Structure, function and latency regulation of a bacterial enterotoxin potentially derived from a mammalian adamalysin/ADAM xenolog

Abstract

Enterotoxigenic Bacteroides fragilis is the most frequent diseasecausing anaerobe in the intestinal tract of humans and livestock and its specific virulence factor is fragilysin, also known as B. fragilis toxin. This is a 21-kDa zinc-dependent metallopeptidase existing in three closely related isoforms that hydrolyze E-cadherin and contribute to secretory diarrhea, and possibly to inflammatory bowel disease and colorectal cancer. Here we studied the function and zymogenic structure of fragilysin-3 and found that its activity is repressed by a ∼170-residue prodomain, which is the largest hitherto structurally characterized for a metallopeptidase. This prodomain plays a role in both the latency and folding stability of the catalytic domain and it has no significant sequence similarity to any known protein. The prodomain adopts a novel fold and inhibits the protease domain via an aspartate-switch mechanism. The catalytic fragilysin-3 moiety is active against several protein substrates and its structure reveals a new family prototype within the metzincin clan of metallopeptidases. It shows high structural similarity despite negligible sequence identity to adamalysins/ADAMs, which have only been described in eukaryotes. Because no similar protein has been found outside enterotoxigenic B. fragilis, our findings support that fragilysins derived from a mammalian adamalysin/ADAM xenolog that was co-opted by B. fragilis through a rare case of horizontal gene transfer from a eukaryotic cell to a bacterial cell. Subsequently, this co-opted peptidase was provided with a unique chaperone and latency maintainer in the time course of evolution to render a robust and dedicated toxin to compromise the intestinal epithelium of mammalian hosts.bacterial endotoxin | human pathogen | zymogen activation

A Novel Mechanism of Latency in Matrix Metalloproteinases

“…Moreover, when expressed from the pKAR6 vector, which attaches an N-terminal glutathione S-transferase fusion protein (see Table 1), Kly18-E156A was obtained with ϳ10 times lower yield than the proprotein (vector pKAR5). We conclude that the propeptide plays a major role in proper folding of Kly18 as previously described for other MPs such as fragilysin (26,27), funnelin metallocarboxypeptidases (79,81,82), and ADAMs/adamalysins (54) but not for mammalian MMPs (83).…”

“…This is reminiscent of the evolutionary origin postulated for fragilysin, which is the only molecular virulence factor described for enterotoxigenic Bacteroides fragilis and for which no similar proteins have been reported, not even from other B. fragilis strains (21). Structural studies supported the view that the catalytic domain of this MP is the result of horizontal gene transfer of a member of the ADAM/adamalysin family, which has 38 orthologs in humans (8, [22][23][24][25], from a mammalian host to this bacterium, which thrives in the intestinal tract (26,27).…”

“…To date, aspartate-switch zymogenic mechanisms have been described only for astacins (7,88) and fragilysins (26), which are only distantly related MPs grouped with MMPs within the metzincins. To verify the function of Asp 25 in latency in pKly18, we used mutant pKly18-Y35A (from pKAR8), as the wild-type form (pKAR7) was insoluble.…”

A Novel Family of Soluble Minimal Scaffolds Provides Structural Insight into the Catalytic Domains of Integral Membrane Metallopeptidases

“…N-terminal sequencing through Edman degradation, peptide mass fingerprinting of tryptic protein digests, and mass spectrometry analyses were carried out at the proteomics facilities of the Centro de Investigaciones Biológicas and Vall d'Hebron Institute of Oncology. Melting temperatures (T m ) through differential scanning fluorimetry (thermofluor) were determined by using Sypro Orange dye (Invitrogen) and an iCycler iQ real time PCR detection system (Bio-Rad) as published previously (51,52). Limited proteolysis trials to remove C-terminal peptides from full-length proabylysin and projannalysin were undertaken in buffer C by overnight incubation at 37 or 80°C with trypsin, subtilisin, thermolysin (all from Sigma), or ulilysin (produced in-house according to Ref.…”

Expression and purification of integral membrane metallopeptidase HtpX

“…maintain the correct active-site architecture and zinc coordination, thus constituting valid models for structural studies. This strategy has been successfully used to determine the structure of numerous MP zymogenic forms, which usually show self-activation at the high protein concentrations required for crystallization [20][21][22]. Accordingly, we constructed HtpX mutant E 140 A to prevent self-cleavage and thus yield a stable form for structural studies.…”