SUBUNIT COMPOSITION OF PRO-PHENOL OXIDASE FROM MANDUCA-SEXTA - MOLECULAR-CLONING OF SUBUNIT PROPO-P1

Phenol oxidase (PO) is known to play an important role in defense mech anisms in insect immunity. It is present as a zymogen in insect hemoly mph, and can be activated by a specific proteolytic reaction that is s timulated by microbial cell wall components. The pro-phenol oxidase (p ro-PO) purified from the larval hemolymph of Manduca sexta contains tw o polypeptides in equal amounts as revealed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). A cDNA for one of the p olypeptides, now designated proPO-p2, has been isolated (Hall et al. ( 1995) Proc. Natl. Acad. Sci. USA, 92, 7764-7768). We purified pro-PO f rom plasma of M. sexta and characterized its subunit composition. A cD NA for M. sexta proPO-p1 was isolated from a larval hemocyte cDNA libr ary. M. sexta proPO-p1 is 78% identical in amino acid sequence to Bomb yx mori proPO-p1, but only 50% to M. sexta or B. mori proPO-p2. Immuno fluorescence labelling and in situ hybridization showed that the pro-P O is synthesized in a single hemocyte type, the oenocytoids. Analysis of pro-PO by size exclusion high-pressure liquid chromatography (HPLC) revealed that pro-PO exists as monomeric, dimeric, trimeric or multim eric structures depending on the ionic strength. All of these isoforms of the protein have phenol oxidase activity upon activation with a de tergent, cetylpyridinium chloride. In analysis by non-denaturing PAGE, the majority of the purified pro-PO was present as two dimers of dist inct mobility (fast and slow forms). Both forms contain proPO-p1 and p roPO-p2, suggesting that they are heterodimers. Individual larvae can contain the slow form, the fast form, or both, which suggests that the slow and fast forms of proPO are allelic variants. These results indi cate that there are two pro-PO genes in M. sexta, which are coordinate ly expressed in oenocytoids, and whose products form predominantly het erodimers in plasma. (C) 1997 Elsevier Science Ltd. All rights reserve d.