Copper-catalyzed oxidation of the recombinant SHa(29-231) prion protein.
Metal-catalyzed oxidation may result in structural damage to proteins and has been implicated in aging and disease, including neurological disorders such as Alzheimer's disease and amyotrophic lateral sclerosis. The selective modification of specific amino acid residues with high metal ion affinity leads to subtle structural changes that are not easy to detect but may have dramatic consequences on physical and functional properties of the oxidized protein molecules. PrP contains a histidine-rich octarepeat domain that binds copper. Because copper-binding histidine residues are particularly prone to metal-catalyzed oxidation, we investigated the effect of this reaction on the recombinant prion protein SHaPrP(29-231). Using Cu2+/ascorbate, we oxidized SHaPrP(29-231) in vitro. Oxidation was demonstrated by liquid chromatography/mass spectrometry, which showed the appearance of protein species of higher mass, including increases in multiples of 16, characteristic of oxygen incorporation. Digestion studies using Lys C indicate that the 29-101 region, which includes the histidine-containing octarepeats, is particularly affected by oxidation. Oxidation was time- and copper concentration-dependent and was evident with copper concentrations as low as 1 microM. Concomitant with oxidation, SHaPrP(29-231) suffered aggregation and precipitation, which was nearly complete after 15 min, when the prion protein was incubated at 37 degrees C with a 6-fold molar excess of Cu2+. These findings indicate that PrP, a copper-binding protein, may be particularly susceptible to metal-catalyzed oxidation and that oxidation triggers an extensive structural transition leading to aggregation.
Requena
J R
JR
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, 50 South Drive, Room 2351 MSC 8012, Bethesda, MD 20892-8012, USA. requena@nih.gov
Groth
D
D
Legname
G
G
Stadtman
E R
ER
Prusiner
S B
SB
Levine
R L
RL
D000595:Amino Acid Sequence; D001205:Ascorbic Acid; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003300:Copper; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D011328:Prions / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure
D010446:Peptide Fragments; D011328:Prions; D011994:Recombinant Proteins; D003300:Copper; D001205:Ascorbic Acid