Abstract
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, remain 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 Förster 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. 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 found in 14 of 63 (22.2%) catalytic antibody structures and 119 of 1602 (7.4%) antibodies with no annotation of catalytic activity. 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.
Highlights
Studies in the 1980s first showed that some natural antibodies were “catalytic” and able to hydrolyze peptide or phosphodiester bonds in antigens
Many variations in orientation and residue composition on the traditional serine protease triad have been described in proteolytic enzymes, and it is possible that either some of these known active sites or completely novel active sites are responsible for the catalytic activity of some Igs [35]
To investigate the basis for catalytic activity and to generate a structural model of 18B7, an accurate amino acid sequence was determined through protease digestion and mass spectrometry of the same antibody stock used in all experiments in this study
Summary
MAb 18B7 Sequencing—Since the 18B7 mAb to GXM was initially isolated and characterized, several studies over the intervening years have found nucleotide and amino acid sequences for the V region containing discrepancies at a number of positions in both H and L chains [24, 37, 38]. Mass chromatogram analysis showed complete coverage of the VH and VL regions with no peptides matching more than one of the possible 18B7 sequences, fully resolving all five VL discrepancies and all five VH discrepancies Based on these data, 18B7 differs from the closely related 3E5 mAb at only 3 amino acids in the VL and 15 positions in the VH (supplemental Fig. S1). Matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis following incubation of P1 with mAb 18B7 resulted in specific cleavage at positions 5 and 6 (Fig. 1A) This result confirms prior NMR data suggesting that mAb 18B7 hydrolyzes P1 in a different pattern from 3E5 and illustrates that small changes in the paratope can affect catalytic activity [28]. The kinetic rate constants Km, Vmax, and Kcat were calculated for each antibody
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