Hyperactivation of human acidic chitinase (Chia) for potential medical use

Abstract

Accumulation of environmental chitin in the lungs can lead to pulmonary fibrosis, characterized by inflammatory infiltration and fibrosis in acidic chitinase (Chia)-deficient mice. Transgenic expression of Chia in these mice ameliorated the symptoms, indicating the potential of enzyme supplementation as a promising therapeutic strategy for related lung diseases. This study focuses on utilizing hyperactivated human Chia, which exhibits low activity. We achieved significant activation of human Chia by incorporating nine amino acids derived from the crab-eating monkey (Macaca fascicularis) Chia, known for its robust chitin-degrading activity. The modified human Chia retained high activity across a broad pH spectrum and exhibited enhanced thermal stability. The amino acid substitutions associated with hyperactivation of human Chia activity occurred species specifically in monkey Chia. This discovery highlights the potential of hyperactivated Chia in treating pulmonary diseases resulting from chitin accumulation in human lungs.

Keywords: acidic chitinase (Chia); amino acid substitutions; chitin; enzyme engineering; evolution; exon swapping; hyperactivation; primate lineage; treating pulmonary diseases.

Figure 1

Hyperactivation of human Chia by substituting multiple exons from monkey Chia.A, schematic representation of Escherichia coli–expressed human WT and R61M Chia, monkey Chia, and chimeric Chia fusion proteins (C1–C6). Amino acid sequences are color-coded: pink for monkey Chia and blue for human R61M Chia. B, chitinolytic activities of human WT and R61M Chia, monkey Chia, and chimeric Chia fusion proteins (C1–C6). Monkey Chia activity at pH 5.0 was set as 100% and depicted as relative activity. Error bars represent the mean ± SD from a triplicate experiment. Chia, acidic chitinase.

Figure 2

Hyperactivation of human Chia by substituting nine amino acids from monkey Chia.A, schematic representation of the Chia chimeric protein. Left: Chia chimera with monkey-derived amino acid sequences substituted in exons 3 to 11 of the Chia coding region. Right: A mutant in which monkey-derived amino acid substitutions were introduced into exons 6 and 8 of human Chia. Amino acids from human or monkey Chia are shown in blue and pink, respectively. B, chitinolytic activities of Chia chimeric proteins. Monkey Chia activity at pH 5.0 was set as 100% and shown as relative activity. Error bars represent the mean ± SD from a triplicate experiment. Chia, acidic chitinase.

Figure 3

Enzymatic properties of M-9, a hyperactivated human Chia mutant, under various conditions.A, optimal pH for M-9 compared with monkey and human Chia R61M. Chitinolytic activities were measured at pH 1.0 to pH 8.0. Monkey Chia activity at pH 5.0 was set as 100% and shown as relative activity. Values represent the average of three measurements. Error bars indicate the mean ± SD from a triplicate experiment. B, temperature dependence of M-9 compared with monkey and human Chia R61M. Temperature profiles from 30 °C to 70 °C for each Chia. Monkey Chia activity was set as 100% and shown as a relative activity. Reaction conditions are color-coded: red for pH 2.0, green for pH 5.0, blue for pH 7.0. C, pH stability of M-9 and parental Chia proteins at 37 °C. The stability of each Chia protein was assessed by pre-incubating them at different pH levels (ranging from pH 1.0 to pH 8.0) for a specified duration. The chitinase activity of each Chia protein after preincubation was measured and expressed as a percentage relative to its maximum activity under optimal conditions. Monkey Chia activity at its optimal pH was set as 100%. Error bars represent the mean ± SD from triplicate experiments. D, thermal stability from 30 °C to 70 °C for each Chia. The maximum activity of each Chia was set as 100% and shown as relative activity. Reaction conditions are color-coded: green for pH 5.0 and blue for pH 7.0. E, chitin degradation activity of hyperactivated Chia for high-molecular-weight chitin. Left: comparison of chitinase activity of each mammalian chitinase on colloidal chitin under pH 2.0, 5.0, and 7.0 conditions. Degradation products were analyzed using the FACE method. Right: quantification data for (GlcNAc)₂. Reaction conditions are color-coded: pink for pH 2.0, yellow for pH 5.0,and blue for pH 7.0. Values are shown relative to the maximum amount of monkey Chia degradation products (pH 2.0) set at 100%. FACE, fluorophore-assisted carbohydrate electrophoresis; Chia, acidic chitinase.

Figure 4

Identification of critical amino acid substitutions for hyperactivation of human Chia.A, sequence diagram illustrating the nine amino acid substitutions involved in hyperactivation. Left: human Chia mutant with single amino acid substitutions among the nine residues associated with hyperactivation. Right: mutant, where one amino acid residue out of the nine involved in hyperactivation is reverted to the human type. B, chitinolytic activities of human R61M Chia and single amino acid substitution human Chia mutants, measured after incubation for 30 min at 37 °C in McIlvaine’s buffer. C, chitinolytic activities of M-9 and eight amino acid substitution human Chia mutants, measured under the same conditions. Monkey Chia activity at pH 5.0 was set as 100% and shown as relative activity. Error bars represent mean ± SD from triplicate experiments. Chia, acidic chitinase.

Figure 5

I173S and V246A are key amino acid substitutions governing the hyperactivation of human Chia.A, sequence diagram showing the nine amino acid substitutions involved in hyperactivation of the generated mutant. B, chitinolytic activities of human R61M, M-2, M-4, M-6, and M-9 Chia proteins, measured after incubation for 30 min at 37 °C in McIlvaine’s buffer. Monkey Chia activity at pH 5.0 was set as 100% and shown as relative activity. Chia, acidic chitinase.

Figure 6

Hyperactivation of human Chia is attributed to amino acids with high activity in monkey Chia.A, sequence diagram comparing the amino acid sequences of exons 6 and 8 of primate Chia proteins. B, conceptual diagram illustrating the divergence of ancestral genes of human and monkey Chia into humans and monkeys. Monkey Chia acquired high chitinase activity during the evolutionary process, with the amino acids responsible for it hyperactivating human Chia. Chia, acidic chitinase.