Abstract
Mouse models have shown the importance of acidic mammalian chitinase activity in settings of chitin exposure and allergic inflammation. However, little is known regarding genetic regulation of AMCase enzymatic activity in human allergic diseases. Resequencing the AMCase gene exons we identified 8 non-synonymous single nucleotide polymorphisms including three novel variants (A290G, G296A, G339T) near the gene area coding for the enzyme active site, all in linkage disequilibrium. AMCase protein isoforms, encoded by two gene-wide haplotypes, and differentiated by these three single nucleotide polymorphisms, were recombinantly expressed and purified. Biochemical analysis revealed the isoform encoded by the variant haplotype displayed a distinct pH profile exhibiting greater retention of chitinase activity at acidic and basic pH values. Determination of absolute kinetic activity found the variant isoform encoded by the variant haplotype was 4-, 2.5-, and 10-fold more active than the wild type AMCase isoform at pH 2.2, 4.6, and 7.0, respectively. Modeling of the AMCase isoforms revealed positional changes in amino acids critical for both pH specificity and substrate binding. Genetic association analyses of AMCase haplotypes for asthma revealed significant protective associations between the variant haplotype in several asthma cohorts. The structural, kinetic, and genetic data regarding the AMCase isoforms are consistent with the Th2-priming effects of environmental chitin and a role for AMCase in negatively regulating this stimulus.
Highlights
All data points are the mean of triplicates measurements, and kinetic parameters are the mean of at least two independent experiments
Asthma Association Analysis of AMCase SNPs—Based on the large gain in enzymatic activity conferred by the variant genetic isoform of the AMCase protein, we hypothesized that the SNP tagging the haplotype, which encoded this variant AMCase protein (G339T), may be associated with asthma
Through haplotype analysis we determined that these SNPs associate to form 6 common gene-wide AMCase haplotypes that code for the different AMCase protein isoforms
Summary
AMCase to be induced strongly in both lung epithelial cells and macrophages among fatal asthmatics [10]. The only genetic studies of the AMCase gene, conducted in Indian and German populations, have found the gene to be polymorphic and observed significant associations between several polymorphisms and asthma disease status [12, 13]. 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]. In this report we have explored the role that genetic variation plays in regulation of AMCase enzymatic activity. We describe all common coding variations and haplotypes in the human AMCase gene. We have biochemically characterized two haplotypic isoforms of the AMCase enzyme differing in amino acids close to the enzyme active site and assessed their effect on the three-dimensional structure of the enzyme. We explored whether the gain-of-function haplotype discovered was associated with asthma risk in several United States ethnic groups
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