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. 2009 Jul 17;284(29):19650-8.
doi: 10.1074/jbc.M109.012443. Epub 2009 May 12.

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

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Free PMC article

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

Max A Seibold et al. J Biol Chem. .
Free PMC article

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.

Figures

FIGURE 1.
FIGURE 1.
The structure of the human AMCase gene, with accompanying common non-synonymous SNPs present among Mexicans, Puerto Ricans, and African Americans. Exons are identified by blue and red block structures and numbered accordingly. The red portion of exons represent untranslated regions and the blue represent the coding portion of exons. All variant mRNA and amino acid positions are reported with regard to mRNA sequence uc001eas.1 (UCSC Genome Browser). Percentages listed refer to minor allele frequencies in Mexicans, Puerto Ricans, and African Americans, respectively. The minor allele and amino acid is listed second.
FIGURE 2.
FIGURE 2.
AMCase protein domain structure and common haplotypes with frequencies in African Americans, Puerto Ricans, and Mexicans. Haplotypes are defined as variant or wild type depending on whether they contained the minor or major alleles of the three linked SNPs (shown in red) in close proximity to the active site (A290G, G296A, G339T), respectively. The haplotype alleles for SNPs A290G and G296A are inferred based on the linkage disequilibrium patterns defined in resequencing. Haplotypes frequencies reported are for SAGE controls and GALA parents.
FIGURE 3.
FIGURE 3.
pH and kinetic characterization of AMCase isoforms. AMCase activity was determined using the synthetic chitin substrate 4-MU-(4-deoxy)-chitobiose. A, pH activity profile of variant (GATGAGCG) and wild type (AGGGAATT) AMCase isoforms. Activity at each pH is expressed as a percentage of activity at optimum pH. Michaelis-Menten kinetic profiles of AMCase isoforms. B, assays conducted at pH 2.2. C, assays conducted at pH 4.6. D, assays conducted at pH 7.0. Substrate concentrations tested were 10, 25, 50, 75, 100, 150, and 200 μm. Curves were fit to data by nonlinear regression using the Michaelis-Menten equation. All graph points are the mean of triplicate measurements and representative of multiple experiments.
FIGURE 4.
FIGURE 4.
Structural implications of the three amino acid changes in the variant isoform compared with wild type AMCase. The wild type AMCase structure is shown in black, the variant isoform structure in red. Only the side chains of those amino acids that are changed in the variant structure compared with wild type and that are implicated in either binding/catalysis or pH specificity (see text) are depicted as sticks (black, wild type; red, variant isoform). The side chains of the three amino acids that are mutated in the variant isoform are also indicated (Asn/Asp-45, Asp/Asn-47, and Arg/Met-61).
FIGURE 5.
FIGURE 5.
Potential causal model describing the interaction of proinflammatory environmental chitin exposures with allergic antigens and the genetic regulation of this process by chitinases. In line with the hygiene hypothesis we predict decreased bacterial populations (and accompanying bacterial chitinases) due to the prevalence of anti-microbial products in developed societies, which may increase environmental chitin exposure. Moreover, in this environment of increased chitin exposures a gain-of-function AMCase variant may be protective against development of allergy and asthma.

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