Next Article in Journal
A Randomized Controlled Prospective Cohort Study on the Efficacy of a Witch Hazel Extract Cream for the Eyelids and Eye Contour Area and a Cleansing Face Cream in Dermatitis of the Eyelids
Next Article in Special Issue
Physicochemical Properties of a Bioactive Polysaccharide Film from Cassia grandis with Immobilized Collagenase from Streptomyces parvulus (DPUA/1573)
Previous Article in Journal
Fractional Ablative Carbon Dioxide Laser versus Fractional Non-Ablative 1410 nm Diode Laser in the Treatment of Acne Scars: A Clinical and Immunohistochemical Study
Previous Article in Special Issue
Effects of Chlorinated Water on Neurite Length of Cultured Dorsal Root Ganglion Neurons and Semaphorin 3A Content of Cultured Epidermal Keratinocytes
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Cosmetics
Volume 11
Issue 3
10.3390/cosmetics11030082
cosmetics-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

N-Acetylneuraminic Acid Inhibits Melanogenesis via Induction of Autophagy

by
Kei Yoshikawa
1 and
Kazuhisa Maeda
2,*
1
Bionics Program, Tokyo University of Technology Graduate School, 1404-1 Katakura, Hachioji, Tokyo 192-0982, Japan
2
School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakura, Hachioji, Tokyo 192-0982, Japan
*
Author to whom correspondence should be addressed.
Cosmetics 2024, 11(3), 82; https://doi.org/10.3390/cosmetics11030082
Submission received: 20 March 2024 / Revised: 30 April 2024 / Accepted: 14 May 2024 / Published: 21 May 2024
(This article belongs to the Special Issue 10th Anniversary of Cosmetics—Recent Advances and Perspectives)
Browse Figures
Versions Notes

Abstract

:
N-acetylneuraminic acid (Neu5Ac) is the predominant form of sialic acid present in the glossy swiftlet (Collocalia esculenta). It is also the only form of sialic acid detected in the human body. In this study, we investigated the mechanism underlying melanogenesis inhibition by Neu5Ac. We discovered that a reduction in tyrosinase protein levels led to an inhibition of melanin production by Neu5Ac. Additionally, the mRNA and protein levels of ubiquitin-specific protease (USP5) and microtubule-associated protein 1 light chain 3 (LC3)-II increased, while those of p62 decreased, indicating enhanced autophagic activity. Lysosomal cathepsin L2 protein levels also increased, and immunostaining revealed colocalization of lysosomal membrane protein (LAMP)-1 and tyrosinase. Additionally, levels of chaperonin containing T-complex polypeptide (CCT), implicated in increased autophagic flux, were elevated. Altogether, these findings suggest that tyrosinase-containing coated vesicles are transported by Neu5Ac into the autophagic degradation pathway, suppressing mature melanosome generation. This process involves increased USP5 levels preventing recognition of polyubiquitin by proteasomes. Furthermore, elevated CCT3 protein levels may enhance autophagic flux, leading to the incorporation of tyrosinase-containing coated vesicles into autophagosomes. These autophagosomes then fuse with lysosomes for cathepsin L2–mediated degradation. Thus, our findings suggest that Neu5Ac reduces tyrosinase activity and inhibits melanosome maturation by promoting selective autophagic degradation of abnormal proteins by p62.

1. Introduction

Research in skin lightening has been primarily directed toward develo** cosmetic ingredients that reduce pigmented spot formation [1]. Additionally, studies have focused on understanding the biological factors involved in pigmentation [2,3], melanogenesis, and the promotion of epidermal turnover [4,5]. Melanin is synthesized from tyrosine via a pathway in which tyrosinase is the primary enzyme. Compounds such as rhododenol, a tyrosinase substrate that produces hydroxyl radicals in melanosomes, have been reported to cause chemical toxicity, resulting in the formation of white spots due to melanosome destruction [6,7]. Therefore, we investigated the mechanism underlying the melanogenesis-inhibitory effect of N-acetylneuraminic acid (Neu5Ac; Figure 1), a nonphenolic structure that does not generate hydroxyl radicals through tyrosinase activity. Instead, Neu5Ac has been found to inhibit tyrosinase activity and melanogenesis [8].
Sialic acid is found in glossy swiftlets (Collocalia esculenta), with approximately 9% of Neu5Ac occurring in the form of sugar chains [9]. Neu5Ac, the most abundant form of sialic acid, is characterized by an acidic amino sugar with nine carbon atoms and an intramolecular carboxyl group. Notably, it is also the only sialic acid detected in the human body [10]. We previously validated the presence of Siglec proteins, which are known to bind sialic acid, in B16 melanoma cells. Sialic acid has been reported to affect intracellular Pmel17 processing and transport [11,12] and has been identified as a major regulator of melanogenesis. Sialylated oligosaccharides were recently found to affect the synthesis and transport of melanosomes to keratinocytes [13]. Furthermore, autophagy may regulate the stability of sialin, a lysosomal transporter of sialic acid, and, consequently, sialic acid levels [14].
The rate-limiting step for tyrosinase activation is glycosylation and transfer to the melanosomes [15]. Glycosylation occurs within the Golgi organ-associated endoplasmic reticulum (GERL) [16,17], followed by maturation [18]. Subsequently, tyrosinase-containing coated vesicles are transported to stage II melanosomes. LAMP-1, which is a glycosylated protein present on the lysosomal membrane, is used as a lysosomal marker [19]. Cathepsin L2 is present in both cytoplasmic vesicles and lysosomes and plays a role in melanosomal proteolysis within lysosomes [20,21,22]. Cells possess two major proteolytic pathways: the ubiquitin–proteasome and macroautophagy–lysosome pathways [23]. The ubiquitin–proteasome system (UPS) degrades most abnormal proteins that have lost their function due to structural breakdown; however, the proteasome cannot destroy aggregated proteins [24,25]. The macroautophagy–lysosome system degrades abnormal proteins that cannot be destroyed by proteasomes [26,27]. The gene clusters involved in these two completely independent degradation systems do not overlap [28]. However, discoveries such as autophagy activation due to ubiquitin–proteasome system inhibition, ubiquitinated protein accumulation in the absence of autophagy, and the identification of a ubiquitin-binding protein group localized to autophagosomes have drawn attention to the crosstalk between the two degradation pathways [29].
Autophagy is a degradation system with low substrate selectivity, as degradation is considered complete when autophagosomes, which can engulf several hundred thousand proteins of average size simultaneously, fuse with lysosomes. In UPS, ubiquitin is added to proteins destined for degradation or denatured via enzymatic reactions involving ubiquitin-activating enzyme (E1), ubiquitin-binding enzyme (E2), and ubiquitin ligase (E3) (ubiquitination). Subsequently, shuttle proteins have the ability to bind ubiquitin chain ubiquitinate proteins and transport them to the proteasome, where they facilitate the degradation of their target proteins [23,26,30]. The ubiquitin-activating enzyme UBE1 (also known as ubiquitin-like modifier-activating enzyme [UBA]1) activates ubiquitin–protein isopeptide bond formation [31,32]. The ubiquitin-specific peptidase ubiquitin-specific protease (USP)5 is a deubiquitinating enzyme that degrades the polyubiquitin chains of aberrant proteins and is involved in ubiquitin recycling [33].
Recently, research has increasingly focused on develo** skin-whitening agents that not only inhibit melanin synthesis in melanocytes but also utilize autophagy, wherein intracellular components are self-degraded within lysosomes [34]. Microtubule-associated protein 1 light chain 3 (LC3) is synthesized in the cytoplasm. Following synthesis, its 22 C-terminal residues are cleaved by Atg4, a cysteine protease. LC3-I is covalently bound to phosphatidylethanolamine (PE), a phospholipid molecule, to form LC3-II, which binds to the sequestration or autophagosome membranes [35,36,37]. p62 (sequestosome 1) is a ubiquitously expressed intracellular protein that is absent in fungi and plants but is retained in multicellular animals; it is a selective autophagy substrate. The N-terminal Phox1 and Bem1p (pB1) domains of p62 have oligomer-forming abilities [38,39]. Consequently, autophagy-deficient cells and tissues accumulate large quantities of p62, forming aggregates of p62 and p62-binding proteins. Activation of autophagy is thus expected to reduce p62 levels, while autophagy inhibitors such as chloroquine are expected to increase their accumulation. p62 has both a C-terminal ubiquitin-binding domain, known as the UBE domain, and a binding domain with LC3, one of the major components of autophagosomes [40]. CCT3, known as chaperonin containing TCP1 subunit 3(γ), is a molecular chaperone. It belongs to the chaperonin family and facilitates the folding of several proteins immediately after synthesis and restores the normal conformations of abnormal proteins [41]. In proliferating cells, high levels of protein synthesis are required for proliferation [42], requiring an increase in CCT levels for the folding of nascent proteins [43]. Knockdown or depletion of CCT leads to an increase in protein aggregation and a decrease in autophagic clearance. Additionally, CCT regulates lysosomal biogenesis via the actin cytoskeleton [44].
The aim of this study is to investigate whether Neu5Ac inhibits melanogenesis through the induction of autophagy.

