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Article: Functional analyses of the chitin-binding domains and the catalytic domain of Brassica juncea chitinase BjCHI1

TitleFunctional analyses of the chitin-binding domains and the catalytic domain of Brassica juncea chitinase BjCHI1
Authors
KeywordsAgglutination
Glycoside hydrolases
Homology modeling
Lectin
Pathogenesis-related protein
Site-directed mutagenesis
Issue Date2004
PublisherSpringer Verlag Dordrecht. The Journal's web site is located at http://springerlink.metapress.com/openurl.asp?genre=journal&issn=0167-4412
Citation
Plant Molecular Biology, 2004, v. 56 n. 2, p. 285-298 How to Cite?
AbstractWe previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains. Synthesis of its mRNA is induced by wounding, methyl jasmonate treatment, Aspergillus niger infection and caterpillar (Pieris rapae) feeding, suggesting that the protein has a role in defense. In that it possesses two chitin-binding domains, BjCHI1 resembles the precursor of Urtica dioica agglutinin but unlike that protein, BjCHI1 retains its chitinase catalytic domain after post-translational processing. To explore the properties of multi-domain BjCHI1, we have expressed recombinant BjCHI1 and two derivatives, which lack one (BjCHI2) or both (BjCHI3) chitin-binding domains, as secreted proteins in Pichia pastoris. Recombinant BjCHI1 and BjCHI2 showed apparent molecular masses on SDS-PAGE larger than calculated, and could be deglycosylated using α-mannosidase. Recombinant BjCHI3, without the proline/ threonine-rich linker region containing predicted O-glycosylation sites, did not appear to be processed by α-mannosidase. BjCHI1's ability to agglutinate rabbit erythrocytes is unique among known chitinases. Both chitin-binding domains are essential for agglutination; this property is absent in recombinant BjCHI2 and BjCHI3. To identify potential catalytic residues, we generated site-directed mutations in recombinant BjCHI3. Mutation E212A showed the largest effect, exhibiting 0% of wild-type specific activity. H211N and R361A resulted in considerable (>91%) activity loss, implying these charged residues are also important in catalysis. E234A showed 36% retention of activity and substitution Y269D, 50%. The least affected mutants were E349A and D360A, with 73% and 68% retention, respectively. Like Y269, E349 and D360 are possibly involved in substrate binding rather than catalysis.
Persistent Identifierhttp://hdl.handle.net/10722/68464
ISSN
2015 Impact Factor: 3.905
2015 SCImago Journal Rankings: 1.915
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorTang, CMen_HK
dc.contributor.authorChye, MLen_HK
dc.contributor.authorRamalingam, Sen_HK
dc.contributor.authorOuyang, SWen_HK
dc.contributor.authorZhao, KJen_HK
dc.contributor.authorUbhayasekera, Wen_HK
dc.contributor.authorMowbray, SLen_HK
dc.date.accessioned2010-09-06T06:04:50Z-
dc.date.available2010-09-06T06:04:50Z-
dc.date.issued2004en_HK
dc.identifier.citationPlant Molecular Biology, 2004, v. 56 n. 2, p. 285-298en_HK
dc.identifier.issn0167-4412en_HK
dc.identifier.urihttp://hdl.handle.net/10722/68464-
dc.description.abstractWe previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains. Synthesis of its mRNA is induced by wounding, methyl jasmonate treatment, Aspergillus niger infection and caterpillar (Pieris rapae) feeding, suggesting that the protein has a role in defense. In that it possesses two chitin-binding domains, BjCHI1 resembles the precursor of Urtica dioica agglutinin but unlike that protein, BjCHI1 retains its chitinase catalytic domain after post-translational processing. To explore the properties of multi-domain BjCHI1, we have expressed recombinant BjCHI1 and two derivatives, which lack one (BjCHI2) or both (BjCHI3) chitin-binding domains, as secreted proteins in Pichia pastoris. Recombinant BjCHI1 and BjCHI2 showed apparent molecular masses on SDS-PAGE larger than calculated, and could be deglycosylated using α-mannosidase. Recombinant BjCHI3, without the proline/ threonine-rich linker region containing predicted O-glycosylation sites, did not appear to be processed by α-mannosidase. BjCHI1's ability to agglutinate rabbit erythrocytes is unique among known chitinases. Both chitin-binding domains are essential for agglutination; this property is absent in recombinant BjCHI2 and BjCHI3. To identify potential catalytic residues, we generated site-directed mutations in recombinant BjCHI3. Mutation E212A showed the largest effect, exhibiting 0% of wild-type specific activity. H211N and R361A resulted in considerable (>91%) activity loss, implying these charged residues are also important in catalysis. E234A showed 36% retention of activity and substitution Y269D, 50%. The least affected mutants were E349A and D360A, with 73% and 68% retention, respectively. Like Y269, E349 and D360 are possibly involved in substrate binding rather than catalysis.en_HK
dc.languageengen_HK
dc.publisherSpringer Verlag Dordrecht. The Journal's web site is located at http://springerlink.metapress.com/openurl.asp?genre=journal&issn=0167-4412en_HK
dc.relation.ispartofPlant Molecular Biologyen_HK
dc.subjectAgglutinationen_HK
dc.subjectGlycoside hydrolasesen_HK
dc.subjectHomology modelingen_HK
dc.subjectLectinen_HK
dc.subjectPathogenesis-related proteinen_HK
dc.subjectSite-directed mutagenesisen_HK
dc.titleFunctional analyses of the chitin-binding domains and the catalytic domain of Brassica juncea chitinase BjCHI1en_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0167-4412&volume=56&spage=285&epage=298&date=2004&atitle=Functional+analysis+of+the+chitin-binding+domains+and+the+catalytic+domain+of+Brassica+juncea+chitinase+BjCHI1en_HK
dc.identifier.emailChye, ML: mlchye@hkucc.hku.hken_HK
dc.identifier.authorityChye, ML=rp00687en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1007/s11103-004-3382-1en_HK
dc.identifier.pmid15604744-
dc.identifier.scopuseid_2-s2.0-12444331392en_HK
dc.identifier.hkuros96634en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-12444331392&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume56en_HK
dc.identifier.issue2en_HK
dc.identifier.spage285en_HK
dc.identifier.epage298en_HK
dc.identifier.isiWOS:000225900500010-
dc.publisher.placeNetherlandsen_HK
dc.identifier.scopusauthoridTang, CM=16640286200en_HK
dc.identifier.scopusauthoridChye, ML=7003905460en_HK
dc.identifier.scopusauthoridRamalingam, S=8709830400en_HK
dc.identifier.scopusauthoridOuyang, SW=7005811045en_HK
dc.identifier.scopusauthoridZhao, KJ=7202071954en_HK
dc.identifier.scopusauthoridUbhayasekera, W=6506474701en_HK
dc.identifier.scopusauthoridMowbray, SL=7004344618en_HK
dc.identifier.citeulike4040152-

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