Contribution of cysteine 158, the glycosylation site threonine 194, the amino- and carboxy-terminal domains of apolipoprotein E in the binding to amyloid peptide beta (1-40).
Recent studies have shown that at physiological conditions (pH 7.6, 37 degrees C), the reactivity of recombinant apoE isoforms secreted by mammalian cells toward amyloid peptide beta (Abeta40) follows the order apoE2 > apoE3 > apoE4 for the apoE monomer and apoE2 > apoE3 for apoE dimer that is formed via that intramolecular disulfide bridges. Different Abeta binding properties have been reported for the plasma-derived apoE and commercially available apoE preparations that differ from the native apoE forms in the degree of their O-glycosylation. To define structural elements of apoE involved in the interaction with Abeta, we have introduced point mutations as well as amino- and carboxy-terminal deletions in the apoE structure. The mutant apoE forms were expressed transiently using the Semliki Forest Virus system, and the culture medium was utilized to study the reactivity of the mutated proteins with Abeta 40. This analysis showed that a mutation in the O-glycosylation site of apoE2 (Thr194-Ala) did not affect the SDS-stable binding of apoE to Abeta. In contrast, introduction of cysteine at position 158 of apoE4 (Arg112, Cys158) increased the SDS-stable binding of apoE to Abeta to the levels similar to those observed in apoE2. Similar analysis showed that apoE truncated at residues 259, 249, 239, and 229 retains the SDS-stable binding to Abeta40, whereas apoE truncated at residues 185 and 165 does not bind to Abeta. The deletion of aminoterminal residues 2-19 reduced the SDS-stable binding of apoE2 to Abeta and deletion of residues 2-81 abolished binding to Abeta. It is also noteworthy that the (Delta2-81) apoE mutant exists predominantly as a dimer, suggesting that removal of residues 2-81 promoted dimerization of apoE. These findings suggest that the amino- and carboxy-terminal residues of apoE are required for SDS-stable binding of apoE to Abeta and that the presence of at least one cysteine contributes to the efficient Abeta binding.
Aleshkov
S B
SB
Section of Molecular Genetics, Center for Advanced Biomedical Research, Department of Medicine, Boston University Medical Center, Massachusetts 02118-2394, USA.
Li
X
X
Lavrentiadou
S N
SN
Zannis
V I
VI
D019943:Amino Acid Substitution / Q000235:genetics; D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001057:Apolipoproteins E / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002236:Carbohydrate Conformation; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D007668:Kidney / Q000166:cytology; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D013912:Threonine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D016229:Amyloid beta-Peptides; D001057:Apolipoproteins E; D046911:Macromolecular Substances; D010446:Peptide Fragments; D020033:Protein Isoforms; C078592:amyloid beta-protein (1-40); D013912:Threonine; D001120:Arginine; D003545:Cysteine