Molecular expression and characterization of erythroid-specific 5-aminolevulinate synthase gain-of-function mutations causing X-linked protoporphyria.
X-linked protoporphyria (XLP) (MIM 300752) is a recently recognized erythropoietic porphyria due to gain-of-function mutations in the erythroid-specific aminolevulinate synthase gene (ALAS2). Previously, two exon 11 small deletions, c.1699_1670ΔAT (ΔAT) and c.1706_1709ΔAGTG (ΔAGTG), that prematurely truncated or elongated the ALAS2 polypeptide, were reported to increase enzymatic activity 20- to 40-fold, causing the erythroid accumulation of protoporphyrins, cutaneous photosensitivity and liver disease. The mutant ΔAT and ΔAGTG ALAS2 enzymes, two novel mutations, c.1734ΔG (ΔG) and c.1642C>T (p.Q548X), and an engineered deletion c.1670-1671TC>GA p.F557X were expressed, and their purified enzymes were characterized. Wild-type and ΔAGTG enzymes exhibited similar amounts of 54- and 52-kDa polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas the ΔAT and p.F557X had only 52-kDa polypeptides. Compared to the purified wild-type enzyme, ΔAT, ΔAGTG and Q548X enzymes had increased specific activities that were only 1.8-, 3.1- and 1.6-fold, respectively. Interestingly, binding studies demonstrated that the increased activity Q548X enzyme did not bind to succinyl-CoA synthetase. The elongated ΔG enzyme had wild-type specific activity, kinetics and thermostability; twice the wild-type purification yield (56 versus 25%); and was primarily a 54-kDa form, suggesting greater stability in vivo. On the basis of studies of mutant enzymes, the maximal gain-of function region spanned 57 amino acids between 533 and 580. Thus, these ALAS2 gain-of-function mutations increased the specific activity (ΔAT, ΔAGTG and p.Q548X) or stability (ΔG) of the enzyme, thereby leading to the increased erythroid protoporphyrin accumulation causing XLP.
Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, Mount Sinai Medical Center, New York, New York 10029, USA.
David F
DF
Bishop
Vassili
V
Tchaikovskii
Irina
I
Nazarenko
Robert J
RJ
Desnick
D000624:5-Aminolevulinate Synthetase / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D004912:Erythrocytes / Q000201:enzymology* / Q000378:metabolism; D005260:Female; D040181:Genetic Diseases, X-Linked / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008297:Male; D009154:Mutation; D046351:Protoporphyria, Erythropoietic / Q000235:genetics*; D013696:Temperature
D000624:5-Aminolevulinate Synthetase