ABSTRACT
We 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 alpha-mannosidase. Recombinant BjCHI3, without the proline/threonine-rich linker region containing predicted O-glycosylation sites, did not appear to be processed by alpha-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.
Subject(s)
Chitin/metabolism , Chitinases/genetics , Mustard Plant/enzymology , Plant Proteins/genetics , Agglutination , Amino Acid Sequence , Binding Sites/genetics , Catalytic Domain/genetics , Chitinases/chemistry , Chitinases/metabolism , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation, Enzymologic , Glycosylation , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Models, Molecular , Molecular Sequence Data , Mustard Plant/genetics , Mutation , Pichia/genetics , Plant Proteins/metabolism , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
The proteins encoded by the Brassica juncea chitinase gene BjCHI1 and its derived genes BjCHI2 and BjCHI3 were expressed by Multi-copy Pichia expression system. The chitinase activity of FPLC purified BjCHI1, BjCHI2 and BjCHI3 were tested and the results showed that all the three proteins degraded both CM-chitin-RBV and colloidal chitin. The Km values of BjCHI1, BjCHI2 and BjCHI3 for CM-chitin-RBV were estimated as 0.799 mg/mL, 0.544 mg/mL and 0.793 mg/mL, respectively. When the colloidal chitin was used as substrate, the Km values were 0.281 mg/mL, 0.388 mg/mL and 1.643 mg/mL, respectively, indicating chitin-binding domain can increase affinity of chitinase to insoluble substrate. In the agglutination activity assay, only BjCHI1 shows activity when the protein concentration was more than 33 micrograms/mL, while BjCHI2 and BjCHI3 without agglutination activity even when the concentration was increased as high as 800 micrograms/mL. This means that the two chitin-binding domains in BjCHI1 are essential for agglutination and BjCHI1 is the first protein which shows both chitinase and agglutination activity identified so far in plants.
