Quantitation of CYP24A1 enzymatic activity with a simple two-hybrid system.
BACKGROUND
Mutations of the CYP24A1 gene encoding the 24-hydroxylase (24OHase) that inactivates metabolites of vitamin D can cause hypercalcemia in infants and adults; in vitro assays of 24OHase activity have been difficult.


OBJECTIVE
We sought an alternative assay to characterize a CYP24A1 mutation in a young adult with bilateral nephrolithiasis and hypercalcemia associated with ingestion of excess vitamin D supplements and robust dairy intake for 5 years.


METHODS
CYP24A1 exons were sequenced from leukocyte DNA. Wild-type and mutant CYP24A1 cDNAs were expressed in JEG-3 cells, and 24OHase activity was assayed by a two-hybrid system.


RESULTS
The CYP24A1 missense mutation L409S was found on only one allele; no other mutation was found in exons or in at least 30 bp of each intron/exon junction. Based on assays of endogenous 24OHase activity and of activity from a transiently transfected CYP24A1 cDNA expression vector, JEG-3 cells were chosen over HepG2, Y1, MA10, and NCI-H295A cells for two-hybrid assays of 24OHase activity. The apparent Michaelis constant, Km(app), was 9.0 ± 2.0 nM for CYP24A1 and 8.6 ± 2.2 nM for its mutant; the apparent maximum velocity, Vmax(app), was 0.71 ± 0.055 d(-1) for the wild type and 0.22 ± 0.026 d(-1) for the mutant. As assessed by Vmax/Km, the L409S mutant has 32% of wild-type activity (P = .0012).


CONCLUSIONS
The two-hybrid system in JEG-3 cells provides a simple, sensitive, quantitative assay of 24OHase activity. Heterozygous mutation of CYP24A1 may cause hypercalcemia in the setting of excessive vitamin D intake, but it is also possible that the patient had another, unidentified CYP24A1 mutation on the other allele.
Department of Pediatrics, University of California, San Francisco, San Francisco, California 94143-1346.
Amy
A
Mugg
Balazs
B
Legeza
Meng Kian
MK
Tee
Izabella
I
Damm
Roger K
RK
Long
Walter L
WL
Miller
D006801:Humans; D006934:Hypercalcemia / Q000201:enzymology* / Q000235:genetics; D008297:Male; D009154:Mutation; D053040:Nephrolithiasis / Q000201:enzymology* / Q000235:genetics; D020798:Two-Hybrid System Techniques; D065668:Vitamin D3 24-Hydroxylase / Q000235:genetics / Q000378:metabolism*; D055815:Young Adult
C585549:CYP24A1 protein, human; D065668:Vitamin D3 24-Hydroxylase