Variant surface glycoprotein from Trypanosoma brucei clone YTat 1.1 contains a latent calmodulin-binding domain.
Calmodulin affinity chromatography and chromatofocusing were used to purify calmodulin-binding proteins of 32-40-kDa from homogenates of Trypanosoma brucei clone YTat1.1. The trypanosome proteins associated with calmodulins from different sources and reversibly inhibited calmodulin-dependent bovine brain phosphodiesterase. Purified 32-kDa protein bound to calmodulin with an approximate Kd of 1.3 nM. Polyclonal antibodies directed against purified 32-kDa protein and monoclonal antibody ECA6 recognized each of the 32-40-kDa proteins. Immunoprecipitation with biotinylated monoclonal antibody ECA6 (Bio-ECA6) or biotinylated calmodulin (Bio-CaM) identified the 32-40-kDa proteins in phenylmethylsulfonyl fluoride-treated lysates of slender forms of YTat1.1, but not procyclic forms of YTat1.1 or slender forms of EATRO110. In the presence of leupeptin, lysates of slender YTat1.1 contained a single protein of 58 kDa that immunoprecipitated with Bio-ECA6. The 58-kDa protein was exposed to the extracellular space as demonstrated by immunolocalization and sensitivity to pronase treatment in intact cells. The protein was identified as variant surface glycoprotein (VSG) based upon immunolocalization, pattern of expression and cross-reactivity of ECA6 with authentic VSG. The amino-terminal 17 residues of 32-kDa protein were identical with the amino-terminus of YTat1.1 VSG. Putative calmodulin-binding domains were identified in other VSGs by computer modeling. The model was tested with CNBr fragments of VSG 117. The fragments reversibly inhibited calmodulin-dependent activation of phosphodiesterase with approximate Kd of 11 nM. We conclude that endogenously generated proteolytic fragments of VSG from clone YTat1.1, and CNBr fragments of VSG 117 bind with high affinity to calmodulin.
Ruben
L
L
Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275.
Ridgley
E L
EL
Haghighat
N G
NG
Chan
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010726:Phosphodiesterase Inhibitors / Q000378:metabolism; D011233:Precipitin Tests; D016415:Sequence Alignment; D014346:Trypanosoma brucei brucei / Q000254:growth & development / Q000378:metabolism*; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010726:Phosphodiesterase Inhibitors; D014643:Variant Surface Glycoproteins, Trypanosoma