2. Materials and Methods

2.1. Effect of Neu5Ac on Proliferation of B16 Melanoma Cells

Mouse B16 melanoma cells (JCRB Cell Bank, Osaka, Japan) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with GlutaMAX (Thermo Fisher Scientific Co., Waltham, MA, USA). After 24 h incubation, 1 µL each of 100, 50, 25, 12.5, 6.25, or 3.125 mg/mL Neu5Ac or PBS (control) was added to different wells, and the cells were cultured for 2 days. Next, 10 µL cell counting kit-8 reagent (Do** Chemical Laboratory Co., Ltd., Kumamoto, Japan) was added to each well; absorbance was measured immediately and 2 h later at 450 nm with a Multi-Detection Microplate Reader (DS Pharma Biomedical Co., Ltd., Osaka, Japan). Experiments were performed with n = 4.

2.2. Effect of Neu5Ac on Tyrosinase Activity in B16 Melanoma Cells

B16 melanoma cells (100,000 cells/mL; 100 µL aliquots) were seeded in 96-well plates; after 24 h culture, 1 µL each of 100, 50, 25, 12.5, 6.25, or 3.125 mg/mL Neu5Ac (fermentation of Escherichia coli, ≥98.0, CABIO Biotech (Wuhan) Co., Ltd., Wuhan, China) or of PBS (control) was added to different wells and the plates were further incubated for 2 days. Subsequently, the medium was completely removed, and 90 µL PBS containing 0.2% Triton X-100 and 10 µL PBS containing 10 mmol/L 3-(3,4-dihydroxyphenyl)-l-alanine (l-DOPA; Wako Pure Chemical Industries, Ltd.) were added. Experiments were performed with n = 4.

2.3. Effect of Neu5Ac on Tyrosinase, Pmel17, and MITF mRNA Levels, Using Real-Time Polymerase Chain Reaction (RT-PCR)

B16 melanoma cells (150,000 cells/mL; 2 mL aliquots) were seeded in a 35 mm dish; after 24 h culture, 200, 100, or 50 mg/mL Neu5Ac or 20 µL PBS (control) was added, and the cells were cultured for 2 days. The medium was completely removed, and RNA was extracted from cells using the RNeasy®Protect Mini Kit (50) (Qiagen Co., Ltd., Tokyo, Japan). The mRNA levels of melanogenesis-related factors (tyrosinase, Pmel17, and MITF) were measured using the Applied Biosystems 7900HT Real-Time PCR System (Thermo Fisher Scientific Co.). β-actin and GADPH were used as housekee** genes. Experiments were performed with n = 3. Data were analyzed using the relative quantification method (ΔΔCt method).

