Synthesis and biological activity of peptides equivalent to the IP22 repeat motif found in proteins from Dictyostelium and Mimivirus.
A novel IP22 repeat motif of unknown function was discovered previously that comprises almost the entire structure of cmbB, a calmodulin-binding protein from Dictyostelium discoideum. An analysis of over 2000 IP22 repeats across 130 different proteins from different species allowed us to define a prototypical IP22 repeat: I/LPxxhxxhxhxxxhxxxhxxxx (where L=leucine, I=isoleucine, h=any hydrophobic amino acid, x=any amino acid). Here we describe the synthesis of three peptide variants of the IP22 motif: IP22-1 (IPNSVTSLKFGDGFNQPLTPGT; 22aa); IP22-2 (LPSTLKTISLSNSTDKKIFKNS; 22aa); and, IP22-3 (IPKSLRSLFLGKGYNQPLEF; 20aa) plus a control peptide from the N-term of cmbB (HNMNPFSPQLDEKKNSHIVEY; 21aa). The structure and purity of synthesized peptides were verified by HPLC and mass spectrometry. The peptides all dose-dependently enhanced random cell motility and cAMP-mediated chemotaxis in Dictyostelium but IP22-3 was most effective peaking in activity around 50 μM. Fluorescein isothiocyanate (FITC)-conjugated IP22 peptides did not penetrate cells suggesting these peptides affect cell motility via cell surface interactions. Treatment of cells with FITC-IP22 peptides also led to enhanced cell motility equivalent to the non-conjugated peptides. Treatment of IP22-3-stimulated cells with 50 μM LY294002, 20 μM quinacrine or both suggests that IP22-3 requires both phosphoinositol 3-kinase and phospholipase A2 signaling to elicit its effects, a mechanism unique from EGFL motility enhancing peptides. The mechanism of action and potential uses of IP22 repeat peptides are discussed.
Catalano
Andrew
A
Department of Cell and Systems Biology, University of Toronto, 25 Harbord st., Toronto, Ontario, Canada, M5S 3G5.
Luo
Wei
W
Wang
Yali
Y
O'Day
Danton H
DH
D000595:Amino Acid Sequence; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002465:Cell Movement; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism*; D056686:Mimiviridae / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D064801:Phospholipase A2 Inhibitors; D054467:Phospholipases A2 / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D002148:Calmodulin-Binding Proteins; D010455:Peptides; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D064801:Phospholipase A2 Inhibitors; D054467:Phospholipases A2; D015800:Protozoan Proteins; D014764:Viral Proteins