Carbohydrate binding specificity of the recombinant chitin-binding domain of human macrophage chitinase.
The chitin-binding domain of human macrophage chitinase was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and assayed for its binding activity. The purified recombinant chitin-binding domain bound to chitin, but not to glucan, xylan, or mannan. The binding of the recombinant chitin-binding domain to chitin was inhibited by N-acetylglucosamine, di-N-acetylchitobiose, and hyaluronan, but not by N-acetylgalactosamine or chondroitin. Furthermore, a solid-phase binding assay showed that the recombinant domain interacts specifically with hyaluronan and hybrid-type N-linked oligosaccharide chains on glycoproteins, and that the oligosaccharide-binding characteristics are similar to those of wheat germ agglutinin, a lectin that binds to chitin. The results suggest that human chitinase chitin-binding domain may be involved in tissue remodeling through binding to polysaccharides or extracellular matrix glycoproteins, and this recombinant protein can be used to elucidate biological functions of the enzyme.
Ujita
Minoru
M
Laboratory of Biological Chemistry, Department of Applied Biological Chemistry, Faculty of Agriculture, Meijo University, Nagoya, Japan. ujita@ccmfs.meijo-u.ac.jp
Sakai
Kaori
K
Hamazaki
Keishi
K
Yoneda
Masahiko
M
Isomura
Shigeki
S
Hara
Akira
A
D001483:Base Sequence; D001665:Binding Sites; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005982:Glutathione Transferase / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008264:Macrophages / Q000201:enzymology*; D009844:Oligosaccharides / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D002686:Chitin; D005982:Glutathione Transferase; D002688:Chitinases