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

Abstract

Acidic mammalian chitinase (AMCase) is implicated in asthma, allergic inflammation, and food processing. Little is known about genetic and evolutional regulation of chitinolytic activity of AMCase. Here, we relate human AMCase polymorphisms to the mouse AMCase, and show that the highly active variants encoded by nonsynonymous single-nucleotide polymorphisms (nsSNPs) are consistent with the mouse AMCase sequence. The chitinolytic activity of the recombinant human AMCase was significantly lower than that of the mouse counterpart. By creating mouse-human chimeric AMCase protein we found that the presence of the N-terminal region of human AMCase containing conserved active site residues reduced the enzymatic activity of the molecule. We were able to significantly increase the activity of human AMCase by amino acid substitutions encoded by nsSNPs (N45, D47, and R61) with those conserved in the mouse homologue (D45, N47, and M61). For abolition of the mouse AMCase activity, introduction of M61R mutation was sufficient. M61 is conserved in most of primates other than human and orangutan as well as in other mammals. Orangutan has I61 substitution, which also markedly reduced the activity of the mouse AMCase, indicating that the M61 is a crucial residue for the chitinolytic activity. Altogether, our data suggest that human AMCase has lost its chitinolytic activity by integration of nsSNPs during evolution and that the enzyme can be reactivated by introducing amino acids conserved in the mouse counterpart.

Spontaneous Chitin Accumulation in Airways and Age-Related Fibrotic Lung Disease

“…We observed no difference in AMCase protein levels in BAL fluid between controls and patients with ILD or asthma (Figure 7B and Figure S7B). AMCase activity can vary with common gene polymorphisms (Seibold et al, 2009; Aminuddin et al, 2012; Okawa et al, 2016), but no differences in exo-or endochitinase activity were observed in BAL fluid between controls and patients with ILD; however, notably, human endochitinase activity in all but one patient sample was comparatively lower than in mouse BAL fluid, and in contrast to normal β-N-acetylglucosaminidase activity (Figure 7C), supporting prior studies describing relatively inefficient enzymatic activity among common human AMCase isoforms (Seibold et al, 2009; Goedken et al, 2011; Okawa et al, 2016). Further, as assessed using CBD-reactive material, significantly increased amounts of chitin polymers were present in BAL fluid from patients with ILD as compared to healthy controls (Figure 7D).…”

Mouse acidic mammalian chitinase exhibits transglycosylation activity at somatic tissue pH

“…We expressed and purified Protein A‐AMCase‐V5‐His from the periplasmic fraction of Escherichia coli as described previously . The protein‐containing fractions were desalted using PD MidiTrap G‐25 (GE Healthcare, Milwaukee, WI, USA) equilibrated with TS buffer [20 m m Tris‐HCl (pH: 7.6), 150 m m NaCl and protein inhibitor (Complete Mini; Roche Diagnostics, Basel, Switzerland)].…”

“…Acidic mammalian chitinase has attracted considerable attention due to its altered expression under certain pathological conditions related to immune response, such as in an induced asthma and antigen‐induced allergic lung inflammation mouse models . Some polymorphisms and haplotypes in the AMCase gene are associated with bronchial asthma in humans and inhibition of its activity has been suggested as a therapeutic strategy against asthma . Furthermore, AMCase has been shown to be involved in eye and stomach diseases .…”

“…Acidic mammalian chitinase has attracted considerable attention due to its altered expression under certain pathological conditions related to immune response, such as in an induced asthma and antigeninduced allergic lung inflammation mouse models [12,13]. Some polymorphisms and haplotypes in the AMCase gene are associated with bronchial asthma in humans [14][15][16] and inhibition of its activity has been Abbreviations 4-MU, 4-methylumbelliferyl; 4-NP, 4-nitrophenyl; AMCase, acidic mammalian chitinase; ANTS, fluorophore 8-aminonaphthalene-1,3, 6-trisulphonic acid; Chit1, chitotriosidase; E. coli, Escherichia coli; FACE, fluorophore-assisted carbohydrate electrophoresis; GlcNAc, N-acetyl-D-glucosamine.…”

Acidic mammalian chitinase is a proteases-resistant glycosidase in mouse digestive system

“…AMCase also functions as a critical initiator of protective type 2 responses to intestinal nematodes18. In addition, several genetic variants of AMCase are associated with bronchial asthma in humans19202122.…”