N -Acetylneuraminic Acid Inhibits Melanogenesis via Induction of Autophagy

: 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.


Introduction
Research in skin lightening has been primarily directed toward developing 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 melanogenesisinhibitory 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].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 ubiquitinproteasome 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 developing 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.

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 (Dojin 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.

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 housekeeping genes.Experiments were performed with n = 3.Data were analyzed using the relative quantification method (∆∆Ct method).

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.
Table 1.Primary and secondary antibodies to melanogenic factors for Western blotting analysis.

Primary Antibodies Secondary Antibodies
Anti-β-actin antibody (AG0297; Proteintech Group, Inc., Rosemont, IL, USA), 1000× dilution Anti-mouse antibody (NA931VS; GE HealthCare Technologies Inc.), 5000× dilution Anti-tyrosinase antibody (T311; Thermo Fisher Scientific Co.), 1000× dilution Anti-mouse antibody (NA931VS; GE Healthcare Technologies Inc.), 5000× dilution 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 housekeeping genes.Each reaction was performed in triplicate.Data were analyzed using the relative quantification method (∆∆Ct 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) 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.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 antirabbit 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).

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.

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.

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 housekeeping gene) using the relative quantification method (∆∆Ct 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.

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. Table 4. Tyrosinase and LAMP-1 primary and secondary antibodies.

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.

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 housekeeping 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.

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.

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.

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).

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).and the p62 protein level significantly decreased at Neu5Ac concentrations of 1 mg/mL and 2 mg/mL.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 anticathepsin L2 antibody and Alexa Fluor 488 secondary antibody (green).The scale bar is 100 µm.

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.

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.

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.

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.

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).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).
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).

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

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 EC 50 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 EC 50 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 ubiquitinproteasome 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.

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.

Figure 2 .
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 3 .
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 antityrosinase 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 .
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 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.

3. 6 .
Effect of Neu5Ac on mRNA and Protein Levels of Ubiquitin-Proteasome System-Related Factors (USP5 and UBE1y)

Table 2 .
Primary and secondary antibodies to ubiquitin-proteasome-related factors for Western blotting analysis.

Table 3 .
Primary and secondary antibodies to autophagy-related factors for Western blotting analysis.

Table 4 .
Tyrosinase and LAMP-1 primary and secondary antibodies.