[Estrogen binding activities of recombinant alpha-fetoproteins expressed in yeast]

Area of Science:

• Biochemistry
• Molecular Biology
• Endocrinology

Background:

• Alpha-fetoprotein (AFP) from rodents binds estrogen, but human AFP does not.
• The estrogen-binding region of rat AFP is localized to amino acid residues 426-467 in its third domain.
• Fifteen amino acid differences exist between rat and human AFP within this region, suggesting these residues are critical for binding.

Purpose of the Study:

• To identify specific amino acid residues responsible for estrogen binding in alpha-fetoprotein.
• To engineer human AFP to bind estrogen by introducing rat-specific amino acid substitutions.
• To explore the potential of modified human AFP in modulating estrogen's biological activity.

Main Methods:

• Site-directed mutagenesis using polymerase chain reaction to create mutant human AFPs.
• Production of 17 mutant human AFPs with varying combinations of rat-specific substitutions.
• Assay of estrogen binding activity in engineered human AFP variants.

Main Results:

• Seven specific substitutions (436Ala-->Val, 437Ala-->Ser, 438Thr-->Ile, 450Leu-->Arg, 451Leu-->Ser, 457Ala-->Leu, and 461Ile-->Tyr) conferred significant estrogen binding activity to human AFP.
• Human AFP was successfully converted into an estrogen-binding variant through these targeted substitutions.
• The engineered human AFP variants demonstrated the ability to bind estrogen.

Conclusions:

• Specific amino acid residues within the 426-467 region are essential for estrogen binding in AFP.
• Human AFP can be engineered to bind estrogen, mimicking the function of rodent AFP.
• These estrogen-binding human AFP variants hold potential for modulating estrogen's biological effects.