Submitted December 23, 2008
Accepted March 26, 2009
Analysis of human 
globin gene mutations that impair binding to the alpha hemoglobin stabilizing protein (AHSP)
Xiang Yu, Todd L. Mollan, Andrew Butler, Andrew J. Gow, John S. Olson, and Mitchell J. Weiss*
Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, PA, United States
Department of Biochemistry and Cell Biology, Rice University, Houston, TX, United States
Department of Pharmacology, Rutgers University, Piscataway, NJ, United States
Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA, United States
* Corresponding author; email: weissmi{at}email.chop.edu.
Alpha Hemoglobin Stabilizing Protein (AHSP) reversibly binds nascent 
globin to maintain its native structure and facilitate its incorporation into hemoglobin A (HbA). Previous studies indicate that some naturally occurring human globin mutations may destabilize the protein by inhibiting its interactions with AHSP. However, these mutations could also affect HbA production through AHSP-independent effects, including reduced binding to 
globin. We analyzed six human globin variants with altered AHSP contact surfaces. Alpha globin amino acid substitutions H103Y, H103R, F117S and P119S impaired interactions with both AHSP and globin. These mutations are destabilizing in biochemical assays and are associated with microcytosis and anemia in humans. By contrast, K99E and K99N globins bind globin normally, but exhibit attenuated binding to AHSP. These mutations impair protein folding and expression in vitro and appear to be mildly destabilizing in vivo. In E. coli and erythroid cells, globin K99E stability is rescued upon coexpression with AHSP mutants in which binding to the abnormal globin chain is restored. Our results better define the biochemical properties of some globin variants and support the hypothesis that AHSP promotes globin chain stability during human erythropoiesis.
