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.
Ihara
Yoshito
Y
Department of Biochemistry, Wakayama Medical University, Wakayama, Japan. y-ihara@wakayama-med.ac.jp
Manabe
Shino
S
Ikezaki
Midori
M
Inai
Yoko
Y
Matsui
In-Sook Lee
IS
Ohta
Yuriko
Y
Muroi
Eiji
E
Ito
Yukishige
Y
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D005454:Fluorescence Polarization; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism*; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D010446:Peptide Fragments / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D019700:Thrombospondin 1 / Q000378:metabolism*; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000099151:MAP Kinase Kinase Kinase 7
D050883:HSC70 Heat-Shock Proteins; D020930:MAP Kinase Kinase Kinases; D008358:Mannose; D010446:Peptide Fragments; D012333:RNA, Messenger; D019700:Thrombospondin 1; D014364:Tryptophan; D014409:Tumor Necrosis Factor-alpha; D000099151:MAP Kinase Kinase Kinase 7; C101500:C(2)-mannosyltryptophan