Identification of two antibody‐interaction sites on the surface of Panulirus interruptus hemocyanin

Abstract

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 hernocyanidantibody 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 p barrel and consists of certain loops, which form connections between /3-strand structures.The epitope for clone J is situated on domain 1 at the surface of an a-helical region and consists mainly of certain a-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 5" for clone E and 35 5 7" for clone J. This observation is clearly related to the difference in orientation of the hexamers for the two complexes.Hemocyanin is an extracellular, copper containing protein that occurs in high concentrations (10-100 mg/ml) in the blood of molluscs and certain arthropods belonging to the chelicerata (spiders, scorpions, horseshoe crabs) and crustacea (spiny lobsters, crabs; Van Holde and van Bruggen, 1971;Van Holde and Miller, 1982;Ellerton et al., 1983;Markl and Decker, 1991). It serves as an oxygen carrier, functionally similar to hemoglobin, but binds oxygen between two copper ions. The architecture of arthropod hemocyanin multimers consists of discrete assemblies of hexameric building blocks (Mr approximately 450000). The threedimensional X-ray structures of the hexameric hemocyanins from the spiny lobster (Panulirus interruptus ; Gaykema et al., 1984Gaykema et al., , 1986Volbeda and Hol, 1989) and of subunit I1 from the horseshoe crab (Limulus polyphemus; Hazes et al., 1993) are known in detail. Roughly, six bean-shaped subunits (Mr approximately 75000) occupy the corners of a trigonal antiprism.Hemocyanin has powerful antigenic properties used in many medical tests. Our interest concerns the structural features which cause these strong immunogenic reactions.Correspondence to E. F. J. van Bruggen, BIOSON Research Institute, Rijksuniversiteit Groningen, Nijenborgh 4, NL-9747 AG

Influence of antibody binding on oxygen binding behavior of Panulirus interruptus hemocyanin

“…For the studies described here monoclonal antibodies D, E and J were used. The epitopes for antibodies E and J have been determined in detail by immuno‐electron microscopy [11]. Antibody E binds to domain 3 at loops between strands β3D‐β3E and between β3G‐β3H, while antibody J binds to a region with the loop between helices α1.4 and α1.5 in domain 1 as the center of the epitope, but also includes the loops between helices α1.2 and α1.3 and between helix α1.6 and strand β1A.…”

“…Antibody E binds to domain 3 at loops between strands β3D‐β3E and between β3G‐β3H, while antibody J binds to a region with the loop between helices α1.4 and α1.5 in domain 1 as the center of the epitope, but also includes the loops between helices α1.2 and α1.3 and between helix α1.6 and strand β1A. The epitope for antibody D is also localized on domain 1 [12], and does not overlap with that for antibody J [11], but has not yet been mapped into more detail.…”