Identification and functional requirement of Cu(I) and its ligands within coagulation factor VIII.
Coagulation factor VIII (FVIII) is a heterodimer consisting of a light chain of 80 kDa (domains A3-C1-C2) in a metal ion-dependent association with a 220-kDa heavy chain (domains A1-A2-B). The nature of the metal ion-dependent association between the heavy and light chains was investigated using atomic absorption spectroscopy, electron paramagnetic resonance spectroscopy (EPR), and site-directed mutagenesis and expression of the FVIII cDNA. Whereas copper ion was not detected in intact recombinant FVIII, EDTA dissociation of the chains yielded an EPR signal consistent with 1 mol of Cu(I)/mol of active protein, supporting the hypothesis that a single molecule of reduced copper ion is buried within intact FVIII and is released and oxidized upon treatment with EDTA. Cu(I), and not Cu(II), was able to reconstitute FVIII activity from dissociated chains, demonstrating a requirement for Cu(I) in FVIII function. Three potential copper ion binding sites exist within FVIII: one type-2 site and two type-1 sites. The importance of these potential copper ion ligands was tested by studying the effect of site-directed mutants. Of the two histidines that compose the type-2 binding site, the His-1957 --> Ala mutant displayed secretion, light and heavy chain assembly, and activity similar to wild-type FVIII, while mutant His-99 --> Ala was partially defective for secretion and had low levels of heavy and light chain association and activity. In contrast, FVIII having the mutation Cys-310 --> Ser within the type-1 copper binding site in the A1 domain was inactive and partially defective for secretion from the cell, and the heavy and light chains of the secreted protein were not associated. Mutant Cys-2000 --> Ser within the A3 domain displayed secretion, assembly, and activity similar to that for wild-type FVIII. These results support the hypothesis that Cu(I) is buried within the type-1 copper binding site within the A1 domain and is required for FVIII chain association and activity.
Tagliavacca
L
L
Department of Biological Chemistry, Ann Arbor, Michigan 48109, USA.
Moon
N
N
Dunham
W R
WR
Kaufman
R J
RJ
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D019556:COS Cells; D003300:Copper / Q000378:metabolism*; D006224:Cricetinae; D017077:Culture Media, Conditioned; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D005169:Factor VIII / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D008024:Ligands; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013054:Spectrophotometry, Atomic
D017077:Culture Media, Conditioned; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008024:Ligands; D046911:Macromolecular Substances; D011994:Recombinant Proteins; C063261:acetylleucyl-leucyl-norleucinal; C042514:cuprous ion; D003300:Copper; D005169:Factor VIII