AUTHOR=Bratanis Eleni , Lood Rolf 

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

JOURNAL=Frontiers in Microbiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.00971

DOI=10.3389/fmicb.2019.00971

ISSN=1664-302X

ABSTRACT=<p>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 <italic>Bdellovibrio bacteriovorus</italic>: 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<sub>2</sub> and completely inhibited by ZnCl<sub>2</sub>. The hydrolysis of pIgA was subsequently utilized for the specific glycan characterization of the released pIgA Fc-tail (Asn<sup>459</sup>). Besides contributing to the basic knowledge of <italic>Bdellovibrio</italic> 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.</p><p>IMPORTANCE</p><p>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 <italic>B. bacteriovorus</italic>, facilitating the study of plasma IgA by cleaving the Fc-tail, including the Asn<sup>459</sup> N-glycan. This further highlights the potential of <italic>B. bacteriovorus</italic> as a source to identify potential novel biotechnological tools.</p>