title: Structural organisation of human bile-salt-activated lipase probed by limited proteolysis and expression of a recombinant truncated variant.
abstract: Bile-salt-activated lipase belongs to the cholinesterase alpha/beta-hydrolase-fold family of proteins. Here, we have investigated the structural organisation of the human isoform by mapping tryptic cleavage sites using limited proteolysis and by expression studies using a recombinant truncated variant. Two accessible regions in the tertiary structure were identified. The first is defined by a tryptic cleavage at Lys429 and lies within the alpha/beta-hydrolase fold in bile-salt-activated lipase between a central beta-sheet and an active-site histidine residue, as deduced from sequence similarity across the cholinesterases and known structural properties. This region exhibits a proteolytic and topological similarity to the lid region in pancreatic lipase. The other accessible region in the tertiary structure is defined by a tryptic cleavage at Arg520 and occurs within a catalytically non-essential segment Leu519-Gln535, as identified by expression of a truncated variant which lacks the C-terminus starting from Leu519. This region is consistent with an interdomain region between the cholinesterase-related part of the protein structure and the unique proline-rich C-terminal repeats. Both protease-sensitive regions appear to occur at domain borders, and, therefore, are consistent with a multi-domain structure. The truncated variant was fully functional as a lipase and as a bile-salt-stimulated esterase. However, compared to the full-length enzyme, the truncated variant showed an increased susceptibility to limited proteolysis, suggesting that the C-terminal repeats may regulate proteolytic degradation of the protein.
authors.affiliation: Biochemistry and Molecular Biology Group, School of Biological Sciences, University of Auckland, New Zealand.
authors.forename: K M
authors.initials: KM
authors.lastname: Loomes
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004795:Enzyme Stability; D006801:Humans; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002787:Sterol Esterase
chemical_list: D018076:DNA, Complementary; D010446:Peptide Fragments; D011994:Recombinant Proteins; C054784:bile salt-stimulated lipase; D002787:Sterol Esterase; D008049:Lipase