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Structural / functional studies on human alkaline phosphatases
Umeå universitet, Medicinsk fakultet, Medicinsk biovetenskap.
2003 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

The recent elucidation of the three-dimensional structure of human placental alkaline phosphatase (PLAP) has enabled me to perform structural studies aimed at characterizing the properties of human PLAP and tissue-nonspecific AP (TNAP) as paradigms for mammalian APs in general, using site-directed mutagenesis, protein expression, kinetic analysis and computer modeling.

In Paper I, we found that a single critical E429G substitution explains the difference in stability and kinetics between the common allelic variants of PLAP and the D allozyme. In Paper II, we demonstrated the role of residue E429 in PLAP in stabilizing the active site metals, elucidated the distinct roles of residues H153 and H317 in catalysis, and the relative importance of five Cys residues in PLAP. We also discovered the significance of Y367, a unique feature of mammalian APs, for enzyme stability and specific inhibition by amino acids. Paper III focused on the identification and mutagenesis analysis of a novel, non-catalytic peripheral binding site of PLAP that appears to mediate a mitogenic effect of PLAP. This site provides indications that PLAP may function as a fetal growth factor.

The last two papers focus on the TNAP isozyme as paradigm. A deficiency in TNAP activity is the cause of the human disease hypophosphatasia, characterized by rickets, osteomalacia and occasionally epileptic seizures. Paper IV has been able to partially explain the variable expressivity of hypophosphatasia traits by examining site-directed mutants of TNAP and performing kinetic analysis using natural substrates PPi and PLP. Finally, Paper V has clarified the mechanism of inhibition of TNAP by uncompetitive inhibitors L-homoarginine, levamisole and theophylline. We identified residues that confer to TNAP its distinct inhibitory properties. These data have significance for future drug design of specific TNAP inhibitors to therapeutically target TNAP as a way of elevating PPi extracellular level and alleviating pathological bone hypermineralization conditions.

sted, utgiver, år, opplag, sider
2003. , s. 128
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 863
Emneord [en]
Biomedicine, mutagenesis, enzyme kinetics, computer modeling, crystallography, natural substrates, uncompetitive inhibition, isozymes, gene families
Emneord [sv]
Biomedicin
HSV kategori
Forskningsprogram
medicinsk genetik
Identifikatorer
URN: urn:nbn:se:umu:diva-136ISBN: 91-7305-542-5 (tryckt)OAI: oai:DiVA.org:umu-136DiVA, id: diva2:140754
Disputas
2003-11-14, E04, 6E, Norrlands Universitetssjukhus, Umeå, 13:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2003-10-29 Laget: 2003-10-29 Sist oppdatert: 2018-01-13bibliografisk kontrollert
Delarbeid
1. Structure, genomic DNA typing and kinetic characterization of the D allozyme of placental alkaline phosphatase
Åpne denne publikasjonen i ny fane eller vindu >>Structure, genomic DNA typing and kinetic characterization of the D allozyme of placental alkaline phosphatase
Vise andre…
2002 (engelsk)Inngår i: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 19, nr 3, s. 258-267Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

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 kcat and Km, 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.

Emneord
alkaline phosphatase, placental; PLAP; ALPP; ALPL; ALPPL2; ALPI; isozyme; negative selection; spontaneous abortion; gene therapy; genetic disease; placental function; SNuPE
Identifikatorer
urn:nbn:se:umu:diva-20842 (URN)10.1002/humu.10052 (DOI)2-s2.0-0036186934 (Scopus ID)
Tilgjengelig fra: 2009-03-26 Laget: 2009-03-26 Sist oppdatert: 2023-03-24
2. Function assignment to conserved residues in mammalian alkaline phosphatases
Åpne denne publikasjonen i ny fane eller vindu >>Function assignment to conserved residues in mammalian alkaline phosphatases
2002 (engelsk)Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, nr 25, s. 22992-22999Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

We have probed the structural/functional relationship of key residues in human placental alkaline phosphatase (PLAP) and compared their properties with those of the corresponding residues in Escherichia coli alkaline phosphatase (ECAP). Mutations were introduced in wild-type PLAP, i.e. [E429]PLAP, and in some instances also in [G429]PLAP, which displays properties characteristic of the human germ cell alkaline phosphatase isozyme. All active site metal ligands, as well as residues in their vicinity, were substituted to alanines or to the homologous residues present in ECAP. We found that mutations at Zn2 or Mg sites had similar effects in PLAP and ECAP but that the environment of the Zn1 ion in PLAP is less affected by substitutions than that in ECAP. Substitutions of the Mg and Zn1 neighboring residues His-317 and His-153 increased kcat and increased Km when compared with wild-type PLAP, contrary to what was predicted by the reciprocal substitutions in ECAP. All mammalian alkaline phosphatases (APs) have five cysteine residues (Cys-101, Cys-121, Cys-183, Cys-467, and Cys-474) per subunit, not homologous to any of the four cysteines in ECAP. By substituting each PLAP Cys by Ser, we found that disrupting the disulfide bond between Cys-121 and Cys-183 completely prevents the formation of the active enzyme, whereas the carboxylterminally located Cys-467-Cys-474 bond plays a lesser structural role. The substitution of the free Cys-101 did not significantly affect the properties of the enzyme. A distinguishing feature found in all mammalian APs, but not in ECAP, is the Tyr-367 residue involved in subunit contact and located close to the active site of the opposite subunit. We studied the A367 and F367 mutants of PLAP, as well as the corresponding double mutants containing G429. The mutations led to a 2-fold decrease in kcat, a significant decrease in heat stability, and a significant disruption of inhibition by the uncompetitive inhibitors L-Phe and L-Leu. Our mutagenesis data, computer modeling, and docking predictions indicate that this residue contributes to the formation of the hydrophobic pocket that accommodates and stabilizes the side chain of the inhibitor during uncompetitive inhibition of mammalian APs.

