Abstract
Mammalian chitinase, a chitinolytic enzyme expressed by macrophages, has been detected in atherosclerotic plaques and is elevated in blood and tissues of guinea pigs infected with Aspergillus. Its normal physiological function is unknown. To understand how the enzyme interacts with its substrate, we have characterized the chitin-binding domain. The C-terminal 49 amino acids make up the minimal sequence required for chitin binding activity. The absence of this domain does not affect the ability of the enzyme to hydrolyze the soluble substrate, triacetylchitotriose, but abolishes hydrolysis of insoluble chitin. Within the minimal chitin-binding domain are six cysteines; mutation of any one of these to serine results in complete loss of chitin binding activity. Analysis of purified recombinant chitin-binding domain revealed the presence of three disulfide linkages. The recombinant domain binds specifically to chitin but does not bind chitosan, cellulose, xylan, beta-1, 3-glucan, beta-1,3-1,4-glucan, or mannan. Fluorescently tagged chitin-binding domain was used to demonstrate chitin-specific binding to Saccharomyces cerevisiae, Candida albicans, Mucor rouxii, and Neurospora crassa. These experiments define structural features of the minimal domain of human chitinase required for both specifically binding to and hydrolyzing insoluble chitin and demonstrate relevant binding within the context of the fungal cell wall.
Highlights
Chitin, a -1,4-linked polymer of N-acetylglucosamine, is one of the most abundant biopolymers in nature
One of the most abundant proteins produced by monocyte-derived macrophages, was identified as a protein highly elevated in the serum of patients with Gaucher disease [10]
By creating a series of nested deletions, we found that the C-terminal 49 amino acids are necessary and sufficient for binding
Summary
Generation of Expression Plasmids—A full-length chitinase clone was isolated from a human macrophage cDNA plasmid library [16]. The PCR was used to generate a chitin-binding domain-encoding fragment (sense primer, 5Ј-AAGAGGTACCTTTGGATAAAAGAAGCCCTGGACAAGACACGTTCTGCC; antisense primer, 5Ј-GAGAGCGGCCGCGACTTAATTCCAGGTGCAGCATTTGCAGG), which was digested with Asp718 I and NotI, ligated into an expression cassette, and cloned into pSAC/VEC, a modified version of the disintegration vector pSAC35 [19]. This shuttle vector is composed of a complete two micron plasmid with a LEU2d selectable marker and two repeated sequences flanking the pUC-derived E. coli origin of replication and -lactamase resistance marker. The chitin was pelleted by Chitin-binding Domain of Human Chitinase centrifugation (5 min at 12,000 ϫ g). Autofluorescence was negligible in control cells incubated in the absence of fluorescent reagents
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