Interaction of beta-1,3-glucan with its recognition protein activates hemolymph proteinase 14, an initiation enzyme of the prophenoloxidase activation system in Manduca sexta.
A serine proteinase pathway in insect hemolymph leads to prophenoloxidase activation, an innate immune response against pathogen infection. In the tobacco hornworm Manduca sexta, recombinant hemolymph proteinase 14 precursor (pro-HP14) interacts with peptidoglycan, autoactivates, and initiates the proteinase cascade (Ji, C., Wang, Y., Guo, X., Hartson, S., and Jiang, H. (2004) J. Biol. Chem. 279, 34101-34106). Here, we report the purification and characterization of pro-HP14 from the hemolymph of bacteria-injected M. sexta larvae. The zymogen, consisting of a single polypeptide with a molecular mass of 68.5 kDa, is truncated at the amino terminus. It is converted to a two-chain active form in the presence of beta-1,3-glucan (a fungal cell wall component) and beta-1,3-glucan recognition protein-2. The 45-kDa heavy chain contains four low-density lipoprotein receptor A repeats, one Sushi domain, and one unique cysteine-rich region, whereas the 30-kDa light chain contains a serine proteinase domain, which was labeled by [(3)H]diisopropyl fluorophosphate. Pro-HP14 in the plasma strongly binds curdlan, zymosan, and yeast and interacts with peptidoglycan and Micrococcus luteus. Addition of autoactivated HP14 elevated phenoloxidase activity level in the larval plasma. Recombinant M. sexta serpin-1I reduced prophenoloxidase activation by inhibiting HP14. These data are consistent with the current model on initiation and regulation of the prophenoloxidase activation cascade upon recognition of pathogen-associated molecular patterns by specific pattern recognition proteins.
Wang
Yang
Y
Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
Jiang
Haobo
H
D000818:Animals; D004156:Catechol Oxidase / Q000378:metabolism; D002473:Cell Wall; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D005656:Fungal Proteins / Q000502:physiology; D006094:Gram-Positive Bacteria / Q000472:pathogenicity; D006458:Hemolymph / Q000502:physiology; D007113:Immunity, Innate; D019476:Insect Proteins / Q000378:metabolism*; D018402:Manduca / Q000201:enzymology* / Q000276:immunology*; D015843:Serpins / Q000502:physiology; D047071:beta-Glucans / Q000378:metabolism*
D004792:Enzyme Precursors; D005656:Fungal Proteins; D019476:Insect Proteins; D015843:Serpins; D047071:beta-Glucans; C033363:beta-1,3-glucan; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D010450:Endopeptidases; C490363:hemolymph proteinase 14, Manduca sexta