title: Expression and characterization of cysteine-modified variants of an amino-terminal fragment of bactericidal/permeability-increasing protein.
abstract: rBPI23 is a biologically active, recombinant N-terminal fragment of human bactericidal/permeability-increasing protein (BPI). While rBPI23 is readily purified from culture supernatants of Chinese hamster ovary (CHO)-K1 transfectants, it is heterogeneous, consisting of monomer and disulfide-linked dimer, characteristics due presumably to the presence of three cysteines within the molecule. We have examined the role of these cysteines in rBPI23 expression, function, and dimer formation by mutating their codons to alanine (C132A), serine (C135S), or alanine (C175A) and expressing analogues of N-terminal fragments ("variants") lacking one, two, or all three cysteines in permanently transfected CHO-K1 cells. We also expressed a variant in which serine 18 was changed to cysteine (S18C), as found in both bovine and rabbit BPI. The C132A variant was readily secreted and purified as a homogeneous, stable monomeric protein species. The C135S and S18C variants were produced as mixtures of monomer and dimer; the C135S variant was poorly secreted, difficult to purify, and unstable on storage. In contrast, the C175A variant and those lacking any two or all three cysteines were expressed but not secreted. Purified rBPI23 and the C132A and S18C variants had comparable bactericidal and lipopolysaccharide (LPS) binding activities and were similarly effective at neutralizing LPS-induced tumor necrosis factor synthesis by THP-1 cells; the purified C135S variant lacked all activities. From these studies with CHO-K1 transfectants, we conclude that (i) cysteines 135 and 175 are both necessary for efficient secretion of a biologically active N-terminal BPI fragment, presumably through the formation of a disulfide bond, (ii) cysteine 132 is responsible for dimer formation, and (iii) only the C132A modification yields a stable, biologically active, N-terminal BPI fragment (designated rBPI21) that is free of dimeric species.
authors.affiliation: XOMA Corporation, 1545 17th Street, Santa Monica, California, 90404, USA.
authors.forename: A H
authors.initials: AH
authors.lastname: Horwitz
authors.forename: S D
authors.initials: SD
authors.lastname: Leigh
authors.forename: S
authors.initials: S
authors.lastname: Abrahamson
authors.forename: H
authors.initials: H
authors.lastname: Gazzano-Santoro
authors.forename: P S
authors.initials: PS
authors.lastname: Liu
authors.forename: R E
authors.initials: RE
authors.lastname: Williams
authors.forename: S F
authors.initials: SF
authors.lastname: Carroll
authors.forename: G
authors.initials: G
authors.lastname: Theofan
mesh_terms: D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides; D001667:Binding, Competitive; D001798:Blood Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D015152:Blotting, Northern; D015153:Blotting, Western; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism; D006224:Cricetinae; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008565:Membrane Proteins; D018832:Molecular Chaperones / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins; D002352:Carrier Proteins; D004220:Disulfides; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D014409:Tumor Necrosis Factor-alpha; C021623:bactericidal permeability increasing protein; D003545:Cysteine