2.4. Effect of Neu5Ac on Tyrosinase, Pmel17, and MITF Protein Levels, Using Western Blotting

B16 melanoma cells (250,000 cells/mL; 10 mL aliquots) were seeded in 100 mL dishes; after 24 h culture, 200, 100, or 50 mg/mL Neu5Ac or 100 µL PBS (control) was added and the cells were cultured for 3 days. The medium was completely removed, and 500 µL 62.5 mmol/L Tris HCl, 2% sodium dodecyl sulfate (SDS) and 1% sodium deoxycholate buffer (pH = 6.8) were added, and the proteins were extracted. A cOmplete Mini EDTA-free tablet (Roche Diagnostics K.K., Tokyo, Japan) was used as the protease inhibitor, and the extracted proteins were reacted for 24 h at 4 °C. Next, 2.5 mL PBS, 2 mL glycerol, 4 mL 10% SDS, 1 mL 2-mercaptoethanol, and 0.5 mL 0.1% bromophenol blue were mixed in a 1:1 ratio with the extracted protein. The solution was then heated at 95 °C for 5 min. The buffer composition was as follows: 3.02 g Tris, 14.40 g glycine, 1 g SDS, and 1000 mL distilled water. Precision Plus Protein Dual Color Standards (Bio-Rad Laboratories, Hercules, CA, USA) were used as molecular weight markers. Three lanes with 20 µL of each sample were placed on a precast gel 4~20% SDS-PAGE mini (Tefco Corporation, Tokyo, Japan), and the voltage device was set to 54 mA for 2 h. The blotting buffer composition was as follows: 3.02 g Tris, 14.40 g glycine, 200 mL 20% methanol, and 1000 mL distilled water. Subsequently, an electric current of 180 mA was applied for 1 h. Blotting was performed at 180 mA, and the membrane was washed thrice with TBS- T (Tris Buffered Saline with Tween 20) for 5 min. Next, the membrane was incubated at 4 °C overnight with a blocking solution of 5% skim milk in TBS-T, followed by primary antibody overnight at 4 °C. The membrane was washed with TBS-T thrice for 5 min and then incubated with a secondary antibody under agitation (160 rpm) for 30 min at room temperature (24). Washing was performed with TBS-T for a total of 1 h, changing the solution at 2, 3, 10, 15, and 30 min. Amersham ECL Select western blotting detection reagent (GE Healthcare Technologies Inc., Chicago, IL, USA) was used as the detection reagent. Each membrane was immersed in 3 mL chromogenic solution, incubated under agitation (160 rpm), and allowed to react for 10 min at room temperature.
The primary and secondary antibodies used are shown in Table 1.

2.5. Effect of Neu5Ac on Ubiquitin-Specific-Processing Protease 5 (USP5) and Ubiquitin-like Modifier-Activating Enzyme 1 Y (UBE1Y) mRNA Levels, Using RT-PCR, and on USP5 and UbE1Y Protein Levels, Using Western Blotting

RNA was extracted from cells as described in Section 2.3. mRNA levels of ubiquitin proteasome-associated factors (USP5 and UBE1Y) were determined using the One Step SYBR® Prime Script RT-PCR Kit II with the Applied Biosystems 7900HT Fast Real-Time PCR System. β-actin and GADPH were used as housekee** genes. Each reaction was performed in triplicate. Data were analyzed using the relative quantification method (ΔΔCt method). Proteins were extracted as described in Section 2.4 and transferred to a membrane via electrophoresis. The primary and secondary antibodies used are shown in Table 2. The UBE1Y was detected using UBE1a/b antibodies and analyzed using mageJ (1.53, National Institutes of Health, Bethesda, MD, USA)

2.6. Effect of Neu5Ac on LC3, p62, CCT3, and LAMP-1 Protein Levels, Using Western Blotting

Proteins were extracted as described in Section 2.4, electrophoresed, and transferred onto membranes. The primary and secondary antibodies used are shown in Table 3. Western blots were photographed with WSE-6100 LuminoGraph I (ATTO Corporation, Tokyo, Japan), and analyzed using ImageJ.

2.7. Effect of Neu5Ac on p62 Localization, Using Fluorescent Immunostaining

B16 melanoma cells (150,000 cells/mL; 2 mL aliquots) were seeded in a 35 mm Matsunami glass bottom dish (Matsunami Glass Ind., Ltd., Osaka, Japan) and incubated for 24 h, followed by further incubation for 2 days after addition 20 μL Neu5Ac (200, 100, 50 mg/mL) or 20 µL PBS (control). The medium was completely removed, and the cells were fixed using 1 mL 4% paraformaldehyde. After the cells were washed thrice with PBS for 5 min each, they were incubated with 500 µL of the primary antibody anti-p62/SQSTMI for 1 h at room temperature. Subsequently, they were washed thrice with PBS for 5 min each. Following the washes, 500 µL of the secondary antibody Alexa Fluor 568 goat anti-rabbit antibody was added, and the dish was wrapped in aluminum foil and incubated for 40 min at room temperature. Next, 1.0 µg/mL H33342 solution (solvent, PBS) was added to 500 µL PBS and allowed to react for 10 min at room temperature. The cells were then observed at 600× with a confocal laser-scanning microscope (FV3000; Olympus Corporation, Tokyo, Japan).

2.8. Effect of Neu5Ac on Tyrosinase and Lysosomal (LAMP-1) Localization, Using Fluorescence Immunostaining (Multiple Staining)

B16 melanoma cells (150,000 cells/mL; 2 mL aliquots) were seeded in a 35 mm Matsunami glass bottom dish and incubated for 24 h, followed by further incubation for 2 days after the addition of 20 µL Neu5Ac (200, 100, or 50 mg/mL) or 20 µL PBS (control). The medium was completely removed, and 4% paraformaldehyde was added 1 mL at a time for cell fixation. After the cells were washed with PBS, 500 µL of a mixture of two primary antibodies was added to the cells, followed by incubation for 1 h at room temperature. Next, the cells were again washed with PBS, following which 500 µL of 1.0 µg/mL H33342 solution (solvent, PBS) was added, and the mixture was incubated for 10 min at room temperature. The cells were then washed with PBS and observed under a confocal laser scanning microscope (FV3000) at 400× and 800× magnification. The primary and secondary antibodies used are shown in Table 4.

2.9. Statistical Analysis

Data were calculated in terms of mean and SD values, and unresponsive t-tests were performed using Microsoft Excel. p < 0.05, p < 0.01, and p < 0.001 were considered statistically significant.

3. Results

3.1. Effect of Neu5Ac on Tyrosinase in B16 Melanoma Cells

B16 melanoma cell proliferation was not affected by the presence of Neu5Ac in culture at concentrations up to 2 mg/mL (Figure 2). Tyrosinase activity inhibition decreased in a concentration-dependent manner upon incubation with 0.5, 1, and 2 mg/mL Neu5Ac, showing a significant difference between the Neu5Ac group and the control. Tyrosinase mRNA levels were examined by RT-PCR (GAPDH housekee** gene) using the relative quantification method (ΔΔCt method). Tyrosinase mRNA levels obtained during culture with Neu5Ac did not significantly differ from those obtained for the control. Tyrosinase protein luminescence bands were analyzed using ImageJ. The tyrosinase protein level significantly decreased after incubation with 1 and 2 mg/mL Neu5Ac. Confocal laser scanning microscopy (magnification, 400×) showed that the number of melanosomes obtained in the presence of 1 and 2 mg/mL Neu5Ac was less than that obtained for the controls.

