Kinetic Characterization of Recombinant Human Acidic Mammalian Chitinase

Abstract

Human acidic mammalian chitinase (AMCase), a member of the family 18 glycosyl hydrolases, is one of the important proteins involved in Th2-mediated inflammation and has been implicated in asthma and allergic diseases. Inhibition of AMCase results in decreased airway inflammation and airway hyper-responsiveness in a mouse asthma model, suggesting that the AMCase activity is a part of the mechanism of Th2 cytokine-driven inflammatory response in asthma. In this paper, we report the first detailed kinetic characterization of recombinant human AMCase. In contrast with mouse AMCase that has been reported to have a major pH optimum at 2 and a secondary pH optimum around 3-6, human AMCase has only one pH optimum for k(cat)/K(m) between pH 4 and 5. Steady state kinetics shows that human AMCase has "low" intrinsic transglycosidase activity, which leads to the observation of apparent substrate inhibition. This slow transglycosylation may provide a mechanism in vivo for feedback regulation of the chitinase activity of human AMCase. HPLC characterization of cleavage of chitooligosaccharides (4-6-mers) suggests that human AMCase prefers the beta anomer of chitooligosaccharides as substrate. Human AMCase also appears to cleave chitooligosaccharides from the nonreducing end primarily by disaccharide units. Ionic strength modulates the enzymatic activity and substrate cleavage pattern of human AMCase against fluorogenic substrates, chitobiose-4-methylumbelliferyl and chitotriose-4-methylumbelliferyl, and enhances activity against chitooligosaccharides. The physiological implications of these results are discussed.

Analysis of productive binding modes in the human chitotriosidase

“…Firstly, there could be binding from À4 to +2 with a b-anomer preference in +2; secondly, one could envisage binding from À2 and towards positive subsites. Both binding modes were confirmed by MALDI-TOF-MS analysis indicating production of (GlcNAc) 8 , a transglycosylation product from combination of (GlcNAc) 6 and (GlcNAc) 2 after productive binding from À2 towards positive subsites, and (GlcNAc) 10 , a transglycosylation product from combination of (GlcNAc) 6 and (GlcNAc) 4 after productive binding from À4 to +2, were 6 , Ç (GlcNAc) 5 , N (GlcNAc) 4 , d (GlcNAc) 3 , j (GlcNAc) 2 . The relative higher abundance of (GlcNAc) 3 compared to (GlcNAc) 2 during (GlcNAc) 5 degradation likely reflects the dominance in (À2 to +3) binding compared to (À3 to +2) resulting in more (GlcNAc) 2 compared to (GlcNAc) 3 serving as sugar donor to (GlcNAc) 5 in transglycosylation reactions.…”

Differential Enzymatic Activity of Common Haplotypic Versions of the Human Acidic Mammalian Chitinase Protein

“…The variants associated with asthma in the German study result in amino acid changes, raising the possibility that coding AMCase polymorphisms may influence chitinase activity and potentially disease risk (12). Biochemical analysis of the human AMCase protein has revealed the enzyme does cleave chitin substrates and that its activity is modified by pH and salt concentration (7). In this report we have explored the role that genetic variation plays in regulation of AMCase enzymatic activity.…”

“…Chitinases are evolutionarily ancient enzymes that hydrolytically cleave the chitin polymer into di-and trisaccharides. Chitinases serve functionally diverse roles across species, including nutrient scavenging, structural remodeling of chitin constituents, parasitism, and innate immunity (6,7).…”

Role of Mammalian Chitinases in Asthma

“…2). Within this barrel, the β 4 strand contains a conserved sequence motif (DXXDXDXE, where D = aspartic acid, E = glutamic acid, and X = any amino acid) that forms the active site of the enzyme, with glutamic acid being the key residue that donates a proton required for hydrolyzing the β(1→4) glycosidic bond in chitin [11]. In chitinase-like proteins, the substitution of this essential glutamic acid to leucine, isoleucine or glutamine accounts for the lack of chitinolytic activity (table 1).…”

“…Chitotriosidase was the first mammalian chitinase to be identified and has since been implicated in Gaucher disease, the most common lysosomal storage disorder in humans caused by an inherited deficiency in glucocerebrosidase [14]. AMCase was subsequently cloned as an acid-stable active enzyme and is highly expressed in the gastrointestinal tract, and to a lesser extent, in the lung and alveolar macrophages in both humans and mice [15, 16], with an optimum activity at pH 4–5 [11]. …”