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_1670 Delta AT (Delta AT) and c.1706_1709 Delta AGTG (Delta AGTG), 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 Delta AT and Delta AGTG ALAS2 enzymes, two novel mutations, c.1734 Delta G (Delta G) 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 Delta AGTG enzymes exhibited similar amounts of 54- and 52-kDa polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas the Delta AT and p.F557X had only 52-kDa polypeptides. Compared to the purified wild-type enzyme, Delta AT, Delta AGTG 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 Delta G 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 (Delta AT, Delta AGTG and p.Q548X) or stability (Delta G) of the enzyme, thereby leading to the increased erythroid protoporphyrin accumulation causing XLP.