3.2. Effect of Neu5Ac on Tyrosinase and LAMP-1 Localization

Fluorescence immunostaining images of tyrosinase and LAMP-1 (Figure 3) were obtained at 400× using a confocal laser scanning microscope (Figure 3). In the tyrosinase and LAMP-1 merged images, cultures grown in the presence of 1 or 2 mg/mL Neu5Ac exhibited LAMP-1 and tyrosinase overlap around the cell nuclei, indicated by yellow dots, along with stronger staining intensity compared to the controls. Neu5Ac at 1 and 2 mg/mL concentrations in the presence of tyrosinase and LAMP-1 were colocalized in the culture. The fluorescence intensity of tyrosinase after treatment with 1 or 2 mg/mL Neu5Ac was lower than that after treatment with the control.

3.3. Effect of Neu5Ac on Cathepsin L2 in B16 Melanoma Cells

Cathepsin L2 was stained using fluorescent immunostaining and observed using a confocal scanning laser microscope (Figure 4). The subcellular localization of cathepsin L2 was examined using immunofluorescence labeling. Cathepsin L2 was detected in cytoplasmic vesicles and lysosomes, and B16 melanoma cells were cultured in the presence of 0.5, 1, or 2 mg/mL. Neu5Ac showed a stronger fluorescence intensity of cathepsin L2 than did the controls (Figure 4).

3.4. Effect of Neu5Ac on Protein Levels of Autophagy-Related Factors (LC3 and p62)

Luminescence band images were obtained for β-actin, LC3, and p62 proteins (Figure 5). Band analysis using ImageJ showed that the LC3-II protein level significantly increased, and the p62 protein level significantly decreased at Neu5Ac concentrations of 1 mg/mL and 2 mg/mL.

3.5. Effect of Neu5Ac on p62 Localization, Using Fluorescence Immunostaining

Fluorescence immunostaining images were obtained for p62 (Figure 6) and observed at 600× with a confocal laser scanning microscope. The results showed that treatment with 1 or 2 mg/mL Neu5Ac reduced p62 levels.

3.6. Effect of Neu5Ac on mRNA and Protein Levels of Ubiquitin-Proteasome System-Related Factors (USP5 and UBE1y)

The USP5 mRNA levels significantly increased upon treatment with 1 or 2 mg/mL Neu5Ac (Figure 7a). UBE1Y mRNA levels increased, but this change was not statistically significant (Figure 7a). Luminescence band images were obtained for the β-actin, USP5, and UBE1Y proteins (Figure 7b,c). Band analysis using ImageJ showed an increase in USP5 protein levels and a decrease in UBE1Y protein levels, but the differences were not statistically significant (Figure 7d).

3.7. Effect of Neu5Ac on Molecular Chaperone (CCT) in B16 Melanoma Cells

CCT3 and CCT5 mRNA levels significantly increased upon treatment with 0.5, 1, and 2 mg/mL Neu5Ac (Figure 8). The CCT4 mRNA level significantly increased on treatment with 2 mg/mL Neu5Ac (Figure 8). Luminescence band images for CCT3 protein levels (normalized to β-actin protein levels) were analyzed using ImageJ. The CCT3 protein level significantly increased on treatment with 0.5, 1, or 2 mg/mL Neu5Ac (Figure 8).

4. Discussion

Previous research has demonstrated that edible bird’s nest peptide (EBNP) containing 6.64% sialic acid has melanin synthesis inhibitory ability, and the melanin synthesis inhibitory EC50 value of EBNP in B16 cells was reported to be 1.48 mg/mL [45]. Sialic acid level varies depending on the source, with edible bird’s nest (EBN) being one of the major sources of sialic acid, primarily comprising Neu5Ac [46]. It has also been reported that digested sialic acid has a significant inhibitory effect on tyrosinase activity in B16 cell experiments, with an EC50 tyrosinase activity value of 0.4 mg/mL [47]. These concentrations are comparable to those used in our present study, where Neu5Ac was examined at concentrations of 0.5 mg/mL, 1 mg/mL, and 2 mg/mL. Importantly, these concentrations were found to be non-cytotoxic in our experiment. However, the mechanism of action of the inhibitory effect of sialic acid on melanogenesis has not been elucidated.
In the current study, Neu5Ac led to a reduction in the levels of tyrosinase protein without affecting the tyrosinase mRNA levels. This suggests that tyrosinase-containing coated vesicles were diverted to the degradation pathway instead of being transported to the melanosomes. Therefore, the localization of tyrosinase and LAMP-1, a marker of lysosomes, was examined using confocal laser scanning microscopy, and colocalization was confirmed. In addition, cathepsin L2 localization and concentration analysis showed that cathepsin L2 expression increased following Neu5Ac treatment. These results suggest that tyrosinase is degraded in lysosomes.
We subsequently investigated whether autophagosomes are involved in the pathway of tyrosinase degradation by lysosomes. We found that Neu5Ac increased the levels of LC3-I and LC3-II, indicating that Neu5Ac promotes autophagosome formation. Additionally, the increase in the ratio of LC3-II to LC3-I and the decrease in p62 levels suggested that Neu5Ac promotes the selective autophagic degradation of abnormal proteins by p62. Furthermore, we observed an increase in CCT3 expression with Neu5Ac treatment, indicating that Neu5Ac promotes autophagic flux.
Proteasomes selectively degrade proteins by recognizing their ubiquitin chains. The increase in USP5 levels observed following Neu5Ac treatment led to the removal of ubiquitin modifications from polyubiquitinated tyrosinases, suggesting that they are less likely to be degraded by proteasomes. Most aberrant proteins that have lost their function owing to protein conformational breakdown are polyubiquitinated and degraded by the ubiquitin–proteasome system [29], whereas aggregated proteins cannot be broken by the proteasome. The ubiquitin-activating enzyme UBE1Y mRNA and protein levels did not show significant changes. These results suggest that Neu5Ac does not affect protein polyubiquitination.
Based on the current results, we discuss the inhibitory effect of Neu5Ac on melanogenesis and its underlying mechanisms. Tyrosinase is redirected to the degradation pathway instead of progressing to stage III melanosomes. The findings indicate that Neu5Ac decreases p62 levels and increases cathepsin L2 levels, suggesting that deubiquitinated tyrosinase binds to p62 as a protein aggregate and is selectively degraded by autophagy. The receptor p62 transports ubiquitin-binding proteins and organelles to autophagosomes [40]. This mechanism underlies the selective degradation of ubiquitinated proteins by autophagy. Phosphorylation of residue 403 of p62 by casein kinase 2 enhances its affinity for the polyubiquitin chain, facilitating the efficient sequestration of polyubiquitinated proteins into sequestosomes [40]. Sequestosomes are thought to be subsequently incorporated into autophagosomes and degraded by lysosomes; therefore, the p62–polyubiquitin–protein complex would be degraded after autophagosome formation and fusion with lysosomes [48,49].
Neu5Ac may impact p62 phosphorylation, facilitating the incorporation of polyubiquitinated aberrant proteins into sequestosomes, the first step of autophagy, and degrading tyrosinase by activating autophagy. Increased CCT3 expression promotes autophagy, and the subsequent decrease in p62 levels suggests its involvement in assisting abnormal proteins in forming precise higher-order structures and complexes.

