Role of C-mannosylation in the secretion of mindin.

BACKGROUND: Mindin (spondin2), a secretory protein related to neural development and immunity, is a member of thrombospondin type I repeat (TSR) superfamily proteins, and has a unique glycosylation of C-mannosylation in its structure. However, it remains unclear whether C-mannosylation plays a functional role in the biosynthesis of mindin in cells. METHODS: Protein C-mannosylation was analyzed by mass spectrometry. Mindin expression was examined by immunoblot and immunofluorescence analyses in COS-7 cells transfected with the expression vectors for wild type (mindin-WT) or C-mannosylation-defective mutant of mindin (mindin-mutF). The redox status was examined in mindin by using 4-acetoamide-4'-maleimidylstilbene-2,2'-disulfonate. RESULTS: When mindin cDNA was expressed in COS-7 cells, C-mannosylation of mindin was confirmed at Trp257 by mass spectrometry. In cells expressing a mindin-mutF, secretion of the mutant was significantly inhibited compared with mindin-WT. In immunofluorescence analysis, mindin-mutF was accumulated in the endoplasmic reticulum (ER), whereas mindin-WT was detected in the Golgi. In addition, mindin-mutF showed an enhanced interaction with calreticulin, an ER-resident chaperone, in cells. In cells, reduced forms were increased in mindin-mutF, compared with a mostly oxidized form of mindin-WT. In the presence of chemical chaperones such as dimethylsulfoxide or 4-phenylbutyrate, inhibited secretion of mindin-mutF was ameliorated in cells, although redox-dependent folding was not affected. CONCLUSIONS: C-Mannosylation of mindin facilitates its secretion especially through modulating disulfide bond formation in mindin in cells. GENERAL SIGNIFICANCE: These results suggest that C-mannosylation plays a functional role in the redox-dependent folding and transport of TSR superfamily proteins in cells.

C-Mannosylated peptides derived from the thrombospondin type 1 repeat interact with Hsc70 to modulate its signaling in RAW264.7 cells.

The thrombospondin type 1 repeat (TSR) is a functional module of proteins called TSR superfamily proteins (e.g., thrombospondin, F-spondin, mindin, etc.) and includes a conserved Trp-x-x-Trp (W-x-x-W) motif, in which the first Trp residue is preferably modified by C-mannosylation. We previously reported that synthesized C-mannosylated TSR-derived peptides (e.g., C-Man-WSPW) specifically enhanced lipopolysaccharide-induced signaling in macrophage-like RAW264.7 cells. In this study, we searched for the proteins that bind to C-mannosylated TSR-derived peptides in RAW264.7 cells and identified heat shock cognate protein 70 (Hsc70). The binding affinity of Hsc70 for C-mannosylated peptides in solution was higher than that for the peptides without C-mannose. The binding was influenced by a nucleotide-induced conformational change of Hsc70, and C-mannosylated peptides preferred the substrate-binding domain of Hsc70. Furthermore, in RAW264.7 cells, addition of Hsc70 stimulated cellular signaling to produce tumor necrosis factor-alpha, via transforming growth factor-beta-activated kinase 1, and the Hsc70-induced signaling was enhanced more in the presence of the peptides with C-mannose than that without C-mannose, suggesting functional interaction between Hsc70 and the C-mannosylated peptides in the cells. Together, these results demonstrate a novel function of the C-mannosylation of TSR-derived peptides in terms of interaction with Hsc70 to regulate cellular signaling.