Inhibition of calcium oxalate crystal growth in vitro by uropontin: another member of the aspartic acid-rich protein superfamily.

Abstract

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...

Inhibitory effect of calcium phosphate‐associated proteins on calcium oxalate crystallization: α2‐HS‐glycoprotein, prothrombin‐F1 and osteopontin

“…Interestingly, one of the amino-terminal sequences of the present peak III protein started at position 19 of human osteopontin. The molecular weight of uropontin cannot be estimated by SDS-PAGE and immunoblotting analysis because the migration of uropontin on SDS-PAGE varies with gel composition [30]. The present results are consistent with osteopontin (uropontin).…”

“…This protein is not stained by CBB but is stained by`Stains-All' [29]. This protein was isolated from urine as an inhibitor of CaOx crystal growth by Shiraga et al [30], who named its uropontin because its N-terminal amino-acid sequence starts at position 19 of osteopontin. Interestingly, one of the amino-terminal sequences of the present peak III protein started at position 19 of human osteopontin.…”

The Role of Osteopontin in Tumor Progression and Metastasis in Breast Cancer

“…Osteopontin was localized to the luminal surfaces in these sites, as well as in human secretions including blood, milk (28,29), and urine (30). Overall, these findings suggested that osteopontin might have a protective role in interactions between epithelial surfaces and the external environment.…”

Detection of osteopontin on Holstein bull spermatozoa, in cauda epididymal fluid and testis homogenates, and its potential role in bovine fertilization

“…OPN contains a calcium-binding site and causes calcium release in osteoclasts through an integrin-stimulated IP 3 pathway (Denhardt & Guo 1993). When secreted into the proximal tubule of the mouse nephron, OPN suppressed the accumulation of calcium oxalate crystals, most likely by interfering with the crystallization process (Shiraga et al 1992). Calcium crystal deposits have also been shown in human rete testis, efferent ducts, and epididymis (Nistal et al 1989(Nistal et al , 1996.…”