title: Complementary Proteomic and Biochemical Analysis of Peptidases in Lobster Gastric Juice Uncovers the Functional Role of Individual Enzymes in Food Digestion.
abstract: 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.
authors.affiliation: Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Calle IPN 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S., 23096, Mexico.
authors.forename: Betsaida
authors.initials: B
authors.lastname: Bibo-Verdugo
authors.affiliation: Department of Pharmaceutical Chemistry, University of California San Francisco, 600 16th Street, San Francisco, CA, 94158, USA.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: O'Donoghue
authors.affiliation: Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Calle IPN 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S., 23096, Mexico.|Center for Discovery and Innovation in Parasitic Diseases, University of California San Francisco, 1700 4th Street, San Francisco, CA, 94158, USA.
authors.forename: Liliana
authors.initials: L
authors.lastname: Rojo-Arreola
authors.affiliation: Department of Pharmaceutical Chemistry, University of California San Francisco, 600 16th Street, San Francisco, CA, 94158, USA.
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
authors.affiliation: Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Calle IPN 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S., 23096, Mexico. fgarcia@cibnor.mx.
authors.forename: Fernando
authors.initials: F
authors.lastname: García-Carreño
mesh_terms: D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins / Q000235:genetics / Q000378:metabolism*; D003080:Cold Temperature; D004063:Digestion / Q000502:physiology; D005750:Gastric Juice / Q000737:chemistry* / Q000201:enzymology; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D058977:Molecular Sequence Annotation; D008121:Nephropidae / Q000201:enzymology* / Q000235:genetics; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D040901:Proteomics; D013379:Substrate Specificity
chemical_list: D060829:Arthropod Proteins; D006026:Glycoside Hydrolases; D010447:Peptide Hydrolases
keywords: Cold-adapted enzyme; Homarus americanus; Peptidase; Substrate specificity