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C‐mannosylation is a rare type of protein glycosylation whereby a single mannose is added to the first tryptophan in the consensus sequence Trp‐Xaa‐Xaa‐Trp/Cys (in which Xaa represents any amino acid). Its consensus sequence is mainly found in proteins containing a thrombospondin type‐1 repeat (TSR1) domain and in type I cytokine receptors. In these proteins, C‐mannosylation affects protein secretion, intracellular localization, and protein stability; however, the role of C‐mannosylation in proteins that are not type I cytokine receptors and/or do not contain a TSR1 domain is less well explored. In this study, we focused on human vitelline membrane outer layer protein 1 homolog (VMO1). VMO1, which possesses two putative C‐mannosylation sites, is a 21‐kDa secreted protein that does not contain a TSR1 domain and is not a type I cytokine receptor. Mass spectrometry analyses revealed that VMO1 is C‐mannosylated at Trp105 but not at Trp44. Although C‐mannosylation does not affect the extracellular secretion of VMO1, it destabilizes the intracellular VMO1. In addition, a structural comparison between VMO1 and C‐mannosylated VMO1 showed that the modification of the mannose changes the conformation of three loops in VMO1. Taken together, our results demonstrate the first example of C‐mannosylation for protein destabilization of VMO1. [ABSTRACT FROM AUTHOR]

Placental extracts have been widely used as skin lightening agents in the Japanese cosmetic market. Here, we show that placental extracts contain factors that can decrease or increase melanin synthesis by normal human melanocytes in vitro in possible association with mitochondrial respiration. When normal human melanocytes were treated with a whole porcine placental extract, melanin synthesis was decreased. In contrast, a porcine placental extract in which exudates and insoluble materials including lipids had been removed increased melanin synthesis. In addition, the amount of tyrosinase, the enzyme critical for melanin synthesis, changed in accordance with the alteration of melanin synthesis. Interestingly, the amount of manganese‐dependent superoxide dismutase (MnSOD), a mitochondrial‐resident antioxidant enzyme, was increased when melanin synthesis was decreased by the whole placental extract. Mitochondrial respiration and glycolysis also changed following treatment with the placental extracts. These results suggest that placental extracts contain factors that can increase or decrease melanin synthesis by normal human melanocytes and that mitochondrial function may be associated with the placental extract‐induced regulation of melanogenesis. [ABSTRACT FROM AUTHOR]

Abstract: To understand a role of UVA radiation in photoaging of the skin, we established a model of photoaging cells using cultured human dermal fibroblasts. Repeated low‐dose UVA radiation for 10 consecutive days induced senescence in fibroblasts, characterized with (1) increased level of senescence‐associated β‐galactosidase, (2) flattened large cell shape, (3) accumulation of reactive oxygen species, (4) yellowish coloration and (5) expression of p16. These were also observed in chronologically aged fibroblasts (doubling times >20), whereas none of these were detected in young cells (doubling times <10). Collectively, we propose that fibroblasts exposed to repetitive UVA radiation may be a good model of aged cells to study the mechanism of aging and photoaging and further to search for novel agents preventing cellular senescence. In addition, H2O2 was produced in the culture medium by a single low dose of UVA irradiation. Further, PAPLAL, a nanoparticle of platinum and palladium having potent catalase‐like activity, significantly delayed the onset of H2O2‐induced cell senescence. The present study strongly indicates that repetitive short‐term UVA irradiation induces aging of cells possibly via H2O2 and may be suppressed by potent anti‐H2O2 agents. [ABSTRACT FROM AUTHOR]

C -mannosylation is a type of protein glycosylation. Human Isthmin-1 (ISM1) is a 52-kDa secreted protein with a thrombospondin type 1 repeat (TSR) domain, containing two consensus C -mannosylation sequences at Trp223 and Trp226. In this study, we sought to examine the role of C -mannosylation in the secretion of ISM1. We established and cultured an ISM1-overexpressing HT1080 cell line and purified recombinant ISM1 for analysis from the conditioned medium by LC-MS/MS. Subcellular localization of ISM1 was observed by confocal fluorescence microscopy. We found that ISM1 is C -mannosylated at Trp223 and Trp226 in the TSR domain. To determine the functions of the C -mannosylation of ISM1, we established a C -mannosylation-defective mutant ISM1-overexpressing HT1080 cell line and measured its secretion of ISM1. The secretion of ISM1 decreased significantly in this mutant ISM1-overexpressing line compared with wild-type cells. Furthermore, ISM1 was N -glycosylated only in these C -mannosylation-defective cells. ISM1 is C -mannosylated in its TSR domain, and the status of the C -mannosylation of ISM1 affects its N -glycosylation. The C -mannosylation of ISM1 regulates its N -glycosylation status. • Isthmin-1 (ISM1) is C -mannosylated at Trp223 and Trp226. • C -mannosylation of ISM1 regulates its secretion and subcellular localization. • N -glycosylation of ISM1 is regulated by its C -mannosylation. [ABSTRACT FROM AUTHOR]

Pigmentation in the dermis is known to be caused by melanophages, defined as melanosome-laden macrophages. In this study, we show that dermal fibroblasts also have an ability to uptake melanosomes and apoptotic melanocytes. We have previously demonstrated that normal human melanocytes constantly secrete melanosome clusters from various sites of their dendrites. After adding secreted melanosome clusters collected from the culture medium of melanocytes, time-lapse imaging showed that fibroblasts actively attached to the secreted melanosome clusters and incorporated them. Annexin V staining revealed that phosphatidylserine (PtdSer), which is known as an 'eat-me' signal that triggers the internalization of apoptotic cells by macrophages, is exposed on the surface of secreted melanosome clusters. Dermal fibroblasts were able to uptake secreted melanosome clusters as did macrophages, and those fibroblasts express TIM4, a receptor for PtdSer-mediated endocytosis. Further, co-cultures of fibroblasts and melanocytes demonstrated that dermal fibroblasts internalize PtdSer-exposed apoptotic melanocytes. These results suggest that not only macrophages, but also dermal fibroblasts contribute to the collection of potentially toxic substances in the dermis, such as secreted melanosome clusters and apoptotic melanocytes, that have been occasionally observed to drop down into the dermis from the epidermis. [ABSTRACT FROM AUTHOR]