Properties of Manduca sexta chitinase and its C-terminal deletions.
Manduca sexta (tobacco hornworm) chitinase is a molting enzyme that contains several domains including a catalytic domain, a serine/threonine-rich region, and a C-terminal cysteine-rich domain. Previously we showed that this chitinase acts as a biopesticide in transgenic plants where it disrupts gut physiology. To delineate the role of these domains further and to identify and characterize some of the multiple forms produced in molting fluid and in transgenic plants, three different forms with variable lengths of C-terminal deletions were generated. Appropriately truncated forms of the M. sexta chitinase cDNA were generated, introduced into a baculovirus vector, and expressed in insect cells. Two of the truncated chitinases (Chi 1-407 and Chi 1-477) were secreted into the medium, whereas the one with the longest deletion (Chi 1-376) was retained inside the insect cells. The two larger truncated chitinases and the full-length enzyme (Chi 1-535) were purified and their properties were compared. Differences in carbohydrate compositions, pH-activity profiles, and kinetic constants were observed among the different forms of chitinases. All three of these chitinases had some affinity for chitin, and they also exhibited differences in their ability to hydrolyze colloidal chitin. The results support the hypothesis that multiple forms of this enzyme occur in vivo due to proteolytic processing at the C-terminal end and differential glycosylation.
Zhu
X
X
Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
Zhang
H
H
Fukamizo
T
T
Muthukrishnan
S
S
Kramer
K J
KJ
D000818:Animals; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018402:Manduca / Q000201:enzymology*; D016296:Mutagenesis; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D013696:Temperature
D021382:Protein Sorting Signals; D002686:Chitin; D002688:Chitinases