Identification of two antibody-interaction sites on the surface of Panulirus interruptus hemocyanin.
Negatively stained complexes of Panulirus interruptus (spiny lobster) hemocyanin with two different monoclonal antibodies, named E and J, were studied by electron microscopy and image processing. The attachment site of the antibodies to the hexameric hemocyanin molecule was deduced from two perpendicular views of hemocyanin/antibody complexes, in which either the threefold axis or one of the twofold axes was oriented perpendicular to the supporting film. Images of complexes in these orientations were searched with reference images simulated from the known X-ray structure of P. interruptus hemocyanin. The two sites were further characterized by combining our results from electron microscopy with structural data obtained by X-ray diffraction and other methods. These two antibodies recognize different non-overlapping epitopes. The epitope for clone E is located on domain 3 at the surface of the beta barrel and consists of certain loops, which form connections between beta-strand structures. The epitope for clone J is situated on domain 1 at the surface of an alpha-helical region and consists mainly of certain alpha-helices connecting loops. The orientation of the hemocyanin hexamers in the two complexes is very different, as is demonstrated most clearly when they form chains. Clone E forms complexes with the threefold axes perpendicular to the chain direction, while for clone J the threefold axes seem to be parallel to the main direction. The angle between the Fab part of an IgG molecule and the threefold axis of the hexamer is 60 +/- 5 degrees for clone E and 35 +/- 7 degrees for clone J. This observation is clearly related to the difference in orientation of the hexamers for the two complexes.
de Haas
F
F
BIOSON Research Institute, Rijksuniversiteit Groningen, The Netherlands.
Perton
F G
FG
van Breemen
J F
JF
Dijkema
J H
JH
Beintema
J J
JJ
van Bruggen
E F
EF
D000818:Animals; D001666:Binding Sites, Antibody; D006433:Hemocyanins / Q000737:chemistry / Q000276:immunology* / Q000648:ultrastructure; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008807:Mice, Inbred BALB C; D016253:Microscopy, Immunoelectron; D008121:Nephropidae; D011487:Protein Conformation; D014961:X-Ray Diffraction
D006433:Hemocyanins