Characterization of a human 20alpha-hydroxysteroid dehydrogenase

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• The enzyme 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) has been proposed as a T-cell differentiation marker in mice.
• Human 20alpha-HSD has been ambiguously associated with short-chain alcohol dehydrogenase family members (17beta-HSD types 1 and 2).
• Other species' 20alpha-HSDs belong to the aldoketo reductase superfamily, including the 3alpha-HSD family.

Purpose of the Study:

• To clone and characterize the human 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD).
• To determine the enzymatic activity and substrate specificity of the cloned human 20alpha-HSD.
• To ascertain the correct enzyme family classification for human 20alpha-HSD.

Main Methods:

• Cloning of a human 20alpha-HSD cDNA from a human skin cDNA library.
• Transfection into human embryonic kidney (HEK-293) cells to assess enzymatic activity.
• Amino acid sequence comparison with known HSD enzymes.
• Analysis of mRNA expression in various human tissues and cell lines.

Main Results:

• A human cDNA was cloned, exhibiting high 20alpha-HSD activity with negligible 3alpha- and 17beta-HSD activities upon transfection.
• Sequence analysis revealed significant identity to other species' 20alpha-HSDs and human 3alpha-HSDs, but low identity to human 17beta-HSDs.
• The enzyme preferentially reduced progesterone to 20alpha-hydroxyprogesterone (Km = 0.6 microM) and preferred NADPH as a cofactor.
• mRNA expression was detected in liver, prostate, testis, adrenal, brain, uterus, mammary gland, and HaCaT cells.

Conclusions:

• The genuine human 20alpha-HSD belongs to the aldoketo reductase family, consistent with orthologs in other species.
• The cloned enzyme demonstrates specific progesterone reduction activity, suggesting a distinct physiological role.
• Widespread tissue expression indicates diverse functions for human 20alpha-HSD beyond T-cell differentiation.