A Novel Broad-Spectrum Elastase-Like Serine Protease From the Predatory Bacterium Bdellovibrio bacteriovorus Facilitates Elucidation of Site-Specific IgA Glycosylation Pattern

Abstract

The increased interest in predatory bacteria due to their ability to kill antibiotic resistant bacteria has also highlighted their inherent plethora of hydrolytic enzymes, and their potential as natural sources of novel therapeutic agents and biotechnological tools. Here, we have identified and characterized a novel protease from the predatory bacterium Bdellovibrio bacteriovorus : BspE (Bdellovibrio elastase-like serine protease). Mapping preferential sites of proteolytic activity showed a single proteolytic cleavage site of native plasma IgA (pIgA) in the Fc-tail; as well as in the secretory component (SC) of secretory IgA (SIgA). Proteolysis of other native immunoglobulins and plasma proteins was either absent (IgG1 and 2, IgM, albumin and orosomucoid) or unspecific with multiple cleavage sites (IgG3 and 4, IgE, IgD). BspE displayed a broad activity against most amino acid bonds in shorter peptides and denatured proteins, with a slight preference for hydrolysis C-terminal of Y, V, F, S, L, R, P, E, and K. BspE autoproteolysis results in numerous cleavage products sustaining activity for more than 6 h. The enzymatic activity remained stable at pH 5.0–9.0 but was drastically reduced in the presence of MnCl 2 and completely inhibited by ZnCl 2 . The hydrolysis of pIgA was subsequently utilized for the specific glycan characterization of the released pIgA Fc-tail (Asn 459 ). Besides contributing to the basic knowledge of Bdellovibrio biology and proteases, we propose that BspE could be used as a potential tool to investigate the importance, and biological function of the pIgA Fc-tail. IMPORTANCE Antibodies are well-established as key components of the immune system, and the importance of antibody glycosylation is steadily gaining recognition. Modifications of antibodies by glycosylation creates a vast repertoire of antibody glycovariants with distinctive and diverse functions in the immune system. Most of the available information regarding antibody glycosylation is based on studies with IgG, which have contributed greatly to the advance of therapeutic antibody treatments. However, much is still unknown regarding the importance of glycosylation and the Fc-structure for the remaining antibody classes. Such research has proven to be technically challenging and demonstrates a need for novel tools to facilitate such investigations. Here we have identified and characterized a novel protease from B. bacteriovorus , facilitating the study of plasma IgA by cleaving the Fc-tail, including the Asn 459 N-glycan. This further highlights the potential of B. bacteriovorus as a source to identify potential novel biotechnological tools.

Biotechnological Potential of Bdellovibrio and Like Organisms and Their Secreted Enzymes

“…Interestingly, B. bacteriovorus is now emerging as a new source for the identification of novel enzymes with biotechnological potential. This potential can be exemplified by the identification and characterization of BspK and BspE with described enzymatic activities on human antibodies (Bratanis et al, 2017;Bratanis and Lood, 2019). BspK specifically hydrolyzes IgG 1 (most common therapeutic antibody) in the hinge, enabling middledown MS analysis of the biological therapeutic (Bratanis et al, 2017).…”

“…Similar enzymes (e.g., Ides, SpeB) are currently being used within the biopharma industry for such purposes. BspE specifically hydrolyzes the Fc-tail from IgA, with its glycan attached (Bratanis and Lood, 2019). While IgA is not commonly used for the development of therapeutic antibodies, BspE is still a valuable tool for the basic research of IgA, Fc-interactions and complement activation -findings that may eventually be translated into products.…”

“…The repertoire of enzymes secreted by this bacterium may further contribute to the reduction of the HGT. Extracellular proteolytic and nuclease activities have been demonstrated in B. bacteriovorus HD100 cultures (Engelking and Seidler, 1974;Gloor et al, 1974;Monnappa et al, 2014;Bratanis et al, 2017;Bratanis and Lood, 2019). Thus, hypothetically, nucleases released into the environment may contribute to the elimination of the "cellfree ARGs, " while extracellular proteases may act on phage particles, leading to their inactivation.…”

“…The revived and growing interest in BALOs and other predatory bacteria has resulted in a rapid increase of information and knowledge that is now readily available. This includes a better overview and understanding of the large diversity of predatory species and their widespread distribution, as well as genome, proteome, secretome, and biochemical data (Schwudke et al, 2001;Rendulic et al, 2004;Dori-Bachash et al, 2008;Song et al, 2009;Pan et al, 2011;Karunker et al, 2013;Avidan et al, 2017;Bratanis et al, 2017;Bratanis and Lood, 2019). Although much of the interest in BALOs relates to the use of predatory bacteria as biological-based therapeutic agents (Willis et al, 2016;Negus et al, 2017), their application is more versatile, as shown by the already existing products on the market.…”

Imaging the Substructures of Individual IgE Antibodies with Atomic Force Microscopy

“…Proteins have attracted huge interest due to their biological functions such as transportation, catalysis, defense, and regulation. − All these properties make protein an indispensable substance in the human body. Antibodies are a kind of the most important proteins in humoral immunity. , Immunoglobulin E (IgE) is the fifth antibody isotype (IgG, IgA, IgM, IgD, and IgE) and is least abundant in serum. , As shown in Figure , the bent Y-shaped IgE shares the same basic molecular structure as other classes of antibodies, consisting of one crystallizable fragment (Fc) as well as two separate and identical antigen-binding fragments (Fab). , The antibody plays an absolutely essential role in allergic responses and can be used for the diagnosis of allergic diseases. − In order to sufficiently understand the function of IgE, it would be interesting to acquire the substructure of IgE adsorbed on a substrate.…”