Sites of iodination in recombinant human brain-derived neurotrophic factor and its effect on neurotrophic activity.
Recombinant human brain-derived neurotrophic factor (BDNF) is now under extensive investigation because of its potential clinical applications. Radioactively labeled proteins are usually required to study receptor binding and pharmacokinetic properties of proteins. This study was undertaken to see if iodination affects the biological and conformational properties of a recombinant BDNF. BDNF was iodinated using a stoichiometric amount of nonradioactive cold NaI to minimize multiple iodinations. Of the four tyrosines present in BDNF--Tyr-52, Tyr-54, Tyr-63, and Tyr-86--only Tyr-63 and Tyr-86 were iodinated under the experimental conditions used. Iodination of Tyr-63 resulted in modification without alteration of the biological activity, whereas iodination of Tyr-86 resulted in a molecule with highly compromised biological activity. Similar inactivation was observed if both Tyr-63 and Tyr-86 were iodinated. These modified proteins exhibited conformation and dimerization apparently identical to those of the native protein, as demonstrated by analytical ultracentrifugation, gel filtration, light scattering, and circular dichroism. From these results, we concluded that Tyr-52 and Tyr-54 are not accessible to the reagent and are probably buried in the hydrophobic core, whereas Tyr-63 and Tyr-86 are exposed on the surface of the molecule; of the two exposed residues, only Tyr-86 contributes to the biological activity.
Rosenfeld
R
R
Amgen Inc., Amgen Center, Thousand Oaks, California 91320-1789.
Philo
J S
JS
Haniu
M
M
Stoney
K
K
Rohde
M F
MF
Wu
G M
GM
Narhi
L O
LO
Wong
C
C
Boone
T
T
Hawkins
N N
NN
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019208:Brain-Derived Neurotrophic Factor; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D005724:Ganglia / Q000187:drug effects / Q000648:ultrastructure; D006801:Humans; D007455:Iodine / Q000378:metabolism*; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D010434:Pepsin A / Q000378:metabolism; D010449:Peptide Mapping; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012974:Sodium Iodide / Q000378:metabolism; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000378:metabolism; D014461:Ultracentrifugation
D019208:Brain-Derived Neurotrophic Factor; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D007455:Iodine; D010434:Pepsin A; D012974:Sodium Iodide