Molecular expression and characterization of erythroid-specific 5-aminolevulinate synthase gain-of-function mutations causing X-linked protoporphyria.

[erythropoietic 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 (ALAS 2 ). Previously , two exon 11 small deletions , c .1699 _1670 ΔAT (ΔAT ) and c .1706 _1709 ΔAGTG (ΔAGTG ), that prematurely truncated or elongated the ALAS 2 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 ALAS 2 enzymes , two novel mutations , c .1734 ΔG (ΔG ) and c .1642 C >T (p .Q 548 X ), and an engineered deletion c .1670 -1671 TC >GA p .F 5 57 X 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 .F 5 57 X had only 52 -kDa polypeptides . Compared to the purified wild-type enzyme , ΔAT , ΔAGTG and Q 548 X 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 Q 548 X 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 ALAS 2 gain-of-function mutations increased the specific activity (ΔAT , ΔAGTG and p .Q 548 X ) or stability (ΔG ) of the enzyme , thereby leading to the increased erythroid protoporphyrin accumulation causing XLP .

Diseases
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Diseases presenting "recently recognized erythropoietic porphyria" symptom

erythropoietic protoporphyria

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