Understanding the biochemical properties and physiological function of the protein syncollin
Syncollin is a 16-kDa protein that was originally isolated from the pancreatic zymogen granule. It is now known also to be expressed in the gut, the spleen and in neutrophils. The protein contains intramolecular disulphide bonds and is present both free within the ZG lumen and tightly associated with the luminal leaflet of the ZG membrane; it is also secreted into the pancreatic juice. Syncollin is able to oligomerize, and assemble into doughnut-shaped structures, which might explain its known pore-forming activity. Syncollin appears to be involved in a number of gut-based disease states. For instance, syncollin expression was found to be down-regulated in the colon when a bacterial suspension was administered to germ-free mice, and in mice with chemically-induced colitis-associated cancer. The available evidence suggests that syncollin plays an important role in the gut, and possibly elsewhere. This dissertation describes my attempts to understand this role and its structural basis. I first assessed various methods for purification of recombinant syncollin. Syncollin was expressed with various epitope tags (including His, GST and Strep) in bacteria, insect cells and mammalian cells. The best results were obtained by expressing syncollin bearing a double-Strep tag at its C terminus (syncollin-Strep) in mammalian (tsA-201) cells and purifying the protein from the cell supernatant using the Strep-Tactin XTTM system. Syncollin-Strep purified in this way contained intra-molecular disulphide bonds and recapitulated the ability of the native protein to bind to syntaxin 2 and permeabilize membranes. In the pancreatic juice, syncollin will encounter an environment rich in proteolytic activity. One might expect, therefore, that its structure would be highly stable. To test this hypothesis, I assessed the thermal stability of the protein using circular dichroism (CD) spectroscopy. The CD spectrum of syncollin-Strep indicated a predominantly beta-sheet structure. When the protein was subjected to a temperature ramp up to 90°C, the spectrum became flattened, although complete unfolding did not occur, indicating that the protein does indeed have a very high thermal stability. A model for syncollin, based on its primary sequence, predicts a predominantly beta-sheet structure, consistent with my CD data, and suggests the presence of intramolecular disulphide bonds. When I disrupted potential bonds by mutation of appropriate cysteine residues, syncollin-Strep retained its antibacterial efficacy, but its thermal stability was reduced, suggesting the involvement of disulphide bonding in stabilizing the structure of the protein. With regard to its potential role in the gut, I found that syncollin-Strep binds to bacterial peptidoglycan, and restricts the growth of representative Gram-positive (Lactococcus lactis) and Gram-negative (Escherichia coli) bacteria. Syncollin induces propidium iodide uptake into E. coli (but not L. lactis), indicating permeabilization of the bacterial membrane. In support of this idea, I confirmed that syncollin-Strep, like native syncollin, has pore-forming properties. Syncollin-Strep causes surface structural damage in both L. lactis and E. coli, as visualized by scanning electron microscopy. In addition, syncollin-Strep had additive effects on L. lactis when combined with either ampicillin (bactericidal) or tetracycline (bacteriostatic) in L. lactis. In light of these results, I propose that syncollin is a previously unidentified member of a large group of antimicrobial polypeptides that control the gut microbiome. I found that expression of syncollin in neutrophils is punctate and granular. Upon activation of the neutrophils, syncollin became mobilized at the plasma membrane, and was also secreted from the cells. Secreted syncollin bound to decondensed chromatin structures characteristic of neutrophil extracellular traps (NETs). Further, when neutrophils were activated in the presence of bacteria, the bacteria became coated with secreted syncollin, consistent with the anti-bacterial role proposed above. In conclusion, the results presented in this dissertation indicate that, through its antibacterial effects, syncollin plays a role in host defence in both the gut and the bloodstream.
Antimicrobial peptide, Host defence, Pancreatic zymogen granule, Membrane permeabilisation, Recombinant protein purification
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