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1.
J Biol Chem ; 294(26): 10042-10054, 2019 06 28.
Article in English | MEDLINE | ID: mdl-31118275

ABSTRACT

Nucleotide sugar transporters (NSTs) regulate the flux of activated sugars from the cytosol into the lumen of the Golgi apparatus where glycosyltransferases use them for the modification of proteins, lipids, and proteoglycans. It has been well-established that NSTs are antiporters that exchange nucleotide sugars with the respective nucleoside monophosphate. Nevertheless, information about the molecular basis of ligand recognition and transport is scarce. Here, using topology predictors, cysteine-scanning mutagenesis, expression of GFP-tagged protein variants, and phenotypic complementation of the yeast strain Kl3, we identified residues involved in the activity of a mouse UDP-GlcNAc transporter, murine solute carrier family 35 member A3 (mSlc35a3). We specifically focused on the putative transmembrane helix 2 (TMH2) and observed that cells expressing E47C or K50C mSlc35a3 variants had lower levels of GlcNAc-containing glycoconjugates than WT cells, indicating impaired UDP-GlcNAc transport activity of these two variants. A conservative substitution analysis revealed that single or double substitutions of Glu-47 and Lys-50 do not restore GlcNAc glycoconjugates. Analysis of mSlc35a3 and its genetic variants reconstituted into proteoliposomes disclosed the following: (i) all variants act as UDP-GlcNAc/UMP antiporters; (ii) conservative substitutions (E47D, E47Q, K50R, or K50H) impair UDP-GlcNAc uptake; and (iii) substitutions of Glu-47 and Lys-50 dramatically alter kinetic parameters, consistent with a critical role of these two residues in mSlc35a3 function. A bioinformatics analysis revealed that an EXXK motif in TMH2 is highly conserved across SLC35 A subfamily members, and a 3D-homology model predicted that Glu-47 and Lys-50 are facing the central cavity of the protein.


Subject(s)
Glutamic Acid/metabolism , Lysine/metabolism , Sodium-Phosphate Cotransporter Proteins, Type IIc/metabolism , Uridine Diphosphate N-Acetylglucosamine/metabolism , Uridine Monophosphate/metabolism , Amino Acid Sequence , Animals , Golgi Apparatus/metabolism , Ion Transport , Mice , Models, Molecular , Protein Conformation , Sequence Homology , Sodium-Phosphate Cotransporter Proteins, Type IIc/chemistry , Sodium-Phosphate Cotransporter Proteins, Type IIc/genetics , Uridine Diphosphate N-Acetylglucosamine/genetics
2.
Bone ; 59: 114-21, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24246249

ABSTRACT

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare metabolic disorder inherited in an autosomal recessive fashion and characterized by hypophosphatemia, short stature, rickets and/or osteomalacia, and secondary absorptive hypercalciuria. HHRH was recently mapped to chromosome 9q34, which contains the gene SLC34A3 which encodes the renal proximal tubular sodium-phosphate cotransporter NaPi-IIc. Here we describe a 29-year-old man with a history of childhood rickets who presented with increased renal phosphate clearance leading to hypophosphatemia, hypercalciuria, low serum parathyroid hormone (PTH), elevated serum 1,25-dihydroxyvitamin D (1,25(OH)2D) and recurrent nephrolithiasis. We performed a mutation analysis of SLC34A3 (exons and adjacent introns) of the proband and his parents to determine if there was a genetic contribution. The proband proved to be compound heterozygous for two missense mutations in SLC34A3: one novel mutation in exon 7 c.571G>C (p.G191R) and one previously identified mutation in exon 13 c.1402C>T (p.R468W). His parents were both asymptomatic heterozygous carriers of one of these two mutations. We also performed an oral phosphate loading test and compared serum phosphate, intact PTH, and intact fibroblast growth factor 23 (iFGF23) in this patient versus patients with other forms of hypophosphatemic rickets, the results of which further revealed that the mechanism of hypophosphatemia in HHRH is independent of FGF23. This is the first report of HHRH in the Chinese population. Our findings of the novel mutation in exon 7 add to the list of more than 20 reported mutations of SLC34A3.


