Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP).
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.
Wennberg
Charlotte
C
Department of Medical Biosciences, Medical Genetics, Umeå University, Umeå, Sweden.
Kozlenkov
Alexey
A
Di Mauro
Sonia
S
Fröhlander
Nils
N
Beckman
Lars
L
Hoylaerts
Marc F
MF
Millán
José Luis
JL
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics; D000483:Alleles; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics; D004247:DNA / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D005260:Female; D058851:GPI-Linked Proteins; D005838:Genotype; D018698:Glutamic Acid / Q000235:genetics; D005998:Glycine / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics*; D007700:Kinetics; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000737:chemistry / Q000235:genetics; D011392:Proline / Q000235:genetics; D020836:Protein Structure, Quaternary / Q000235:genetics
D018076:DNA, Complementary; D058851:GPI-Linked Proteins; D007527:Isoenzymes; D011257:Pregnancy Proteins; D018698:Glutamic Acid; D004247:DNA; D001120:Arginine; D011392:Proline; D000469:Alkaline Phosphatase; C019839:alkaline phosphatase, placental; D005998:Glycine