Regulation of phenoloxidase activity by high- and low-molecular-weight inhibitors from the larval hemolymph of Manduca sexta.
Insect phenoloxidases (POs) generate quinones and other reactive intermediates to immobilize and kill invading pathogens and parasites. Due to the presumed cytotoxicity of these compounds, PO activity and its proteolytic activation have to be regulated as a local, transient reaction against nonself in order to minimize damage to the host tissues and cells. We identified a Manduca sexta cDNA encoding a polypeptide sequence with its carboxyl-terminal 33 residues similar to the housefly phenoloxidase inhibitor (POI). The recombinant POI, secreted into the Escherichia coli periplasmic space along with its fusion partner DsbC, was released by osmotic shock and isolated by nickel affinity chromatography. Following enterokinase digestion and protein separation, the POI was purified to near homogeneity in a soluble form which inhibited M. sexta PO at a high concentration. We then produced the inhibitor using a modified baculovirus-insect cell system and isolated the glycoprotein from the conditioned medium. Deglycosylation coupled with inhibition assay revealed that O-glycosylation only moderately increased its inhibitory activity. While this led us to speculate the role of Tyr(64) hydroxylation, we were unable to modify the recombinant protein with tyrosine hydroxylase or purify M. sexta POI (Tyr(64)dopa) from the larval plasma. Instead, we isolated a low-M(r), heat-stable compound which strongly inhibited PO. The wavelength of maximum absorbance is 257 nm for the inhibitor. These data suggest that the down-regulation of PO activity in M. sexta is achieved by two mechanisms at least.
Lu
Zhiqiang
Z
Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, USA.
Jiang
Haobo
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006458:Hemolymph / Q000737:chemistry*; D019476:Insect Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007814:Larva / Q000201:enzymology / Q000378:metabolism; D018402:Manduca / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment
D004791:Enzyme Inhibitors; D019476:Insect Proteins; D014442:Monophenol Monooxygenase