analysis. Thus, we performed HPLC/MS for further analysis. Three distinct peaks (detection wavelength of 280 nm) were observed (Figure 3a), and each peak was analyzed using webconnected MS equipment. The result showed that P1 primarily exhibited histidine-dopamine crosslinks (m/z = 307) (Figure 3b). The role of catechol-histidine adducts for hardening structure has been reported in nature.8, 20, 21 Thus, histidinedopamine is the key molecule in strengthening the structure of mandibles. P2 was identified as arterenone (m/z = 168) and its sodium adduct (m/z = 190), which commonly originated from dopamine (Figure 3c). In particular, the peak with the highest intensity (P3) was dopamine (m/z = 154) and its fragment (m/z = 137) (Figure 3d). Amine-associated fragmentation is often observed in other compounds, such as serotonin, in which the terminal primary amine group was freed from serotonin without additional fragmentation (Figure S4). We performed nano-indentation experiments to determine the hardness of grasshopper mandibles according to their pigmentation levels. We considered representative load displacement curves in each pigmentation state (Figure S5) to obtain the mechanical parameters of Young’s modulus and hardness by applying the Oliver-Pharr method.35, 36 The untanned region in the grasshopper mandibles exhibited 0.30 ± 0.01 GPa for hardness and 3.8 ± 0.6 GPa for Young’s modulus. In contrast, the tanned area showed 0.36 ± 0.03 GPa for hardness and 6.1 ± 0.4 GPa for Young’s modulus. Thus, dopamine-mediated tanning reactions positively contribute to mechanical strength. The mechanical properties of both tanned and untanned areas exhibit a decrease in hydrated condition (untanned: 0.28 ± 0.01 GPa for hardness and 2.5 ± 0.1 GPa for Young’s modulus, tanned: 0.30 ± 0.01 GPa for hardness and 5.47 ± 0.2 GPa for Young’s modulus) suggesting the significant role of water contents in determining the rigidity somewhat similar to squid beaks.6 In general, the hardness of the tanned region is relatively higher than previously reported hardness values for insect cuticles.7
Figure 3. HPLC/MS analysis of the solution after acid hydrolysis followed by phenylboronate separation of grasshopper mandibles. (a) Three major peaks were detected at 280 nm. (b) MS data of peak 1 (P1), (c) peak 2 (P2), and (d) peak3 (P3).
In addition to the mechanical properties, the surface morphology of the mandibles changes. Hexagonal arrays were observed (SEM), which could originate from densified chitins.
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rapid build-up of the hard, organic solids that are essential to tooth function directly following birth from caterpillars.
ASSOCIATED CONTENT Supporting Information. Experimental procedures and additional figures. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author * E-mail:
[email protected] ACKNOWLEDGMENT This work is supported from National Research Foundation of South Korea: Mid-career Scientist Grant (2015021564), and Molecular-Level Interface Research Center (20090083525).
REFERENCES
Figure 4. (a) Young’s modulus (X-axis) and hardness (Y-axis) of untanned and tanned regions of grasshopper mandibles. (b) The typical mechanical property map for hard tissues (functioning as tools) with low degree of mineralization.7
Treatment with alkaline peroxide proved that the hexagonal arrays are from chitin. Alkaline peroxide solubilizes proteins and tanned pigments; however, chitin remains intact. Figure S6 clearly shows the hexagonal structures of unsolubilized chitins. Interestingly, diagonal lengths of the chitin hexagon decrease from untanned to tanned areas, which indicate densification mediated by dopamine crosslinking (Figure 2, 3b, S7). We calculated the average length of those structures in each scan region (P1: ~ 12 µm, P2: ~ 10 µm and P3: ~ 8 µm, respectively) (Figure S7e-g). Accordingly, we hypothesized that the chemically unprotected dopamine facilitates crosslinking between the protein/chitin components in mandible chitins upon air exposure during the tanning process. Herein, we are the first to report that dopamine is a sclerotization element in insects. Crosslinking chemistry in the tanned areas of grasshopper mandibles is present in the form of histidine-dopamine but neither NADA- nor NBAD-histidine. The mandibles show strong mechanical hardness compared with other cuticles. The surface morphology of the mandible revealed that the superior hardness originated from the dopamine based tanning process, which leads to chitin densification. The rapid oxidation of dopamine compared with the reported catecholamines of NADA and NBAD may explain the
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