Dundee, Ninewells Hospital, Dundee, DDl 9SY and IDivision of. Parasitology, NIMR, London, NW7 1AA. Malaria, a disease caused by parasites of the genus ...
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Biochemical Society Transactions (2002) Volume 30, Part 3
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Anti-malaria immune effector mechanisms recruited by human IgGl and IgAl monoclonal antibodies R.!. Pleass, D.H. McGuinness, IA.A. Holder, and J.M. Woof Department of Molecular & Cellular Pathology, University of Dundee, Ninewells Hospital, Dundee, D D l 9SY and IDivision of Parasitology, NIMR, London, NW7 1AA Malaria, a disease caused by parasites of the genus Plasmodium, is a major cause of morbidity worldwide, and estimates put the number of deaths at 2-3 million annually. The succcss of passive immunization to treat both humans and animals suggest that antibody-based strategies could have value as alternative therapeutic approaches. We have generated a unique panel of human chimeric IgGl and IgAl antibodies with identical V domains against the C-terminal -19 kDa portion of Plasmodium yoelii merozoite surface protein 1 (MSP-I 19), a major target of protective immune responses and a leading vaccine candidate. Experiments using immunofluorescence microscopy show a distinctive pattern of reactivity with schizonts and merozoites, consistent with recognition of an antigen present on the parasite surface. As a more physiologically relevant test of function, we have shown that both IgGl and IgAI, when attached to MSP-119 coated microtiter plates, can elicit potent oxidative bursts via Fc receptors on human neutrophils. The ability of these antibodies to kill malaria parasites is being investigated in vitro using phagocytosis assays and in vivo using both normal mice and mice transgenic for human Fc receptors.
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Amino acid sequence requirements in the human IgAl hinge for cleavage by streptococcal IgAl proteases B.W. Senior, M.R. Batten, IM. Kilian, and J.M. Woof Department of Molecular and Cellular Pathology, University of Dundee Medical School, Ninewells Hospital, Dundee D D l 9SI:
U.K. and ‘Department of Medical Microbiology and Immunology, Rartholin Building, University of Aarhus, DK-8000, Aarhus C, Denmark Several mutant IgAl antibodies with substitutions at proline 227 and threonine 228, the residues flanking the cleavage site for all streptococcal IgAl proteases, were constructed. The substitutions were without major effect upon the structure o r functional abilities of the antibodies. However they had a major effect upon their sensitivity to cleavage by some streptococcal IgAl proteases. S.pneumoniae IgAl protease showed no absolute requirement for either prolinc or threonine at residues 227-228. However, the IgAl proteases of S.oralis, S.sanguis and S.mitis had an absolute requirement for prolinc at 227 but not for threonine at 228 which could be substituted with valine. There was evidence with S.mitis and S.sanguzs that proteases from different strains had different amino acid requirements for cleavage. The protease of one S.sanguis strain appeared to be able to cleave the hinge at an alternative distant site if cleavage of the wild-type site was prevented by amino acid substitution. These studies mark a preliminary step towards the design of specific inhibitors of these enzymes.
0 2002 Biochemical Society
85 Chimeric human antibodies directed against a major surface antigen of Pseudomonas aeruginosa D.H. McGuinness, R.J. Pleass, B.W. Senior, and J.M. Woof Department of Molecular and Cellular Pathology, University of Dundee Medical School, Ninewells Hospital, Dundee, DDI 9Sl:
U.K.
Pseudomonas aeruginosa is an important opportunistic pathogen, present in a significant percentage of fatal nosocomial infections, and a cause of particular concern to cystic fibrosis patients and immuno-compromised individuals. These problems, coupled with the fact that I? aeruginosa is now recognised as one of the most antibiotic resistant bacteria, have kindled an upsurge in research into novel therapeutic approaches. Specific human antibodies may have potential in this arena. In this study we describe the generation and functional analysis of the first human chimeric IgGl and IgAl antibodies directed against lipoprotein I, a major surface antigen of I? aeruginosa. The antibodies have been shown to bind to intact bacteria and to trigger Fc receptor-mediated respiratory bursts in human effector cells.
86 A solution structural model of human IgA2 P.W. Whitty, *A. Robertson, M.A. Kerr, J.M. Woof, and ‘S.J. Perkins Department of Molecular and Cellular Pathology, University of Dundee, Ninewells Hospital, Dundee DD1 9SI: U.K. and .‘Department of Biochemistry, Royal Free and University College Medical School, London NW3 2PE U.K. There are two subclasses of human immunoglobulin A, IgAl and IgA2. Human IgA2 has a short hinge region of 8 amino acids linking the Fab arms and the Fc region, while IgAl possesses a much more extended hinge of 23 residues. X-ray and neutron scattering analysis of recombinant monomeric IgA2 (of IgA2m( 1) allotype) revealed a radius of gyration RG of 5.1 nm, which is significantly smaller than that of IgAl at 6.1-6.2 nm. For IgA2, the distance distribution function P(r) comprised a single peak and a maximum dimension of 17 nm, while IgAl gave two peaks and a 21 nm maximum dimension. An automated curve fit search analysis of a homology model for IgA2 was conducted, in which random IgA2 hinge structures connect the Fab and Fc fragments in any orientation. Around 50 out of 10,000 models fitted the scattering and ultracentrifugation data. This approach produced an IgA2 structure that is significantly more compact than that of IgA1. The IgA2 Fab and Fc arrangement resembles that in the crystal structure of the hinge-deleted human IgGl Mcg, which shares the characteristic of an inter-light chain disulphide bond.
