Essential binding and functional domains of human bleomycin hydrolase.
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.
Koldamova
R P
RP
Department of Pharmacology, University of Pittsburgh, School of Medicine, and University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15261, USA.
Lefterov
I M
IM
Gadjeva
V G
VG
Lazo
J S
JS
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
D017931:DNA Primers; D011994:Recombinant Proteins; D001761:Bleomycin; D003546:Cysteine Endopeptidases; C031262:bleomycin hydrolase