title: Essential binding and functional domains of human bleomycin hydrolase.
abstract: Bleomycin hydrolase (BH) is unusual among cysteine proteinases because it appears to form multihomomeric structures, inactivates the antitumor glycopeptide bleomycin, and contains a unique C-terminal amino acid sequence. We now demonstrate intrinsic endopeptidase activity associated with human BH (hBH) using artificial substrates and intracellular dimerization of hBH using a yeast two-hybrid assay. To determine domains important for homomeric interactions and catalysis, we constructed N- and C-terminal deletion mutants and identified an N-terminal region (hBH1-82) that interacted with two nonoverlaping hBH domains: one near the N-terminus (hBH14-103) and another neighboring the C-terminus (hBH358-455). In vitro hBH aggregated with a molecular mass of 235 kD corresponding to a homotetramer and the C-terminus was critical for this oligomerization since no tetramers were found when the last 40 amino acids were deleted. The penultimate 8 amino acids, which constitute a unique and highly conserved bleomycin hydrolase-like domain (BHYD), were essential for BH and aminopeptidase activity but not for endopeptidase activity or oligomer formation. Thus, the C-terminus of hBH has two independent roles controlling both the catalytic activity and oligomerization of hBH.
authors.affiliation: Department of Pharmacology, University of Pittsburgh, School of Medicine, and University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: R P
authors.initials: RP
authors.lastname: Koldamova
authors.forename: I M
authors.initials: IM
authors.lastname: Lefterov
authors.forename: V G
authors.initials: VG
authors.lastname: Gadjeva
authors.forename: J S
authors.initials: JS
authors.lastname: Lazo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001761:Bleomycin; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D001761:Bleomycin; D003546:Cysteine Endopeptidases; C031262:bleomycin hydrolase