The majority of human urinary stones are primarily composed of calcium salts. Although normal urine is frequently supersaturated with respect to calcium oxalate, most humans do not form stones. Inhibitors are among the multiple factors that may influence the complex process of urinary stone formation. We have isolated an inhibitor of calcium oxalate crystal growth from human urine by monoclonal antibody immunoaffinity chromatography. The N-terminal amino acid sequence and acidic amino acid content of this aspartic acid-rich protein, uropontin, are similar to those of other pontin proteins from bone, plasma, breast milk, and cells. The inhibitory effect of uropontin on calcium oxalate crystal growth in vitro supports the concept that pontins may have a regulatory role. This function would be analogous to that of other members of the aspartic acid-rich protein superfamily, which stereospecifically regulate the mineralization fronts of calcium-containing crystals.Urinary tract stone disease is a common human malady, and the vast majority of stones formed in the urinary space are mineralized with calcium salts (1, 2). The elements contained in urine also provide a potential model system for evaluating the biologic control of mineralization in other body fluids. Although normal urine is frequently supersaturated with respect to calcium oxalate, most humans do not form stones. Urinary stone formation is a complex process involving multiple factors, and the precise role of the inhibitors that are present within urine is uncertain. The majority of the inhibition of crystal growth observed in normal urine is due to the presence of protein macromolecules rather than to the presence of lower molecular weight molecules (3). We approached the problem of identifying other crystal inhibitor proteins by preparing monoclonal antibodies from rats immunized with the main inhibitory peak of human urine protein (3). One of these monoclonal antibodies was used to purify an inhibitor of calcium oxalate crystal growth from human urine by immunoaffinity chromatography. METHODS Protein Purification. Human urine samples were carried through all procedures in the presence of 0.02% sodium azide and two protease inhibitors, 0.5 mM phenylmethanesulfonyl fluoride and 1.0 mM N-ethylmaleimide, and were partially depleted of the most abundant protein in normal urine, Tamm-Horsfall protein (TH), by salt precipitation followed by centrifugation at 5000 x g for 30 min (4). TH-depleted urine was adsorbed to DEAE-cellulose, batch eluted, and fractionated by DEAE-cellulose column chromatography, using a 0.1-0.4 M NaCl linear gradient in Tris buffer (3). The TH depletion step was performed since we found that TH is present within the main inhibitory peak from DEAE-cellulose and does not inhibit crystal growth in the assay used in the present study (5). Inhibitory activity of fractions was assayed by measuring the inhibition of incorporation of [14C]oxalate (Amersham) into calcium oxalate monohydrate seed crystals from a metastable calcium oxalat...

Fibulin-1 is a modular glycoprotein with amino-terminal anaphylatoxin-like modules followed by nine epidermal growth factor (EGF)-like modules and, depending on alternative splicing, four possible carboxyl termini. Fibulin-1 has been shown to self-associate as well as to bind calcium, fibronectin (FN), laminin, nidogen, and fibrinogen. To map ligand-binding sites within fibulin-1, polypeptides corresponding to various regions of fibulin-1 were expressed recombinantly and evaluated for their capacity to bind calcium, FN, or fibulin-1. A calcium-binding site(s) was mapped to EGF-like modules 5-9. A fibulin-1 self-association site was localized to EGF-like modules 5 and 6 (amino acid residues 356 -440), as was a binding site for FN. The self-association interaction mediated by this pair of modules involved calcium since divalent cation chelators reduced the binding affinity of the interaction. By contrast, FN binding to EGF-like modules 5 and 6 was unaffected by the presence of divalent cation chelators. It can be concluded that EGF-like modules 5 and 6 bind calcium and mediate homotypic interaction between EGF-like modules 5 and 6 present in different fibulin-1 molecules and heterotypic interaction between EGF-like modules 5 and 6 and type III repeats 13 and 14 in FN. While additional binding sites for calcium or FN were not detected, another fibulin-1 self-association site was found within amino acid residues 30 -173. However, unlike the self-association site in EGF-like modules 5 and 6, which was functional in the native protein, the amino-terminal site was cryptic and revealed only after the protein was denatured.