Subject(s)
Asian People/genetics , Familial Hypophosphatemic Rickets/complications , Familial Hypophosphatemic Rickets/genetics , Hypercalciuria/complications , Hypercalciuria/genetics , Mutation/genetics , Sodium-Phosphate Cotransporter Proteins, Type IIc/genetics , Adult , Amino Acid Sequence , Base Sequence , China , Familial Hypophosphatemic Rickets/blood , Familial Hypophosphatemic Rickets/diagnostic imaging , Fibroblast Growth Factor-23 , Fibroblast Growth Factors/blood , Heterozygote , Humans , Hypercalciuria/blood , Hypercalciuria/diagnostic imaging , Male , Molecular Sequence Data , Parathyroid Hormone/blood , Phosphates/blood , Radiography , Sodium-Phosphate Cotransporter Proteins, Type IIc/chemistry
3.
Pflugers Arch ; 465(9): 1261-79, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23515872

ABSTRACT

The SLC34 family of Na(+)-dependent inorganic phosphate cotransporters comprises two electrogenic isoforms (NaPi-IIa, NaPi-IIb) and an electroneutral isoform (NaPi-IIc). Both fulfill essential physiological roles in mammalian phosphate homeostasis. By substitution of three conserved amino acids, found in all electrogenic isoforms, at corresponding sites in NaPi-IIc, electrogenicity was re-established and the Na(+)/P i stoichiometry increased from 2:1 to 3:1. However, this engineered electrogenic construct (AAD-IIc) had a reduced apparent P i affinity and different presteady-state kinetics from the wild-type NaPi-IIa/b. We investigated AAD-IIc using electrophysiology and voltage clamp fluorometry to elucidate the compromised behavior. The activation energy for cotransport was threefold higher than for NaPi-IIc and 1.5-fold higher than for NaPi-IIa and the temperature dependence of presteady-state charge displacements suggested that the large activation energy was associated with the empty carrier reorientation. AAD-IIc shows a weak interaction of external Na(+) ions with the electric field, and thus retains the electroneutral cooperative interaction of two Na(+) ions preceding external P i binding of NaPi-IIc. Most of the presteady-state charge movement was accounted for by the empty carrier (in the absence of external P i ), and the cytosolic release of one Na(+) ion (in the presence of P i ). Simulations using a kinetic model recapitulated the presteady-state and steady-state behavior and allowed identification of two critical partial reactions: the final release of Na(+) to the cytosol and external P i binding. Fluorometric recordings from AAD-IIc mutants with Cys substituted at functionally important sites established that AAD-IIc undergoes substrate- and voltage-dependent conformational changes that correlated qualitatively with its presteady-state kinetics.


Subject(s)
Sodium-Phosphate Cotransporter Proteins, Type IIc/metabolism , Sodium/metabolism , Static Electricity , Action Potentials , Amino Acid Sequence , Amino Acid Substitution , Animals , Kinetics , Mice , Molecular Sequence Data , Mutation , Phosphates/metabolism , Protein Binding , Sodium/chemistry , Sodium-Phosphate Cotransporter Proteins, Type IIc/chemistry , Sodium-Phosphate Cotransporter Proteins, Type IIc/genetics , Xenopus
4.
J Bone Miner Metab ; 25(6): 407-13, 2007.
Article in English | MEDLINE | ID: mdl-17968493

