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

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.

Loss and Gain of Human Acidic Mammalian Chitinase Activity by Nonsynonymous SNPs

“…Consistently with previous reports, recombinant mouse AMCase had the highest activity at pH 2.0 (Boot et al 2001; Kashimura et al 2013), whereas the recombinant human AMCase was most active at around pH 4.0–5.0 (Chou et al 2006; Seibold et al 2009; Goedken et al 2011) with the human enzyme being significantly less active as compared with the mouse AMCase (fig. 1 C ; supplementary table S1_1 and S1_2, Supplementary Material online; P < 0.01).…”

“…Seibold et al (2009) described nonsynonymous SNPs (nsSNPs) that variably endowed isoforms of human AMCase with differential enzymatic activity and showed that a haplotype encoding an isoform of AMCase with heightened enzyme activity was associated with protection from asthma in humans. No detailed knowledge is available regarding genetic and evolutional regulation of chitinolytic activity of AMCase.…”

Evolutionary and biochemical differences between human and monkey acidic mammalian chitinases

“…So far, we have observed a substantial increase in catalytic efficiency of MACase compared to AMCase. In our study, we compared MACase to a variant of human AMCase that is catalytically more active than the originally discovered version (AF290004) and is shown to be associated with protection from asthma (Seibold et al, 2009). It could be possible that macaques needed an even more active enzyme to efficiently remove chitin, and therefore prevent an allergic response (Reese et al, 2007).…”

“…Our version of the gene was slightly different from the variant (accession number AF290004) described in the original publication (Boot et al, 2001). At the protein level it contained N45D, D47N and R61 M substitutions which are known genetic variations (Seibold et al, 2009). The crystal structure of our variant has been solved recently (Olland et al, 2009).…”

“…However, in another study, mice over-expressing AMCase were shown to have a reduced innate immune response caused by chitin (Reese et al, 2007). Recently, an isoform of human AMCase with increased enzymatic activity has been shown to be associated with protection from asthma in humans (Seibold et al, 2009).…”