5. Conclusions

The current findings, along with previous research, suggest that Neu5Ac reduces tyrosinase activity, inhibits melanosome maturation, and suppresses melanin production by promoting its degradation through selective autophagy.

Author Contributions

Conceptualization, K.Y. and K.M.; methodology, K.Y. and K.M.; investigation, K.Y. and K.M.; data curation, K.Y.; writing—original draft preparation, K.Y.; writing—review and editing, K.M.; visualization, K.Y.; supervision, K.M.; funding acquisition, K.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Maeda, K. Large melanosome complex is increased in keratinocytes of solar lentigo. Cosmetics 2017, 4, 49. [Google Scholar] [CrossRef]
  2. Hirobe, T. Role of keratinocyte-derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes. Pigment Cell Res. 2005, 18, 2–12. [Google Scholar] [CrossRef] [PubMed]
  3. Maeda, K. Mechanism of action of topical tranexamic acid in the treatment of melasma and sun-induced skin hyperpigmentation. Cosmetics 2022, 9, 108. [Google Scholar] [CrossRef]
  4. Maeda, K. New method of measurement of epidermal turnover in humans. Cosmetics 2017, 4, 47. [Google Scholar] [CrossRef]
  5. Maeda, K. Timeline of the development of skin-lightening active ingredients in Japan. Molecules 2022, 27, 4774. [Google Scholar] [CrossRef] [PubMed]
  6. Gu, L.; Zeng, H.; Takahashi, T.; Maeda, K. In vitro methods for predicting chemical leukoderma caused by quasi-drug cosmetics. Cosmetics 2017, 4, 31. [Google Scholar] [CrossRef]
  7. Gu, L.; Maeda, K. Metabolism of enantiomers of rhododendrol in human skin homogenate. Metabolites 2022, 12, 412. [Google Scholar] [CrossRef]
  8. Wong, Z.C.F.; Chan, G.K.L.; Wu, K.Q.Y.; Poon, K.K.M.; Chen, Y.; Dong, T.T.X.; Tsim, K.W.K. Complete digestion of edible bird’s nest releases free N-acetylneuraminic acid and small peptides: An efficient method to improve functional properties. Food Funct. 2018, 9, 5139–5149. [Google Scholar] [CrossRef] [PubMed]
  9. Kathan, R.H.; Weeks, D.I. Structure studies of collocalia mucoid. I. Carbohydrate and amino acid composition. Arch. Biochem. Biophys. 1969, 134, 572–576. [Google Scholar] [CrossRef]
  10. Wang, B.; Brand-Miller, J. The role and potential of sialic acid in human nutrition. Eur. J. Clin. Nutr. 2003, 57, 1351–1369. [Google Scholar] [CrossRef]
  11. Valencia, J.C.; Rouzaud, F.; Julien, S.; Chen, K.G.; Passeron, T.; Yamaguchi, Y.; Abu-Asab, M.; Tsokos, M.; Costin, G.E.; Yamaguchi, H.; et al. Sialylated core 1 O-glycans influence the sorting of Pmel17/gp100 and determine its capacity to form fibrils. J. Biol. Chem. 2007, 282, 11266–11280. [Google Scholar] [CrossRef] [PubMed]
  12. Valencia, J.C.; Hearing, V.J. The Role of Glycosylation in the Control of Processing and Cellular Transport of the Functional Amyloid PMEL17. In Glycosylation; Stefana Petrescu, S., Ed.; IntechOpen: London, UK, 2012. [Google Scholar]
  13. Mishima, Y. Molecular and biological control of melanogenesis through tyrosinase genes and intrinsic and extrinsic regulatory factors. Pigment Cell Res. 1994, 7, 376–387. [Google Scholar] [CrossRef] [PubMed]
  14. Huang, C.; Seino, J.; Wang, L.; Haga, Y.; Suzuki, T. Autophagy regulates the stability of sialin, a lysosomal sialic acid transporter. Biosci. Biotechnol. Biochem. 2015, 79, 553–557. [Google Scholar] [CrossRef]
  15. Diwakar, G.; Klump, V.; Lazova, R.; Pawelek, J. Evidence for glycosylation as a regulator of the pigmentary system: Key roles of sialyl(α2-6)gal/GalNAc-terminated glycans in melanin synthesis and transfer. Glycoconj J. 2015, 32, 413–420. [Google Scholar] [CrossRef] [PubMed]
  16. Ogura, H.; Mishima, Y. [GERL-coated vesicle system in the melanin synthesizing cell: Its three-dimensional ultrastructure and tyrosinase activity (author’s transl)]. Nihon Hifuka Gakkai Zasshi. 1977, 87, 883–901. [Google Scholar] [PubMed]
  17. Imokawa, G.; Mishima, Y. Isolation and biochemical characterization of tyrosinase-rich GERL and coated vesicle in melanin synthesizing cells. Br. J. Dermatol. 1981, 104, 169–178. [Google Scholar] [CrossRef] [PubMed]
  18. Wasmeier, C.; Hume, A.N.; Bolasco, G.; Seabra, M.C. Melanosomes at a glance. J. Cell Sci. 2008, 121, 3995–3999. [Google Scholar] [CrossRef] [PubMed]
  19. Kabuta, T.; Fujiwara, Y.; Wada, K. Do lysosomes take up various macromolecules independently of membrane traffic? Seikagaku 2013, 85, 1057–1066. (In Japanese) [Google Scholar]
  20. Ebanks, J.P.; Koshoffer, A.; Wickett, R.R.; Schwemberger, S.; Babcock, G.; Hakozaki, T.; Boissy, R.E. Epidermal keratinocytes from light vs. dark skin exhibit differential degradation of melanosomes. J. Investig. Dermatol. 2011, 131, 1226–1233. [Google Scholar] [CrossRef]
  21. Chen, N.; Seiberg, M.; Lin, C.B. Cathepsin L2 levels inversely correlate with skin color. J. Investig. Dermatol. 2006, 126, 2345–2347. [Google Scholar] [CrossRef]
