Differences in the chitinolytic activity of mammalian chitinases on soluble and crystalline substrates

Abstract

Chitin is an abundant polysaccharide used by a large range of organisms for structural rigidity and water repulsion. As such, the insoluble crystalline structure of chitin poses significant challenges for enzymatic degradation. Vertebrates do not produce chitin, but do express chitin degrading enzymes. Acidic mammalian chitinase, the primary enzyme involved in the degradation of environmental chitin in mammalian lungs, is a processive glycosyl hydrolase that may be able to make multiple hydrolysis events for each binding event. Mutations to acidic mammalian chitinase have been associated with risk factors for asthma, and genetic deletion of the enzyme in mice results in significantly increased morbidity and mortality with age. Aside from the local catalytic mechanism at the active site, the mechanisms by which acidic mammalian chitinase binds, orients, and processes on crystalline chitin are poorly understood. Previous efforts to quantify activity have been limited either by the use of short oligomeric substrates or by signal-to-noise considerations that limited throughput or quantifiability. Here, we develop new methods to quantify chitinase activity using a colloidal chitin source and a fluorogenic enzyme-coupled assay, which allows us to dissect total activity into catalysis and binding, with sufficient throughput to quantify the effects of multiple mutations. We use this technique to measure the effect of the carbohydrate-binding domain on activity, to quantify differences in activity between acidic mammalian chitinase and chitotriosidase, the other chitinase expressed by mammals, and to characterize the effects of both naturally occurring mutations and mutations identified by a directed evolution screen to increase activity with oligomeric substrates. Our results underscore the importance of measuring activity using crystalline chitin substrates to understand the mechanisms of chitin degradation, and provide a new tool to facilitate effective quantification of binding and catalysis with these complex substrates. We anticipate that this approach will be useful for further studies of chitinase function, as well as for any future efforts at engineering glycosyl hydrolases.

Distribution and Biotechnological Applications of Chitinase: A Review

“…True chitinases also possess a carboxyl terminal chitin binding domain [74]. Chitotrisidase and acidic mammalian chitinase (AMCase) belong to true chitinases category and both contain a catalytic and chitin binding domains [5]. Chitotrisidase is antifungal in nature and is produced in macrophages and expressed in Gauchers cells while AMCase is produced by eosinophils, macrophages and epithelial cells [14,28].…”

“…Chitin is world's second most abundant carbon reserve in nature [3]. It is a renewable natural source and is an inelastic linear homopolymer having N-acetyl glucosaminyl residues linked through β-1,4 linkages [4,5]. It is the main structural component of exoskeleton in invertebrates like insects and crustaceans and has also been found in the cell wall of many fungi [6,7].…”