Manduca sexta serpin-4 and serpin-5 inhibit the prophenol oxidase activation pathway: cDNA cloning, protein expression, and characterization.
Infection stimulates the innate immune responses of insects, including activation of prophenol oxidase (pro-PO) in plasma as the last step of a serine protease cascade. To investigate the roles of protease inhibitors in regulating this pathway, we cloned cDNAs for two new serpins (serpin-4 and serpin-5) from the tobacco hornworm, Manduca sexta. Serpin-4 and serpin-5 mRNAs are constitutively expressed at a low level in larval hemocytes and fat body and increased dramatically upon bacterial challenge. These serpins are present in larval plasma at approximately 3 (serpin-4) and approximately 1 mug/ml (serpin-5) and increased 3-8-fold by 24 h after injection of bacteria or fungi. Recombinant serpin-4 and serpin-5 decreased pro-PO activation when added to plasma, but they did not directly inhibit the pro-PO-activating proteases. Instead, they apparently regulate the pathway by inhibiting one or more target proteases upstream of the pro-PO-activating proteases.
Tong
Youren
Y
Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
Kanost
Michael R
MR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000502:physiology*; D004156:Catechol Oxidase / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D016147:Genes, Tumor Suppressor / Q000502:physiology*; D015151:Immunoblotting; D007313:Insecta; D007814:Larva / Q000502:physiology; D018402:Manduca / Q000378:metabolism*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015843:Serpins / Q000502:physiology*; D012965:Sodium Chloride / Q000737:chemistry; D013997:Time Factors
D002352:Carrier Proteins; D018076:DNA, Complementary; D004792:Enzyme Precursors; D012333:RNA, Messenger; D011994:Recombinant Proteins; C085344:SERPIN-B5; D015843:Serpins; C051324:kallistatin; D012965:Sodium Chloride; C038451:pro-phenoloxidase; D004156:Catechol Oxidase