Ca2+-dependent and phospholipid-independent binding of annexin 2 and annexin 5.
Annexins are a family of homologous proteins that associate with anionic phospholipid (aPL) in the presence of Ca(2+). Evidence that the function of one annexin type may be regulated by another was recently reported in studies investigating cytomegalovirus-aPL interactions, where the fusogenic function of annexin 2 (A2) was attenuated by annexin 5 (A5). This observation suggested that A2 may bind directly to A5. In the present study, we demonstrated this interaction. The A2-A5 complex was first detected utilizing (covalently linked) fluorescein-labelled A5 (F-A5) as a reporter group. The interaction required concentrations of Ca(2+) in the millimolar range, had an apparent dissociation constant [ K (d)(app)] of 1 nM at 2 mM Ca(2+) and was independent of aPL. A2 bound comparably with F-A5 pre-equilibrated with an amount of aPL that could bind just the F-A5 or to an excess amount of aPL providing sufficient binding sites for all of F-A5 and A2. A2-A5 complex formation was corroborated in an experiment, where [(125)I]A2 associated in a Ca(2+)-dependent manner with A5 coated on to polystyrene. Surface plasmon resonance was used as a third independent method to demonstrate the binding of A2 and A5 and, furthermore, supported the conclusion that the monomeric and tetrameric forms of A2 bind equivalently to A5. Together these results demonstrate an A2-A5 interaction and provide an explanation as to how A5 inhibits the previously reported A2-dependent enhancement of virus-aPL fusion.
Research and Development Department, Canadian Blood Services, 1800 Alta Vista Drive, Ottawa, ON, Canada K1G 4J5.
Nicole D
ND
Brooks
Jean E
JE
Grundy
Nadine
N
Lavigne
Mélanie C
MC
Derry
Christina M
CM
Restall
C Roger
CR
MacKenzie
David M
DM
Waisman
Edward L G
EL
Pryzdial
D017306:Annexin A2 / Q000378:metabolism*; D017304:Annexin A5 / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D020349:Surface Plasmon Resonance
D017306:Annexin A2; D017304:Annexin A5; D010743:Phospholipids; D002118:Calcium