title: Recombinant and native zymogen forms of human complement factor D.
abstract: We have expressed a full-length cDNA clone encoding human factor D by using a baculovirus expression system. The purified recombinant protein reacted with Ab against native factor D, but was hemolytically inactive and slightly larger than factor D. These results suggested that the recombinant protein was the elusive zymogen of factor D. Amino acid sequencing demonstrated that the recombinant factor D consisted of two proenzyme forms with respective activation peptides, AAPPRGR and APPRGR. Catalytic amounts of trypsin converted recombinant profactor D to its enzymatically active form, exhibiting SDS-PAGE mobility and specific hemolytic activity similar to those of native factor D. About 90% of trypsin-activated recombinant profactor D had the same NH2-terminus as factor D. Human thrombin, kallikrein, and plasmin could also activate recombinant profactor D, but relatively high concentrations of these enzymes were required and the specific hemolytic activity of the "activated" profactor D was about one-third that of native factor D. Trypsin-activatable profactor D was also purified from the urine of a patient with Fanconi's syndrome. This native profactor D represented less than 1.0% of the total antigenic factor D in the patient's urine and had a Gly-Arg dipeptide as the activation peptide. Apparently, urine profactor D was produced by cleavage of pre-profactor D at Arg-(-3) by a serine protease with trypsin-like specificity, which probably is different from the putative leader peptidase that produces the recombinant profactor D. Urine profactor D was inhibited by diisopropyl fluorophosphate although the recombinant proenzyme was resistant to this inhibitor.
authors.affiliation: Department of Medicine, University of Alabama at Birmingham 35294.
authors.forename: Y
authors.initials: Y
authors.lastname: Yamauchi
authors.forename: J W
authors.initials: JW
authors.lastname: Stevens
authors.forename: K J
authors.initials: KJ
authors.lastname: Macon
authors.forename: J E
authors.initials: JE
authors.lastname: Volanakis
mesh_terms: D000595:Amino Acid Sequence; D011416:Complement Factor D / Q000737:chemistry* / Q000378:metabolism / Q000652:urine; D010450:Endopeptidases / Q000494:pharmacology; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry; D006461:Hemolysis; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D004792:Enzyme Precursors; D011994:Recombinant Proteins; D010450:Endopeptidases; C493488:CFD protein, human; D011416:Complement Factor D