Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP) |
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Authors: | Wennberg Charlotte Kozlenkov Alexey Di Mauro Sonia Fröhlander Nils Beckman Lars Hoylaerts Marc F Millán José Luis |
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Institution: | 1. Department of Medical Biosciences, Medical Genetics, Ume? University, Ume?, Sweden;2. Chemistry Department, Moscow State University, Moscow, Russian Federation;3. UNESP, Campus de Jaboticabal, S?o Paulo, Brazil;4. The Burnham Institute, La Jolla, California;5. Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium;6. The Burnham Institute, La Jolla, CaliforniaThe Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037 |
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Abstract: | 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. |
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Keywords: | alkaline phosphatase placental PLAP ALPP ALPL ALPPL2 ALPI isozyme negative selection spontaneous abortion gene therapy genetic disease placental function SNuPE |
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