MOLECULAR-CLONING, EXPRESSION, AND PARTIAL CHARACTERIZATION OF 2 NOVEL MEMBERS OF THE OVALBUMIN FAMILY OF SERINE PROTEINASE-INHIBITORS

A human placental lambda gt11 cDNA Library was screened for sequences encoding proteins related to human proteinase inhibitor 6 (PIG), and t wo plaques were identified that displayed weak hybridization at high s tringency, Isolation and characterization of the DNA inserts revealed two novel sequences encoding proteins composed of 376 and 374 amino ac ids with predicted molecular masses of similar to 42 kDa, The novel pr oteins displayed all of the structural features unique to the ovalbumi n family of intracellular serpins including the apparent absence of a cleavable N-terminal signal sequence, The degree of amino acid sequenc e identity between the novel serpins and PI6 (63-68%) significantly ex ceeds that of any other combination of known intracellular serpins, Th e two novel serpins encoded by the two novel cDNA sequences have been designated as proteinase inhibitor 8 (PI8) and proteinase inhibitor 9 (PI9), The putative reactive center P-1-P-1' residues for PI8 and PI9 Arg(339)-Cys(340) and Glu(340)-Cys(341), respectively, PI9 appears to be unique in that it is the first human serpin identified with an acid ic residue in the reactive center P, position, In addition, the reacti ve center loop of PI9 exhibits 54% identity with residues found in the reactive center loop of the cowpox virus CrmA serpin. Two PI8 transcr ipts of 1.4 kilobases (kb) and 3.8 kb were detected by Northern analys is in equal and greatest abundance in liver and lung, while the 1,4-kb mRNA was in excess over the 3,8-kb mRNA in skeletal muscle and heart. Two PI9 transcripts of 3.4 and 4.4 kb were detected in equal and grea test abundance in lung and placenta and were weakly detected in all ot her tissues. PI8 and PI9 were expressed in baby hamster kidney and yea st cells, respectively, Immunoblot analyses using rabbit anti-PIG IgG indicated the presence of .PI8 in the cytosolic fraction of stably tra nsfected cells that formed an SDS stable 67-kDa complex with human thr ombin. PI9 was purified to homogeneity from the yeast cell lysate by a combination of heparin agarose chromatography and Mono Q fast protein liquid chromatography and migrated as a single band in SDS-polyacryla mide gel electrophoresis with an apparent molecular mass of 42 kDa, Pu rified recombinant PI9 failed to inhibit the amidolytic activities of trypsin, papain, thrombin, or Staphylococcus aureus endoproteinase Glu -C and did not form an SDS-stable complex when incubated with thrombin . The cognate intracellular proteinases that interact with PI8 and PI9 are unknown.