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Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP)
Author(s) -
Wennberg Charlotte,
Kozlenkov Alexey,
Di Mauro Sonia,
Fröhlander Nils,
Beckman Lars,
Hoylaerts Marc F.,
Millán José Luis
Publication year - 2002
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.10052
Subject(s) - placental alkaline phosphatase , biology , alkaline phosphatase , dna , genomic dna , typing , genetics , microbiology and biotechnology , biochemistry , computational biology , enzyme
The D allozyme of placental alkaline phosphatase (PLAP) displays enzymatic properties at variance with those of the common PLAP allozymes. We have deduced the amino acid sequence of the PLAP D allele by PCR cloning of its gene, ALPP. Two coding substitutions were found in comparison with the cDNA of the common PLAP F allele, i.e., 692C>G and 1352A>G, which translate into a P209R and E429G substitution. A single nucleotide primer extension (SNuPE) assay was developed using PCR primers that enable the amplification of a 1.9 kb PLAP fragment. Extension primers were then used on this PCR fragment to detect the 692C>G and 1352A>G substitution. The SNuPE assay on these two nucleotide substitutions enabled us to distinguish the PLAP F and D alleles from the PLAP S/I alleles. Functional studies on the D allozyme were made possible by constructing and expressing a PLAP D cDNA, i.e., [Arg209, Gly429]PLAP, into wild‐type Chinese hamster ovary cells. We determined the k cat and K m , of the PLAP S, F, and D allozymes using the non‐physiological substrate p‐nitrophenylphosphate at an optimal pH (9.8) as well as two physiological substrates, i.e., pyridoxal‐5′‐phosphate and inorganic pyrophosphate at physiological pH (7.5). We found that the biochemical properties of the D allozyme of PLAP are significantly different from those of the common PLAP allozymes. These biochemical findings suggest that a suboptimal enzymatic function by the PLAP D allozyme may be the basis for the apparent negative selective pressure of the PLAP D allele. The development of the SNuPE assay will enable us to test the hypothesis that the PLAP D allele is subjected to intrauterine selection by examining genomic DNA from statistically informative population samples. Hum Mutat 19:258–267, 2002. © 2002 Wiley‐Liss, Inc.