Catalytic activity in monoclonal antibodies to Cryptococcus neoformans glucuronoxylomannan
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Studies in the 1980s first showed that some natural antibodies were "catalytic" and able to hydrolyze peptide or phosphodiester bonds in antigens. Many naturally occurring catalytic antibodies have since been isolated from human sera and associated with positive and negative outcomes in autoimmune disease and infection. The function and prevalence of these antibodies, however, remains unclear. A previous study suggested that the 18B7 monoclonal antibody against glucuronoxylomannan (GXM)---the major component of the Cryptococcus neoformans polysaccharide capsule---hydrolyzed a peptide antigen mimetic. Using mass spectrometry and Forster resonance energy transfer techniques, we confirm and characterize the hydrolytic activity of 18B7 against peptide mimetics and show that 18B7 is able to hydrolyze an oligosaccharide substrate, providing the first example of a naturally occurring catalytic antibody for polysaccharides. Additionally, we show that the catalytic 18B7 antibody increases release of capsular polysaccharide from fungal cells. We also determine kinetic parameters for 18B7's peptidase activity and demonstrate the effects of serine protease and competitive inhibitors. A serine protease inhibitor blocked peptide and oligosaccharide hydrolysis by 18B7 and a putative serine-protease-like active site was identified in the light chain variable region of the antibody. An algorithm was developed to detect similar sites present in unique antibody structures in the Protein Data Bank. The putative site was enriched in catalytic antibody structures and found in 119 of 1602 (7.4%) antibodies with no annotation of catalytic activity. To validate this algorithm, we obtained another antibody with the putative catalytic motif and showed that it was able to hydrolyze a peptide substrate. The ability of many antibodies to cleave antigen, albeit slowly, supports the notion that this activity is an important immunoglobulin function in host defense. The discovery of GXM hydrolytic activity suggests new therapeutic possibilities for polysaccharide-binding antibodies. In the final part of this thesis, we describe related experiments detailing the passive release of capsular polysaccharide by C. neoformans, shedding light on the dynamics of polysaccharide production and secretion by the organism. We also describe studies investigating the structural epitope of mAbs to GXM using glycan microarrays, which provide a better understanding for mAb activity and GXM structure.
Source: Dissertation Abstracts International, Volume: 78-09(E), Section: B.;Advisors: Arturo Casadevall.