Kinetic characterization of recombinant human acidic mammalian chitinase.
Identifieur interne : 002D12 ( Main/Exploration ); précédent : 002D11; suivant : 002D13Kinetic characterization of recombinant human acidic mammalian chitinase.
Auteurs : Yi-Te Chou [États-Unis] ; Shihua Yao ; Robert Czerwinski ; Margaret Fleming ; Rustem Krykbaev ; Dejun Xuan ; Huanfang Zhou ; Jonathan Brooks ; Lori Fitz ; James Strand ; Eleonora Presman ; Laura Lin ; Ann Aulabaugh ; Xinyi HuangSource :
- Biochemistry [ 0006-2960 ] ; 2006.
Descripteurs français
- KwdFr :
- MESH :
English descriptors
- KwdEn :
- MESH :
- chemical , metabolism : Chitinases, Recombinant Proteins.
- Animals, CHO Cells, Cricetinae, Cricetulus, Humans, Hydrogen-Ion Concentration, Kinetics, Osmolar Concentration.
Abstract
Human acidic mammalian chitinase (AMCase), a member of the family 18 glycosyl hydrolases, is one of the important proteins involved in Th2-mediated inflammation and has been implicated in asthma and allergic diseases. Inhibition of AMCase results in decreased airway inflammation and airway hyper-responsiveness in a mouse asthma model, suggesting that the AMCase activity is a part of the mechanism of Th2 cytokine-driven inflammatory response in asthma. In this paper, we report the first detailed kinetic characterization of recombinant human AMCase. In contrast with mouse AMCase that has been reported to have a major pH optimum at 2 and a secondary pH optimum around 3-6, human AMCase has only one pH optimum for k(cat)/K(m) between pH 4 and 5. Steady state kinetics shows that human AMCase has "low" intrinsic transglycosidase activity, which leads to the observation of apparent substrate inhibition. This slow transglycosylation may provide a mechanism in vivo for feedback regulation of the chitinase activity of human AMCase. HPLC characterization of cleavage of chitooligosaccharides (4-6-mers) suggests that human AMCase prefers the beta anomer of chitooligosaccharides as substrate. Human AMCase also appears to cleave chitooligosaccharides from the nonreducing end primarily by disaccharide units. Ionic strength modulates the enzymatic activity and substrate cleavage pattern of human AMCase against fluorogenic substrates, chitobiose-4-methylumbelliferyl and chitotriose-4-methylumbelliferyl, and enhances activity against chitooligosaccharides. The physiological implications of these results are discussed.
DOI: 10.1021/bi0525977
PubMed: 16584180
Affiliations:
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USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Chemical and Screening Sciences, Wyeth Research, 500 Arcola Road, Collegeville, Pennsylvania 19426</wicri:regionArea><wicri:noRegion>Pennsylvania 19426</wicri:noRegion></affiliation></author><author><name sortKey="Yao, Shihua" sort="Yao, Shihua" uniqKey="Yao S" first="Shihua" last="Yao">Shihua Yao</name></author><author><name sortKey="Czerwinski, Robert" sort="Czerwinski, Robert" uniqKey="Czerwinski R" first="Robert" last="Czerwinski">Robert Czerwinski</name></author><author><name sortKey="Fleming, Margaret" sort="Fleming, Margaret" uniqKey="Fleming M" first="Margaret" last="Fleming">Margaret Fleming</name></author><author><name sortKey="Krykbaev, Rustem" sort="Krykbaev, Rustem" uniqKey="Krykbaev R" first="Rustem" last="Krykbaev">Rustem Krykbaev</name></author><author><name sortKey="Xuan, Dejun" sort="Xuan, Dejun" uniqKey="Xuan D" first="Dejun" last="Xuan">Dejun Xuan</name></author><author><name sortKey="Zhou, Huanfang" sort="Zhou, Huanfang" uniqKey="Zhou H" first="Huanfang" last="Zhou">Huanfang Zhou</name></author><author><name sortKey="Brooks, Jonathan" sort="Brooks, Jonathan" uniqKey="Brooks J" first="Jonathan" last="Brooks">Jonathan Brooks</name></author><author><name sortKey="Fitz, Lori" sort="Fitz, Lori" uniqKey="Fitz L" first="Lori" last="Fitz">Lori Fitz</name></author><author><name sortKey="Strand, James" sort="Strand, James" uniqKey="Strand J" first="James" last="Strand">James Strand</name></author><author><name sortKey="Presman, Eleonora" sort="Presman, Eleonora" uniqKey="Presman E" first="Eleonora" last="Presman">Eleonora Presman</name></author><author><name sortKey="Lin, Laura" sort="Lin, Laura" uniqKey="Lin L" first="Laura" last="Lin">Laura Lin</name></author><author><name sortKey="Aulabaugh, Ann" sort="Aulabaugh, Ann" uniqKey="Aulabaugh A" first="Ann" last="Aulabaugh">Ann Aulabaugh</name></author><author><name sortKey="Huang, Xinyi" sort="Huang, Xinyi" uniqKey="Huang X" first="Xinyi" last="Huang">Xinyi Huang</name></author></analytic><series><title level="j">Biochemistry</title><idno type="ISSN">0006-2960</idno><imprint><date when="2006" type="published">2006</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>CHO Cells</term><term>Chitinases (metabolism)</term><term>Cricetinae</term><term>Cricetulus</term><term>Humans</term><term>Hydrogen-Ion Concentration</term><term>Kinetics</term><term>Osmolar Concentration</term><term>Recombinant Proteins (metabolism)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Animaux</term><term>Cellules CHO</term><term>Chitinase (métabolisme)</term><term>Cinétique</term><term>Concentration en ions d'hydrogène</term><term>Concentration osmolaire</term><term>Cricetinae</term><term>Cricetulus</term><term>Humains</term><term>Protéines recombinantes (métabolisme)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Chitinases</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Chitinase</term><term>Protéines recombinantes</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>CHO Cells</term><term>Cricetinae</term><term>Cricetulus</term><term>Humans</term><term>Hydrogen-Ion Concentration</term><term>Kinetics</term><term>Osmolar Concentration</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Cellules CHO</term><term>Cinétique</term><term>Concentration en ions d'hydrogène</term><term>Concentration osmolaire</term><term>Cricetinae</term><term>Cricetulus</term><term>Humains</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Human acidic mammalian chitinase (AMCase), a member of the family 18 glycosyl hydrolases, is one of the important proteins involved in Th2-mediated inflammation and has been implicated in asthma and allergic diseases. Inhibition of AMCase results in decreased airway inflammation and airway hyper-responsiveness in a mouse asthma model, suggesting that the AMCase activity is a part of the mechanism of Th2 cytokine-driven inflammatory response in asthma. In this paper, we report the first detailed kinetic characterization of recombinant human AMCase. In contrast with mouse AMCase that has been reported to have a major pH optimum at 2 and a secondary pH optimum around 3-6, human AMCase has only one pH optimum for k(cat)/K(m) between pH 4 and 5. Steady state kinetics shows that human AMCase has "low" intrinsic transglycosidase activity, which leads to the observation of apparent substrate inhibition. This slow transglycosylation may provide a mechanism in vivo for feedback regulation of the chitinase activity of human AMCase. HPLC characterization of cleavage of chitooligosaccharides (4-6-mers) suggests that human AMCase prefers the beta anomer of chitooligosaccharides as substrate. Human AMCase also appears to cleave chitooligosaccharides from the nonreducing end primarily by disaccharide units. Ionic strength modulates the enzymatic activity and substrate cleavage pattern of human AMCase against fluorogenic substrates, chitobiose-4-methylumbelliferyl and chitotriose-4-methylumbelliferyl, and enhances activity against chitooligosaccharides. The physiological implications of these results are discussed.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country></list><tree><noCountry><name sortKey="Aulabaugh, Ann" sort="Aulabaugh, Ann" uniqKey="Aulabaugh A" first="Ann" last="Aulabaugh">Ann Aulabaugh</name><name sortKey="Brooks, Jonathan" sort="Brooks, Jonathan" uniqKey="Brooks J" first="Jonathan" last="Brooks">Jonathan Brooks</name><name sortKey="Czerwinski, Robert" sort="Czerwinski, Robert" uniqKey="Czerwinski R" first="Robert" last="Czerwinski">Robert Czerwinski</name><name sortKey="Fitz, Lori" sort="Fitz, Lori" uniqKey="Fitz L" first="Lori" last="Fitz">Lori Fitz</name><name sortKey="Fleming, Margaret" sort="Fleming, Margaret" uniqKey="Fleming M" first="Margaret" last="Fleming">Margaret Fleming</name><name sortKey="Huang, Xinyi" sort="Huang, Xinyi" uniqKey="Huang X" first="Xinyi" last="Huang">Xinyi Huang</name><name sortKey="Krykbaev, Rustem" sort="Krykbaev, Rustem" uniqKey="Krykbaev R" first="Rustem" last="Krykbaev">Rustem Krykbaev</name><name sortKey="Lin, Laura" sort="Lin, Laura" uniqKey="Lin L" first="Laura" last="Lin">Laura Lin</name><name sortKey="Presman, Eleonora" sort="Presman, Eleonora" uniqKey="Presman E" first="Eleonora" last="Presman">Eleonora Presman</name><name sortKey="Strand, James" sort="Strand, James" uniqKey="Strand J" first="James" last="Strand">James Strand</name><name sortKey="Xuan, Dejun" sort="Xuan, Dejun" uniqKey="Xuan D" first="Dejun" last="Xuan">Dejun Xuan</name><name sortKey="Yao, Shihua" sort="Yao, Shihua" uniqKey="Yao S" first="Shihua" last="Yao">Shihua Yao</name><name sortKey="Zhou, Huanfang" sort="Zhou, Huanfang" uniqKey="Zhou H" first="Huanfang" last="Zhou">Huanfang Zhou</name></noCountry><country name="États-Unis"><noRegion><name sortKey="Chou, Yi Te" sort="Chou, Yi Te" uniqKey="Chou Y" first="Yi-Te" last="Chou">Yi-Te Chou</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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