Molecular cloning and characterization of a 42-kDa protein phosphatase of Leishmania chagasi.
Using rabbit serum raised against a potent T cell-stimulating antigen fraction of Leishmania chagasi promastigotes, we have cloned and expressed the Leishmania type 2C serine/threonine protein phosphatase, LcPP2C. LcPP2C was shown to be present as a 42-kDa protein in both the infective promastigote and tissue amastigote stages of L. chagasi and Leishmania amazonensis. DNA hybridization studies established the close conservation of LcPP2C among eight of eight geographically diverse species of Leishmania which cause a spectrum of human diseases. To support the relationship between LcPP2C and mammalian type 2C protein phosphatases observed through predicted amino acid sequence comparisons, we expressed enzymatically active rLcPP2C in Escherichia coli. We demonstrated that purified rLcPP2C readily dephosphorylated [32P]casein, an activity dependent on Mg2+ and insensitive to okadaic acid. In agreement with studies of rat liver PP2C, activity was maintained when Mg+2 was replaced with Mn+2 but not with Ca2+. As these parameters are characteristic of the eukaryotic type 2C serine/threonine protein phosphatases, LcPP2C can be classified as a member of this protein family. We further showed that of the four major classes of eukaryotic serine/threonine protein phosphatases, PP2C-and PP1-like activities, are readily detectable in Leishmania.
Seattle Biomedical Research Institute, Washington 98109.
J M
JM
Burns
M
M
Parsons
D E
DE
Rosman
S G
SG
Reed
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D016054:DNA, Protozoan; D015151:Immunoblotting; D007891:Leishmania / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000953:Antigens, Protozoan; D016054:DNA, Protozoan; D010749:Phosphoprotein Phosphatases