Structural and functional definition of the human chitinase chitin-binding domain.
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.
Tjoelker
L W
LW
ICOS Corp., Bothell, Washington 98021, USA. ltjoelker@icos.com
Gosting
L
L
Frey
S
S
Hunter
C L
CL
Trong
H L
HL
Steiner
B
B
Brammer
H
H
Gray
P W
PW
D000595:Amino Acid Sequence; D001665:Binding Sites; D002176:Candida albicans / Q000737:chemistry / Q000648:ultrastructure; D002473:Cell Wall / Q000737:chemistry / Q000648:ultrastructure; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D005658:Fungi; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009089:Mucor / Q000737:chemistry / Q000648:ultrastructure; D017354:Point Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014312:Trisaccharides / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D014312:Trisaccharides; D002686:Chitin; C041161:chitotriose; D002688:Chitinases; D003545:Cysteine