GAP43 shows partial co-localisation but no strong physical interaction with prolyl oligopeptidase.
It has recently been proposed that prolyl oligopeptidase (POP), the cytosolic serine peptidase with neurological implications, binds GAP43 (Growth-Associated Protein 43) and is implicated in neuronal growth cone formation, axon guidance and synaptic plasticity. We investigated the interaction between GAP43 and POP with various biophysical and biochemical methods in vitro and studied the co-localisation of the two proteins in differentiated HeLa cells. GAP43 and POP showed partial co-localisation in the cell body as well as in the potential growth cone structures. We could not detect significant binding between the recombinantly expressed POP and GAP43 using gel filtration, CD, ITC and BIACORE studies, pull-down experiments, glutaraldehyde cross-linking and limited proteolysis. However, glutaraldehyde cross-linking suggested a weak and transient interaction between the proteins. Both POP and GAP43 interacted with artificial lipids in our in vitro model system, but the presence of lipids did not evoke binding between them. In native polyacrylamide gel electrophoresis, GAP43 interacted with one of the three forms of a polyhistidine-tagged prolyl oligopeptidase. The interaction of the two proteins was also evident in ELISA and we have observed co-precipitation of the two proteins during co-incubation at higher concentrations. Our results indicate that there is no strong and direct interaction between POP and GAP43 at physiological conditions.
Szeltner
Zoltán
Z
Institute of Enzymology, BRC, Hungarian Academy of Sciences, Budapest, H-1113, Karolina út 29, Hungary. szeltner@enzim.hu
Morawski
Markus
M
Juhász
Tünde
T
Szamosi
Ilona
I
Liliom
Károly
K
Csizmók
Veronika
V
Tölgyesi
Ferenc
F
Polgár
László
L
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D002454:Cell Differentiation; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D019922:GAP-43 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005976:Glutaral / Q000737:chemistry; D020439:Growth Cones / Q000378:metabolism / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D000085822:Prolyl Oligopeptidases; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003432:Cross-Linking Reagents; D019922:GAP-43 Protein; D008051:Lipid Bilayers; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C000705287:PREPL protein, human; D000085822:Prolyl Oligopeptidases; D005976:Glutaral