Complementary Proteomic and Biochemical Analysis of Peptidases in Lobster Gastric Juice Uncovers the Functional Role of Individual Enzymes in Food Digestion

Crustaceans are a diverse group, distributed in widely variable environmental conditions for which they show an equally extensive range of biochemical adaptations. Some digestive enzymes have been studied by purification/characterization approaches. However, global analysis is crucial to understand how digestive enzymes interplay. Here, we present the first proteomic analysis of the digestive fluid from a crustacean (Homarus americanus) and identify glycosidases and peptidases as the most abundant classes of hydrolytic enzymes. The digestion pathway of complex carbohydrates was predicted by comparing the lobster enzymes to similar enzymes from other crustaceans. A novel and unbiased substrate profiling approach was used to uncover the global proteolytic specificity of gastric juice and determine the contribution of cysteine and aspartic acid peptidases. These enzymes were separated by gel electrophoresis and their individual substrate specificities uncovered from the resulting gel bands. This new technique is called zymoMSP. Each cysteine peptidase cleaves a set of unique peptide bonds and the S2 pocket determines their substrate specificity. Finally, affinity chromatography was used to enrich for a digestive cathepsin D1 to compare its substrate specificity and cold-adapted enzymatic properties to mammalian enzymes. We conclude that the H. americanus digestive peptidases may have useful therapeutic applications, due to their cold-adaptation properties and ability to hydrolyze collagen.     

Resurrection of an ancient inflammatory locus reveals switch to caspase-1 specificity on a caspase-4 scaffold

Pyroptosis is a mechanism of inflammatory cell death mediated by the activation of the prolytic protein gasdermin D by caspase-1, caspase-4, and caspase-5 in human, and caspase-1 and caspase-11 in mouse. In addition, caspase-1 amplifies inflammation by proteolytic activation of cytokine interleukin-1β (IL-1β). Modern mammals of the order Carnivora lack the caspase-1 catalytic domain but express an unusual version of caspase-4 that can activate both gasdermin D and IL-1β. Seeking to understand the evolutionary origin of this caspase, we utilized the large amount of data available in public databases to perform ancestral sequence reconstruction of an inflammatory caspase of a Carnivora ancestor. We expressed the catalytic domain of this putative ancestor in Escherichia coli, purified it, and compared its substrate specificity on synthetic and protein substrates to extant caspases. We demonstrated that it activates gasdermin D but has reduced ability to activate IL-1β. Our reconstruction suggests that caspase-1 was lost in a Carnivora ancestor, perhaps upon a selective pressure for which the generation of biologically active IL-1β by caspase-1 was detrimental. We speculate that later, a Carnivora encountered selective pressures that required the production of IL-1β, and caspase-4 subsequently gained this activity. This hypothesis would explain why extant Carnivora possess an inflammatory caspase with caspase-1 catalytic function placed on a caspase-4 scaffold.