ABSTRACT

Two cases of hereditary hypophosphatemic rickets with hypercalciuria (HHRH) were reported in Japanese female siblings. Both of them manifested short stature and bowed legs, and biochemical examination revealed hypophosphatemia, phosphaturia, and hypercalciuria. The serum concentrations of 1,25-dihydroxyvitamin D (1,25(OH)(2)D) were elevated. In the oral phosphate loading test, serum phosphate levels were markedly increased in the HHRH patients, and the elevation was much higher than that in patients affected with X-linked hypophosphatemic rickets (XLH), suggesting the increased gastrointestinal absorption of phosphate in HHRH. Bone histology studies showed increased osteoid surface and width in HHRH, which was compatible with osteomalacia. In the HHRH patients, there were no hypomineralized periosteocytic lesions, which was a hallmark of XLH in bone histology. In one of the HHRH patients, phosphate administration alone almost completely cured the osteomalacia within a year, although pharmacological doses of 1,25(OH)(2)D(3) had little effect. In osteoblasts isolated from a HHRH patient, basal alkaline phosphatase (ALP) activities and osteocalcin syntheses by a physiological concentration of 1,25(OH)(2)D(3) were not stimulated by the increased medium phosphate concentrations from 0.5 to 4 mM. In contrast, these two parameters were stimulated by the increased medium phosphate concentrations both in normal and XLH osteoblasts, although the regulatory patterns of increased osteocalcin syntheses were different from normal to XLH osteoblasts; 2 and 4 mM of phosphate concentrations at least were necessary for normal and XLH osteoblasts, respectively. The gene analysis of phosphate transporter revealed a novel heterozygous mutation (R564C) in the exon of phosphate transporter NPT type IIc. These lines of evidence suggested that the pathogenesis of osteomalacia in HHRH was different from XLH in terms of the utility of phosphate in osteoblasts. These abnormalities were speculated to be associated with the abnormal functions of phosphate transporter gene type IIc, although the exact roles of this phosphate transporter in the human osteoblast are still unknown.


Subject(s)
Familial Hypophosphatemic Rickets/complications , Genetic Diseases, X-Linked , Hypercalciuria/complications , Osteoblasts/metabolism , Osteoblasts/pathology , Sodium-Phosphate Cotransporter Proteins, Type IIc/genetics , Alkaline Phosphatase/metabolism , Amino Acid Sequence , Bone and Bones/drug effects , Bone and Bones/pathology , Case-Control Studies , Cell Separation , DNA Mutational Analysis , Diagnostic Tests, Routine , Familial Hypophosphatemic Rickets/genetics , Familial Hypophosphatemic Rickets/pathology , Female , Humans , Middle Aged , Molecular Sequence Data , Osteoblasts/drug effects , Osteoblasts/enzymology , Osteocalcin/biosynthesis , Phosphates/pharmacology , Sodium-Phosphate Cotransporter Proteins, Type IIc/chemistry
5.
Kidney Int ; 70(9): 1548-59, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16955105

ABSTRACT

Members of the SLC34 gene family of solute carriers encode for three Na+-dependent phosphate (P i) cotransporter proteins, two of which (NaPi-IIa/SLC34A1 and NaPi-IIc/SLC34A3) control renal reabsorption of P i in the proximal tubule of mammals, whereas NaPi-IIb/SCLC34A2 mediates P i transport in organs other than the kidney. The P i transport mechanism has been extensively studied in heterologous expression systems and structure-function studies have begun to reveal the intricacies of the transport cycle at the molecular level using techniques such as cysteine scanning mutagenesis, and voltage clamp fluorometry. Moreover, sequence differences between the three types of cotransporters have been exploited to obtain information about the molecular determinants of hormonal sensitivity and electrogenicity. Renal handling of P i is regulated by hormonal and non-hormonal factors. Changes in urinary excretion of P i are almost invariably mirrored by changes in the apical expression of NaPi-IIa and NaPi-IIc in proximal tubules. Therefore, understanding the mechanisms that control the apical expression of NaPi-IIa and NaPi-IIc as well as their functional properties is critical to understanding how an organism achieves P i homeostasis.


Subject(s)
Kidney Tubules, Proximal/metabolism , Phosphates/metabolism , Sodium-Phosphate Cotransporter Proteins, Type IIa/metabolism , Sodium-Phosphate Cotransporter Proteins, Type IIb/metabolism , Sodium-Phosphate Cotransporter Proteins, Type IIc/metabolism , Animals , Homeostasis , Humans , Mice , Parathyroid Hormone/physiology , Sodium-Phosphate Cotransporter Proteins, Type IIa/chemistry , Sodium-Phosphate Cotransporter Proteins, Type IIa/genetics , Sodium-Phosphate Cotransporter Proteins, Type IIb/chemistry , Sodium-Phosphate Cotransporter Proteins, Type IIb/genetics , Sodium-Phosphate Cotransporter Proteins, Type IIc/chemistry , Sodium-Phosphate Cotransporter Proteins, Type IIc/genetics , Structure-Activity Relationship
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