Identifikatorer
urn:nbn:se:umu:diva-20852 (URN)10.1074/jbc.M202298200 (DOI)
Tilgjengelig fra: 2009-03-27 Laget: 2009-03-27 Sist oppdatert: 2017-12-13
3. Structural and functional analysis of a non-catalytic binding site in human placental alkaline phosphatase
Åpne denne publikasjonen i ny fane eller vindu >>Structural and functional analysis of a non-catalytic binding site in human placental alkaline phosphatase
Vise andre…
(engelsk)Manuskript (Annet vitenskapelig)
Identifikatorer
urn:nbn:se:umu:diva-20853 (URN)
Tilgjengelig fra: 2009-03-27 Laget: 2009-03-27 Sist oppdatert: 2018-06-09
4. Kinetic characterization of hypophosphatasia mutations with physiological substrates
Åpne denne publikasjonen i ny fane eller vindu >>Kinetic characterization of hypophosphatasia mutations with physiological substrates
2002 (engelsk)Inngår i: Journal of bone and mineral research, ISSN 0884-0431, Vol. 17, nr 8, s. 1383-1391Artikkel i tidsskrift (Fagfellevurdert) Published
Identifikatorer
urn:nbn:se:umu:diva-2562 (URN)
Tilgjengelig fra: 2009-03-26 Laget: 2009-03-26bibliografisk kontrollert
5. Residues determining the specific binding of uncompetitive inhibitors to mammalian alkaline phosphatases
Åpne denne publikasjonen i ny fane eller vindu >>Residues determining the specific binding of uncompetitive inhibitors to mammalian alkaline phosphatases
Vise andre…
(engelsk)Manuskript (Annet vitenskapelig)
Abstract [en]

Recent data have suggested that the activity of human tissue-nonspecific alkaline phosphatase (TNAP) could be targeted therapeutically to ameliorate soft-tissue ossification abnormalities resulting from insufficient production of inorganic pyrophosphate (1). Thus, understanding the mechanism of action and precise binding site for inhibitors of TNAP function is paramount. We compared the modeled three-dimensional structure of TNAP with the 3D structure of human placental alkaline phosphatase (PLAP), and identified the residues that differ between these two isozymes within a 12 Å radius of the active site. We then used site-directed mutagenesis to substitute TNAP residues to their respective homologues in PLAP or in the related germ cell (GCAP) isozyme or to Ala. In addition, we mutagenized most of the corresponding residues in PLAP to their TNAP homologues, and studied the role of a conserved residue Y371 in TNAP using the A371 mutation. All mutants were characterized for their sensitivity towards the uncompetitive inhibitors Lhomoarginine (L-hArg), levamisole, theophylline and L-phenylalanine. We found that the identity of residue108 in TNAP largely determines the specificity of inhibition by LhArg. The conserved Tyr-371 is also necessary for binding of L-hArg. The selectivity of inhibition by L-Phe was determined by the identity of residues 108 and 109 in TNAP (or corresponding residues 107 and 108 in PLAP). In contrast, the binding of levamisole to TNAP is mostly dependent on His-434 and Tyr-371, but not on residues 108 or 109. Substitutions H434E (as in PLAP), H434Q (as in chicken TNAP), H434S (as in the intestinal isozyme), H434G (as in GCAP), or H434A, all lead to a significant decrease in inhibition. The reciprocal E429H mutation in PLAP improved the inhibition by levamisole by 10-fold. The main determinant of sensitivity to theophylline is His-434. The H434E substitution (as in PLAP) causes a 50-fold reduction in inhibition, making TNAP even less inhibited than wt PLAP. The reciprocal E429H mutation in PLAP improves the Ki for theophylline inhibition 30 times, close to the level of wt TNAP. Interestingly, theophylline inhibition in TNAP could be further improved by introducing an E108F mutation. Thus, we have clarified the location of the binding sites for all three TNAP inhibitors and we have also been able to exchange inhibitor specificities between TNAP and PLAP. These data will help us in drug design efforts aimed at discovering novel and better inhibitors of TNAP for clinical use.

Identifikatorer
urn:nbn:se:umu:diva-20868 (URN)
Tilgjengelig fra: 2009-03-27 Laget: 2009-03-27 Sist oppdatert: 2018-06-09

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