  22. Zeng, H.; Koizumi, S.; Maeda, K. Involvement of cathepsin K and cathepsin L2 in the digestion of melanosome complexes in epidermal keratinocytes by fennel extract. Dermatol. Cosmet. JOJ 2021, 4, 1–12. [Google Scholar]
  23. Komatsu, M.; Ichimura, Y. [Involvement of ubiquitin system in mammalian autophagy]. Seikagaku 2012, 84, 472–478. [Google Scholar] [PubMed]
  24. Korolchuk, V.I.; Mansilla, A.; Menzies, F.M.; Rubinsztein, D.C. Autophagy inhibition compromises degradation of ubiquitin-proteasome pathway substrates. Mol. Cell 2009, 33, 517–527. [Google Scholar] [CrossRef] [PubMed]
  25. Hara, T.; Nakamura, K.; Matsui, M.; Yamamoto, A.; Nakahara, Y.; Suzuki-Migishima, R.; Yokoyama, M.; Mishima, K.; Saito, I.; Okano, H.; et al. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature 2006, 441, 885–889. [Google Scholar] [CrossRef]
  26. Johansen, T.; Lamark, T. Selective autophagy mediated by autophagic adapter proteins. Autophagy 2011, 7, 279–296. [Google Scholar] [CrossRef] [PubMed]
  27. Kirkin, V.; McEwan, D.G.; Novak, I.; Dikic, I. A role for ubiquitin in selective autophagy. Mol. Cell 2009, 34, 259–269. [Google Scholar] [CrossRef]
  28. Rubinsztein, D.C. Autophagy induction rescues toxicity mediated by proteasome inhibition. Neuron 2007, 4, 854–856. [Google Scholar] [CrossRef] [PubMed]
  29. Kocaturk, N.M.; Gozuacik, D. Crosstalk Between Mammalian Autophagy and the Ubiquitin-Proteasome System. Front Cell Dev. Biol. 2018, 6, 128. [Google Scholar] [CrossRef]
  30. Weidberg, H.; Shvets, E.; Elazar, Z. Biogenesis and cargo selectivity of autophagosomes. Annu. Rev. Biochem. 2011, 80, 125–156. [Google Scholar] [CrossRef]
  31. Komatsu, M.; Waguri, S.; Koike, M.; Sou, Y.S.; Ueno, T.; Hara, T.; Mizushima, N.; Iwata, J.; Ezaki, J.; Murata, S.; et al. Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell 2007, 131, 1149–1163. [Google Scholar] [CrossRef]
  32. Nezis, I.P.; Simonsen, A.; Sagona, A.P.; Finley, K.; Gaumer, S.; Contamine, D.; Rusten, T.E.; Stenmark, H.; Brech, A. Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain. J. Cell Biol. 2008, 180, 1065–1071. [Google Scholar] [CrossRef] [PubMed]
  33. Avvakumov, G.V.; Walker, J.R.; Xue, S.; Allali-Hassani, A.; Asinas, A.; Nair, U.B.; Fang, X.; Zuo, X.; Wang, Y.X.; Wilkinson, K.D.; et al. Two ZnF-UBP domains in isopeptidase T (USP5). Biochemistry 2012, 51, 1188–1198. [Google Scholar] [CrossRef]
  34. Lee, K.W.; Ryu, H.W.; Oh, S.S.; Park, S.; Madhi, H.; Yoo, J.; Park, K.H.; Kim, K.D. Depigmentation of α-melanocyte-stimulating hormone-treated melanoma cells by β-mangostin is mediated by selective autophagy. Exp. Dermatol. 2017, 26, 585–591. [Google Scholar] [CrossRef] [PubMed]
  35. Pankiv, S.; Clausen, T.H.; Lamark, T.; Brech, A.; Bruun, J.A.; Outzen, H.; Øvervatn, A.; Bjørkøy, G.; Johansen, T. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J. Biol. Chem. 2007, 282, 24131–24145. [Google Scholar] [CrossRef] [PubMed]
  36. Ichimura, Y.; Kumanomidou, T.; Sou, Y.S.; Mizushima, T.; Ezaki, J.; Ueno, T.; Kominami, E.; Yamane, T.; Tanaka, K.; Komatsu, M. Structural basis for sorting mechanism of p62 in selective autophagy. J. Biol. Chem. 2008, 283, 22847–22857. [Google Scholar] [CrossRef]
  37. Mizushima, N.; Yoshimori, T. How to interpret LC3 immunoblotting. Autophagy 2007, 3, 542–545. [Google Scholar] [CrossRef] [PubMed]
  38. Ichimura, Y.; Komatsu, M. Selective degradation of p62 by autophagy. Semin. Immunopathol. 2010, 32, 431–436. [Google Scholar] [CrossRef] [PubMed]
  39. Thirumalai, D.; Lorimer, G.H. Chaperonin-mediated protein folding. Annu. Rev. Biophys. Biomol. Struct. 2001, 30, 245–269. [Google Scholar] [CrossRef] [PubMed]
  40. Matsumoto, G.; Wada, K.; Okuno, M.; Kurosawa, M.; Nukina, N. Serine 403 phosphorylation of p62/SQSTM1 regulates selective autophagic clearance of ubiquitinated proteins. Mol. Cell 2011, 44, 279–289. [Google Scholar] [CrossRef]
  41. Liu, W.; Lu, Y.; Yan, X.; Lu, Q.; Sun, Y.; Wan, X.; Li, Y.; Zhao, J.; Li, Y.; Jiang, G. Current understanding on the role of CCT3 in cancer research. Front Oncol. 2022, 12, 961733. [Google Scholar] [CrossRef]
  42. Wang, K.; He, J.; Tu, C.; Xu, H.; Zhang, X.; Lv, Y.; Song, C. Upregulation of CCT3 predicts poor prognosis and promotes cell proliferation via inhibition of ferroptosis and activation of AKT signaling in lung adenocarcinoma. BMC Mol. Cell. Biol. 2022, 23, 25. [Google Scholar] [CrossRef] [PubMed]
  43. Eldridge, A.G.; O’Brien, T. Therapeutic strategies within the ubiquitin proteasome system. Cell Death Differ. 2010, 17, 4–13. [Google Scholar] [CrossRef] [PubMed]
