Characterization of calcium-binding sites in the kidney stone inhibitor glycoprotein nephrocalcin with vanadyl ions: electron paramagnetic resonance and electron nuclear double resonance spectroscopy.

Abstract

This inhibitor protein has been isolated in four isoforms from urine of normal subjects (1) and kidney stone patients (2), from surgically removed calcium oxalate kidney stones (3), and from kidney tissue of nine species of vertebrates (4). Among these nine species, the dissociation constant of these inhibitor proteins for binding COM crystals varies from 6 x 10-6 to 4 x 10-8 M. Purification of NC from bovine kidney tissue by DEAE-cellulose chromatography showed that it also consists offour isoforms, which have been designated as fractions A-D (4-6). Fractions A-D of NC were also obtained similarly from other species, as described earlier (1-4). Fractions A and B, the "strong" inhibitors, are abundant in normal subjects (1), whereas stone formers excrete less of fractions A and B and more of C and D, which are "weak" inhibitors (2). NC is an acidic glycoprotein containing 33% acidic amino acid residues (Asp and Glu) and about 5% aromatic and basic amino acid residues (5). Fractions A-D differ according to the content of carbohydrate and phosphate. Also, the strong inhibitors contain three or four y-tcarboxyglutamic acid residues per molecule, which are not present in NC of stone-forming patients (5).We have previously shown that, on the basis of 31P NMR studies of Ca2+-NC complexes and by equilibrium dialysis with 45Ca, a total of 4 mol of Ca2+ are bound per mol of NC (6). In this study we characterized the metal-binding properties of normal NCt (fraction A) by EPR and electron nuclear double resonance (ENDOR) using the vanadyl ion (VO2+) as a paramagnetic probe. The VO2+ ion has been shown to be an effective paramagnetic substitute for many divalent metal ions in metalloproteins and metalloenzymes (7-10) and is, in particular, an effective substitute for Ca2+ and Mg2+ (11,12). VO2+ is an ideal substitute for Ca2+ because VO2+, like Ca2+, has a high affinity for binding to oxygen-donor ligands (7). By EPR spectrometric titration, we have shown that VO2+ binds to NC-A with a stoichiometry of 4 mol/mol of protein. Furthermore, we have also demonstrated that VO2+ competes with Ca2+ in binding to NC. An unusual result of this preliminary study of VO2+-NC complexes obtained by ENDOR spectroscopy showed that the coordinating ligands to the metal ions are from protein residues with complete exclusion of solvent water from the inner coordination sphere. EXPERIMENTAL PROCEDURES Materials. NC was isolated from fresh bovine kidney tissue and separated into four fractions by DEAE-cellulose chromatography (Whatman DE-52), as described (1). These fractions were eluted by varying the ionic strength: NC-A at 10-15 inS, NC-B at 16-23 mS, NC-C at 24-27 mS, and NC-D at 32-38 inS, using a linear gradient ofNaCl from 0.1 M to 0.5 M in 0.05 M Tris HCl at pH 7.3. Fraction A (NC-At), which exhibits the highest affinity for Ca2+ binding, was used in this study. This fraction of protein has been found in normal (non-kidney stone) subjects and can be used as a model system for a strong inhibitor protein. Fraction A of NC was first eluted...

Structural Origins of the Insulin-mimetic Activity of Bis(acetylacetonato)oxovanadium(IV)

“…Only in aqueous solution is the fourth species D observed. Its spectrum yields g-and A-values identical to those of the [VO(H 2 O) 5 ] 2ϩ ion (9,26,28). However, no previous study has shown the equivalence of species A, B, and C in aqueous solution to AЈ, BЈ, and CЈ, respectively, in methanol.…”

“…EPR and ENDOR spectra were recorded with an X-band Bruker ESP 300E spectrometer equipped with a cylindrical TE 011 ENDOR cavity, an Oxford Instruments ESR910 liquid helium cryostat, and Bruker ENDOR digital accessory as described previously (25,26). The ESP 300E spectrometer was equipped with a complete computer interface (Bruker ESP3220 data system) for spectrometer control and data acquisition and processing.…”

Fatal hemorrhage in mice lacking γ-glutamyl carboxylase

“…However, the identification of additional substrates with diverse functions such as the bone proteins osteocalcin and MGP, the kidney proteins nephrocalcin A-D, and the signaling molecule GAS6, [13][14][15][16] together with the expression of ␥-carboxylase in multiple tissues, 3 all suggest a broader function for this posttranslational modification, in addition to its role in forming the membrane-binding domains of the vitamin K-dependent blood coagulation and regulatory proteins. 37 Further support for this notion comes from the identification of the conservation of the ␥-carboxylase gene in Ciona intestinalis and other chordates, 5 as well as in Drosophila and Conus snail species, 6,7 organisms which arose well before the evolution of the known vitamin K-dependent coagulation factors.…”

“…Of note, knockout mice with complete deficiency of prothrombinase or prothrombin exhibit an embryonic lethal phenotype in approximately one-half of animals, possibly due to loss of a required early developmental signal through the thrombin receptor, with the remaining mice dying at term of massive hemorrhage. [9][10][11][12] Other mammalian proteins known to undergo ␥-carboxylation include osteocalcin, matrix Gla protein (MGP), 13 Gas6, 14 nephrocalcin A-D, 15 the proline-rich ␥-carboxylated proteins (PRGP-1 and PRGP-2), 16 and the transmembrane proteins TMG3 and TMG4. 17 ␥-Carboxylation is absolutely required for function of the vitamin K-dependent blood coagulation factors.…”

Urinary Kidney Stone Inhibitors. What Is the New?

“…Nephrocalcin (NC) also inhibits CaOx nucleation and aggregation, and crystal adhesion to renal cells [6,9]. One mole of NC binds 4 mol of Ca 2+ , and its binding sites differ completely from those in other Ca 2+ -binding proteins [10]. NC is produced in the human kidney by the proximal tubule and the thick ascending limb of Henle's loop.…”