The a-ketoglutarate-dependent dioxygenase aspartyl (asparaginyl) J-hydroxylase (EC 1.14. (18,19). A cDNA fragment (nt 1-1033) was deleted from bovine Asp (Asn) /3hydroxylase cDNA by digestion with Pst I and ligated to form pNC3d-1. A 1.4-kb cDNA insert [nt 1034-2498, containing a 233-bp 3' untranslated region (19)] resulting from Xba I/Xho I digestion was isolated and then cloned into an E. coli expression vector, pFLAG-1 (IBI) (20), to form p52-1.Since this 1.4-kb cDNA insert lacked nt 931-1033, a synthetic double-stranded DNA fragment containing these nucleotides, a HindIII site at the 5' end, and a Pst I site at the 3' end was inserted into p52-1 to form the expression plasmid p52. The cDNA reading frame (nt 931-2265) ofp52 was confirmed as follows: p52 was then used to transform E. coli DHSaF'IQ (BRL); it was then purified and the 5' ends ofthe cDNA insert were sequenced. Purified p52 was introduced into both a normal E. coli strain and a protease-deficient strain (BL21; Novagen).Expression of P52. A p52 transformant was grown at 37TC in 3 x LB medium (30 g oftryptone, 15 g of yeast extract, and 5 g of NaCl per liter, pH 7.5) to an OD595 of 0.4-0.6. The culture was diluted 1:40 with 3x LB and incubated at 3rC until an OD595 of 1.5-1.8 was attained. Expression was induced with 1 mM isopropyl P-D-thiogalactopyranoside for 4 hr at 28C.For the purification of P52 (Table 1), 300 g of frozen cellswere resuspended in 1.2 liters of ice-cold lysis buffer (50 mM Tris-HCI, pH 7.5/0.1% Nonidet P-40/3 mM EDTA with protease inhibitors) and lysed. Protease inhibitor concentrations were as follows: aprotinin, 3.0 Ig/ml; leupeptin, 1.5 gg/ml; pepstatin, 2.1 pg/ml; phenylmethylsulfonyl fluoride, 0.6 mM; soybean trypsin inhibitor, 0.01%; benzamidine, 10Abbreviations: EGF, epidermal growth factor; P52, 52-kDa recombinant Asp (Asn) P-hydroxylase.

Factor VII (F.VII) is a vitamin-K-dependent serine protease required in the early stages of blood coagulation. We describe here a patient with severe F.VII deficiency, with a normal plasma F.VII antigen level (452 ng/mL) and F.VII activity less than 1%, who is homozygous for two defects: a G-->A transition at nucleotide 6055 in exon 4, which results in an Arg-->Gln change at amino acid 79 (R79Q); and a G-->A transition at nucleotide 8961 in exon 6, which results in an Arg-->Gln substitution at amino acid 152 (R152Q). The R79Q mutation occurs in the first epidermal growth factor (EGF)-like domain, which has previously been implicated in binding to tissue factor. The R152Q mutation occurs at a site (Arg 152-Ile 153) that is normally cleaved to generate activated F.VII (F.VIIa). Analysis of purified F.VII from patient plasma shows that the material cannot be activated by F.Xa and cofactors. In addition, in an in vitro binding assay using relipidated recombinant tissue factor, patient plasma showed markedly reduced binding to tissue factor at all concentrations tested. In an effort to separate the contributions of the two mutations, three recombinant variants, wild-type, R79Q, and R152Q, were prepared and analyzed. The R152Q variant had markedly reduced activity in a clotting assay, whereas R79Q showed a milder, concentration-dependent reduction. The R152Q variant exhibited nearly normal binding in the tissue factor binding assay, whereas the R79Q variant had markedly reduced binding. The time course of activation of the R79Q variant was slowed compared with wild-type. Our results suggest that the first EGF-like domain is required for binding to tissue factor and that the F.VII zymogen lacks activity and requires activation for expression of biologic activity.

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