  44. Sternlicht, H.; Farr, G.W.; Sternlicht, M.L.; Driscoll, J.K.; Willison, K.; Yaffe, M.B. The t-complex polypeptide 1 complex is a chaperonin for tubulin and actin in vivo. Proc. Natl. Acad. Sci. USA 1993, 90, 9422–9426. [Google Scholar] [CrossRef] [PubMed]
  45. Bai, W.; Liu, X.; Fan, Q.; Lian, J.; Guo, B. Study of the antiaging effects of bird’s nest peptide based on biochemical, cellular, and animal models. J. Funct. Foods 2023, 103, 105479. [Google Scholar] [CrossRef]
  46. Ling, A.J.W.; Chang, L.S.; Babji, A.S.; Latip, J.; Koketsu, M.; Lim, S.J. Review of sialic acid’s biochemistry, sources, extraction and functions with special reference to edible bird’s nest. Food Chem. 2022, 367, 130755. [Google Scholar] [CrossRef] [PubMed]
  47. Fan, Q.; Lian, J.; Liu, X.; Zou, F.; Wang, X.; Chen, M. A study on the skin whitening activity of digesta from edible bird’s nest: A mucin glycoprotein. Gels 2022, 8, 24. [Google Scholar] [CrossRef]
  48. Majeski, A.E.; Dice, J.F. Mechanisms of chaperone-mediated autophagy. Int. J. Biochem. Cell Biol. 2004, 36, 2435–2444. [Google Scholar] [CrossRef]
  49. Cuervo, A.M. Chaperone-mediated autophagy: Dice’s ‘wild’ idea about lysosomal selectivity. Nat. Rev. Mol. Cell Biol. 2011, 12, 535–541. [Google Scholar] [CrossRef]
Cosmetics 11 00082 g001
Figure 1. Chemical structure of N-acetylneuraminic acid (Neu5Ac).
Figure 1. Chemical structure of N-acetylneuraminic acid (Neu5Ac).
Cosmetics 11 00082 g001
Cosmetics 11 00082 g002aCosmetics 11 00082 g002b
Figure 2. Effect of Neu5Ac on cell proliferation and tyrosinase and melanin levels in B16 melanoma cells. (a) Effect of Neu5Ac on cell number and tyrosinase activity. (b) Effect of Neu5Ac on tyrosinase mRNA and protein levels. Mean ± SD, n = 4 or n = 3, *: p < 0.05, **: p < 0.01. (c) Effect of Neu5Ac on melanin levels.
Figure 2. Effect of Neu5Ac on cell proliferation and tyrosinase and melanin levels in B16 melanoma cells. (a) Effect of Neu5Ac on cell number and tyrosinase activity. (b) Effect of Neu5Ac on tyrosinase mRNA and protein levels. Mean ± SD, n = 4 or n = 3, *: p < 0.05, **: p < 0.01. (c) Effect of Neu5Ac on melanin levels.
Cosmetics 11 00082 g002aCosmetics 11 00082 g002b
Cosmetics 11 00082 g003
Figure 3. Effect of Neu5Ac on tyrosinase and LAMP-1 localization, using fluorescence immunostaining (multiple staining). Nuclei were stained with DAPI (blue), tyrosinase was labeled with an anti-tyrosinase antibody, and Alexa Fluor 488 secondary antibody (green), and LAMP-1 was labeled with an anti-LAMP1 antibody and Alexa Fluor 568 secondary antibody (red). Tyrosinase and LAMP-1 colocalization areas are indicated in yellow. The scale bar is 100 μm.
Figure 3. Effect of Neu5Ac on tyrosinase and LAMP-1 localization, using fluorescence immunostaining (multiple staining). Nuclei were stained with DAPI (blue), tyrosinase was labeled with an anti-tyrosinase antibody, and Alexa Fluor 488 secondary antibody (green), and LAMP-1 was labeled with an anti-LAMP1 antibody and Alexa Fluor 568 secondary antibody (red). Tyrosinase and LAMP-1 colocalization areas are indicated in yellow. The scale bar is 100 μm.
Cosmetics 11 00082 g003
Cosmetics 11 00082 g004
Figure 4. Effect of Neu5Ac on cathepsin L2 levels, using fluorescence immunostaining (multiple staining method). The nuclei were stained with DAPI (blue). Cathepsin L2 was labeled with an anti-cathepsin L2 antibody and Alexa Fluor 488 secondary antibody (green). The scale bar is 100 μm.
Figure 4. Effect of Neu5Ac on cathepsin L2 levels, using fluorescence immunostaining (multiple staining method). The nuclei were stained with DAPI (blue). Cathepsin L2 was labeled with an anti-cathepsin L2 antibody and Alexa Fluor 488 secondary antibody (green). The scale bar is 100 μm.
Cosmetics 11 00082 g004
Cosmetics 11 00082 g005aCosmetics 11 00082 g005b
Figure 5. (a) Effect of Neu5Ac on LC3. (b) LC3-II/β-actin ratio was calculated by measuring the brightness of LC3-II and β-actin bands with ImageJ. mean ± SD, n = 3. (c) Effect of Neu5Ac on p62. (d) The brightness of p62 and β-actin bands was measured with ImageJ. The p62/β-actin ratio was calculated and plotted. mean ± SD, n = 3, *: p < 0.05 vs. control, **: p < 0.01 vs. control.
Figure 5. (a) Effect of Neu5Ac on LC3. (b) LC3-II/β-actin ratio was calculated by measuring the brightness of LC3-II and β-actin bands with ImageJ. mean ± SD, n = 3. (c) Effect of Neu5Ac on p62. (d) The brightness of p62 and β-actin bands was measured with ImageJ. The p62/β-actin ratio was calculated and plotted. mean ± SD, n = 3, *: p < 0.05 vs. control, **: p < 0.01 vs. control.
Cosmetics 11 00082 g005aCosmetics 11 00082 g005b
Cosmetics 11 00082 g006aCosmetics 11 00082 g006b
Figure 6. Effect of Neu5Ac on p62 localization, using fluorescence immunostaining. The nuclei were stained with DAPI (blue). P62 was labeled with an anti-p62/SQSTM1antibody and Alexa Fluor 568 secondary antibody (red). The scale bar was 50 μm.
