The melanization road more traveled by: Precursor substrate effects on melanin synthesis in cell-free and fungal cell systems

Abstract

Natural brown-black eumelanin pigments confer structural coloration in animals and potently block ionizing radiation and antifungal drugs. These functions also make them attractive for bioinspired materials design, including coating materials for drugdelivery vehicles, strengthening agents for adhesive hydrogel materials, and free-radical scavengers for soil remediation. Nonetheless, the molecular determinants of the melanin "developmental road traveled" and the resulting architectural features have remained uncertain because of the insoluble, heterogeneous, and amorphous characteristics of these complex polymeric assemblies. Here, we used 2D solid-state NMR, EPR, and dynamic nuclear polarization spectroscopic techniques, assisted in some instances by the use of isotopically enriched precursors, to address several open questions regarding the molecular structures and associated functions of eumelanin. Our findings uncovered: 1) that the identity of the available catecholamine precursor alters the structure of melanin pigments produced either in Cryptococcus neoformans fungal cells or under cell-free conditions; 2) that the identity of the available precursor alters the scaffold organization and membrane lipid content of melanized fungal cells; 3) that the fungal cells are melanized preferentially by an L-DOPA precursor; and 4) that the macromolecular carbon-and nitrogen-based architecture of cell-free and fungal eumelanins includes indole, pyrrole, indolequinone, and open-chain building blocks that develop depending on reaction time. In conclusion, the availability of catecholamine precursors plays an important role in eumelanin development by affecting the efficacy of pigment formation, the melanin molecular structure, and its underlying scaffold in fungal systems. 13 C, 15 N]tyrosine precursor. The 1.5-day melanins were examined in a 10-h experiment (dark blue contours) run on a Bruker AV3 DNP spectrometer operating at a 1 H frequency of 600 MHz, temperature of 100 K, magic-angle spinning frequency of 12.5000 Ϯ 0.0005 kHz, and TEDOR evolution period of 1.6 ms corresponding to one-bond pairwise 13 C-15 N correlations. The 3.0-day melanins were examined with a 46-h experiment (red contours) run on a Varian (Agilent) DD2 spectrometer operating at a 1 H frequency of 600 MHz, temperature of 234 K, magic-angle spinning frequency of 20.00 Ϯ 0.02 kHz, and TEDOR evolution period of 1.4 ms. Spinning sidebands that are incidental to the structural analysis are denoted by asterisk symbols. The 13 C-15 N cross-peaks designated by uppercase letters are identified provisionally with directly bound C-N pairs within the illustrated chemical structures, drawing on the corresponding ACD-predicted chemical shifts and previously proposed melanin structural constituents (11,13,16,41). The predicted chemical shift values, typically quoted with average error limits of 3-10 ppm, are shown for 13 C (red) and 15 N (blue). Cell-free and fungal melanin development

The structural unit of melanin in the cell wall of the fungal pathogenCryptococcus neoformans

“…The differences between C. neoformans derived particles and those from auto-polymerized L-DOPA imply different synthesis, as fungal particles showed a unique electron microscopic appearance and size. We have shown previously that C. neoformans and synthetic melanins shared similar chemical groups in the aromatic core (18); however, differences between these materials could stem from the fact that the production of fungal particles is vesicle-associated and catalyzed by a laccase, whereas autopolymerization is a spontaneous oxidative process that occurs in solution. Moreover, C. neoformans melanin granules isolated from the extracellular media are spherical shaped and biologically synthesized particles of up to 200 nm in diameter, similar to those isolated from natural eumelanins such as Sepia officinales (43,67), and are found covalently bound to proteins as also reported for mammalian melanin (68).…”

“…The typical 90 degree pulse_lengths for the 13C cross-polarization (CPMAS) experiments were 1.65 μs for 1 H and 2.25 μs for 13 C, respectively. As described previously (18), 1 H decoupling using the small phase incremental alternation pulse sequence (SPINAL) with a field strength of ~180 kHz was applied during signal acquisition. The 2D 13 C-13 C through-space dipolar assisted rotational resonance (DARR) correlation experiments were collected using a 50 ms mixing time with similar 1 H and 13 C 90 degree pulselengths and decoupling field strength as described for the 13 C CPMAS experiments.…”

“…The higher order of structure of eumelanin is considered 'disordered', since different planar structures are oriented in diverse geometries to one another resulting from diversity of DHI/DHICA oligomers stabilized by hydrogen bonding, cation-pi and Van der Waals interactions (48). The aggregation of these simpler units lead to formation of the granular structure that we refer to as melanin granules, which have functional groups that interact with cell wall and cell membrane components such as lipids [(CH2)n, depicted in green] proteins and polysaccharides (C=C, depicted in pink) (18). B) Model representing the architecture of the plasma membrane represented by a lipid bilayer, that interact with melanin nanoparticles that aggregate to form larger melanin granules (21).…”

“…Once in the cell wall, aggregation of melanosomes into up to 200 nm melanin granules might be promoted by laccase enzyme delivered to the cell wall via MVB fusion with the plasma membrane (46,81,89) (E), while their arrangement within the fungal cell wall may use as a scaffold concentric membranous sheets reported in basidiomycetes (49,93,94) (F, white arrows). Melanosomes anchor to the cell wall via multiple interactions: i) associative and covalent interactions between them and cell-wall constituents (chitin, chitosan, glucans, lipids), possibly mediated by Blp1-chitin and CNAG_05312glucans/chitin/chitosan (16,18,19), and ii) electrostatic interactions modulated by chitosan-melanin charges (14,31,32,34). Regular cell growth requires the cell-wall remodeling to allow budding inducing the melanin scaffold degradation/alteration via secretion of of peptidases (96), chitinases (97), and glucanase (62) thus releasing melanin granules and strung-like granules (G) into the extracellular environment.…”

“…neoformans is a unique system for the study of melanin biology because unlike other melanotic fungi, only exogenously provided catecholamines or diphenolic substrates, such as dihydroxyphenylalanine (L-DOPA), are utilized for melanin synthesis and not as an energy source (11)(12)(13). Consequently, it is possible to selectively label the melanin polymer with NMRor radioactive isotopes, a property that has been exploited for melanin structural studies (14)(15)(16)(17)(18)(19). Melanization of C. neoformans results in the deposition of the polymer at the innermost surface of the cell wall, which becomes filled throughout over time (20).…”