Transglycosidase activity of chitotriosidase - Improved enzymatic assay for the human macrophage chitinase

被引:130
作者
Aguilera, B
Ghauharali-van der Vlugt, K
Helmond, MTJ
Out, JMM
Donker-Koopman, WE
Groener, JEM
Boot, RG
Renkema, GH
van der Marel, GA
van Boom, JH
Overkleeft, HS
Aerts, JMFG
机构
[1] Univ Amsterdam, Acad Med Ctr, Dept Biochem, NL-1105 AZ Amsterdam, Netherlands
[2] Leiden Univ, Leiden Inst Chem, Gorlaeus Labs, NL-2300 RA Leiden, Netherlands
[3] Univ Tampere, Inst Med Technol, FIN-33014 Tampere, Finland
关键词
D O I
10.1074/jbc.M301804200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Chitotriosidase is a chitinase that is massively expressed by lipid-laden tissue macrophages in man. Its enzymatic activity is markedly elevated in serum of patients suffering from lysosomal lipid storage disorders, sarcoidosis, thalassemia, and visceral Leishmaniasis. Monitoring of serum chitotriosidase activity in Gaucher disease patients during progression and therapeutic correction of their disease is useful to obtain insight in changes in body burden on pathological macrophages. However, accurate quantification of chitotriosidase levels by enzyme assay is complicated by apparent substrate inhibition, which prohibits the use of saturating substrate concentrations. We have therefore studied the catalytic features of chitotriosidase in more detail. It is demonstrated that the inhibition of enzyme activity at excess substrate concentration can be fully explained by transglycosylation of substrate molecules. The potential physiological consequences of the ability of chitotriosidase to hydrolyze as well as transglycosylate are discussed. The novel insight in transglycosidase activity of chitotriosidase has led to the design of a new substrate molecule, 4-methylumbelliferyl-(4-deoxy) chitobiose. With this substrate, which is no acceptor for transglycosylation, chitotriosidase shows normal Michaelis-Menten kinetics, resulting in major improvements in sensitivity and reproducibility of enzymatic activity measurements. The novel convenient chitotriosidase enzyme assay should facilitate the accurate monitoring of Gaucher disease patients receiving costly enzyme replacement therapy.
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页码:40911 / 40916
页数:6
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