Figure 6. Effect of Neu5Ac on p62 localization, using fluorescence immunostaining. The nuclei were stained with DAPI (blue). P62 was labeled with an anti-p62/SQSTM1antibody and Alexa Fluor 568 secondary antibody (red). The scale bar was 50 μm.
Cosmetics 11 00082 g006aCosmetics 11 00082 g006b
Cosmetics 11 00082 g007aCosmetics 11 00082 g007b
Figure 7. Effect of Neu5Ac on mRNA (a) and protein levels (b–d) of the ubiquitin-proteasome system-related factors USP5 and UBE1Y. Mean ± SD, n = 3, *: p < 0.05 vs. control, **: p < 0.01 vs. control.
Figure 7. Effect of Neu5Ac on mRNA (a) and protein levels (b–d) of the ubiquitin-proteasome system-related factors USP5 and UBE1Y. Mean ± SD, n = 3, *: p < 0.05 vs. control, **: p < 0.01 vs. control.
Cosmetics 11 00082 g007aCosmetics 11 00082 g007b
Cosmetics 11 00082 g008aCosmetics 11 00082 g008b
Figure 8. Effect of Neu5Ac on mRNA levels (a) of molecular chaperone CCT and protein levels of CCT3 (b,c). Mean ± SD, n = 3, *: p < 0.05 vs. control, **: p < 0.01 vs. control.
Figure 8. Effect of Neu5Ac on mRNA levels (a) of molecular chaperone CCT and protein levels of CCT3 (b,c). Mean ± SD, n = 3, *: p < 0.05 vs. control, **: p < 0.01 vs. control.
Cosmetics 11 00082 g008aCosmetics 11 00082 g008b
Table 1. Primary and secondary antibodies to melanogenic factors for Western blotting analysis.
Table 1. Primary and secondary antibodies to melanogenic factors for Western blotting analysis.
Primary AntibodiesSecondary Antibodies
Anti-β-actin antibody (AG0297; Proteintech Group, Inc., Rosemont, IL, USA), 1000× dilutionAnti-mouse antibody (NA931VS; GE HealthCare Technologies Inc.), 5000× dilution
Anti-tyrosinase antibody (T311; Thermo Fisher Scientific Co.), 1000× dilutionAnti-mouse antibody (NA931VS; GE Healthcare Technologies Inc.), 5000× dilution
Table 2. Primary and secondary antibodies to ubiquitin-proteasome-related factors for Western blotting analysis.
Table 2. Primary and secondary antibodies to ubiquitin-proteasome-related factors for Western blotting analysis.
Primary AntibodiesSecondary Antibodies
Anti–beta-actin antibody (AG0297; Proteintech Group, Inc.), 1000× dilutionAnti-mouse antibody (NA931VS; GE Healthcare Technologies Inc.), 5000× dilution
Anti-USP5/IsoT antibody (A301-542A1; Bethyl Laboratories, Inc., Montgomery, TX, USA), 1000× dilutionAnti-rabbit antibody (NA934VS; GE Healthcare Technologies Inc.), 5000× dilution
Anti-UBE1a/b rabbit antibody (4891; Cell Signaling Technology, Inc., Danvers, MA, USA), 1000× dilutionAnti-rabbit antibody (NA934VS.; GE Healthcare Technologies Inc.), 5000× dilution
Table 3. Primary and secondary antibodies to autophagy-related factors for Western blotting analysis.
Table 3. Primary and secondary antibodies to autophagy-related factors for Western blotting analysis.
Primary AntibodiesSecondary Antibodies
Anti-beta-actin antibody (AG0297; Proteintech Group, Inc.), 1000× dilutionAnti-mouse antibody (NA931VS; GE Healthcare Technologies Inc.), 5000× dilution
Anti-LC3A/B (D3U4C; #12741; Cell Signaling Technology Inc.), 1000× dilutionAnti-rabbit antibody (NA934VS; GE Healthcare Technologies Inc.), 5000× dilution
Anti-P62/SQSTM1 antibody (Proteintech Group, Inc.), 1000× dilutionAnti-rabbit antibody (NA934VS; GE Healthcare Technologies Inc.), 5000× dilution
Anti-CCT3 antibody (10571-1-AP; Proteintech Group, Inc.), 1000× dilutionAnti-rabbit antibody (NA934VS; GE Healthcare Technologies Inc.), 5000× dilution
Anti-LAMP-1 antibody (bs-1970R; Funakoshi), 500× dilutionAnti-rabbit antibody (NA934VS; GE Healthcare Technologies Inc.), 5000× dilution
Table 4. Tyrosinase and LAMP-1 primary and secondary antibodies.
Table 4. Tyrosinase and LAMP-1 primary and secondary antibodies.
Primary AntibodiesSecondary Antibodies
Anti-LAMP-1 polyclonal antibody (bs-1970R; Thermo Fisher Scientific Co.), 200× dilutionAlexa Fluor 568 goat anti-rabbit antibody (A11011; Thermo Fisher Scientific Co.), 1000× dilution
Anti-tyrosinase antibody (T311; Thermo Fisher Scientific Co.), 1000× dilutionAlexa Fluor 488 goat anti-mouse antibody (A11006; Thermo Fisher Scientific Co.), 1000× dilution
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Yoshikawa, K.; Maeda, K. N-Acetylneuraminic Acid Inhibits Melanogenesis via Induction of Autophagy. Cosmetics 2024, 11, 82. https://doi.org/10.3390/cosmetics11030082

AMA Style

Yoshikawa K, Maeda K. N-Acetylneuraminic Acid Inhibits Melanogenesis via Induction of Autophagy. Cosmetics. 2024; 11(3):82. https://doi.org/10.3390/cosmetics11030082

Chicago/Turabian Style

Yoshikawa, Kei, and Kazuhisa Maeda. 2024. "N-Acetylneuraminic Acid Inhibits Melanogenesis via Induction of Autophagy" Cosmetics 11, no. 3: 82. https://doi.org/10.3390/cosmetics11030082

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop