The effect of glycation on the structure, function and biological fate of human serum albumin as revealed by recombinant mutants.
Recombinant wild-type human serum albumin (rHSA), the single-residue mutants K199A, K439A and K525A and the triple-residue mutant K199A/K439A/K525A were produced using a yeast expression system. Portions of the rHSA were glycated to different degrees (2.5-250 mM D-glucose). As detected by far-UV and near-UV CD, intrinsic tryptophan-fluorescence and probed by 1,1'-bis(4-anilino)naphthalene-5,5-disulfonic acid, the single-residue mutations had no effect on albumin conformation, whereas the triple-residue mutation and glycation caused conformational changes. The triple-residue mutation and glycation had comparable increased effects on high-affinity binding of warfarin (site I), but decreased effects on high-affinity binding of dansylsarcosine (site II) and the esterase-like activity of albumin. The relation between plasma half-lives in rats were found to be glycated rHSA (50 mM glucose)<triple-residue mutated rHSA<rHSA. The opposite trend was found for liver and kidney uptakes in mice. Even though the functional and the in vivo properties of rHSA could be effected differently by the minor conformational changes caused by the triple-residue mutation and glycation, the present findings indicate that the effect of glycation can be partly explained by blockage of the positive charges of lysine at positions 199, 439 and 525.
Nakajou
Keisuke
K
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, 862-0973 Kumamoto, Japan.
Watanabe
Hiroshi
H
Kragh-Hansen
Ulrich
U
Maruyama
Toru
T
Otagiri
Masaki
M
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D004274:DNA, Recombinant / Q000235:genetics; D004950:Esterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008297:Male; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012709:Serum Albumin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D004274:DNA, Recombinant; D008024:Ligands; D011994:Recombinant Proteins; D012709:Serum Albumin; D004950:Esterases