Identification of Uronic‐Acid‐Rich Protein as Urinary Bikunin, the Light Chain of Inter‐α‐Inhibitor

Abstract

Uronic-acid-rich protein (UAP) is a urinary glycoprotein that inhibits calcium oxalate crystallization in vitro. It shows a structural similarity to bikunin, a component of inter-a-inhibitor (Id) known for its inhibition of the action of many serine proteinases like trypsin and chymotrypsin. To clarify the relationship between these macromolecules, UAP, IaI, urinary bikunin, and plasma bikunin were purified and studied. Their calcium oxalate crystallization inhibitory activity was assayed before and after treatment with chondroitinase AC and pronase. Their molecular mass was determined by using SDS/PAGE before and after these treatments. Polyclonal bikunin antibody was used on Western blots for immunological identification. The partial amino acid sequence of UAP before and after chondroitinase treatment was determined. Also, the antitryptic activity of UAP was measured and compared to that of bikunin, which is responsible for the antiprotease activity of IaI. UAP exhibited a strong calcium oxalate crystallization inhibitory activity. I d and both bikunins were less inhibitory. Chondroitinase AC had no effect on inhibitory activity of these proteins even when their molecular mass changed. However, after pronase treatment, the inhibitory activity of both bikunins and UAP was completely destroyed. The antitryptic activity of UAP was found to be 0.78 U/mg which is lower than that of bikunin which is about 1.9 U/mg. On Western blotting, bikunin antibody immunoreacted with UAP and both urinary and plasma bikunins. Partial amino acid sequence confirmed the identity of UAP as urinary bikumin.Keywords: nephrolithiasis ; calcium oxalate ; inter-a-inhibitor ; uronic-acid-rich protein ; bikunin.Kidneys excrete a urine normally supersaturated with respect to many salts including calcium oxalate, the most common compound found in kidney stones [l, 21. But normal urine does not support crystallization because of the presence of many natural inhibitors. Purification, identification, and characterization of such macromolecules have been the object of extensive studies [3 -91. Among these inhibitors is uronic-acid-rich protein (UAP), a glycoprotein of 35 kDa isolated from human [3] and rat urine [ 101 which strongly inhibits calcium oxalate crystallization in vitro. The same inhibitor isolated from the urine of stone formers showed less inhibitory activity when compared to that purified from the urine of healthy subjects [ll]. This result suggests a structural abnormality of UAP obtained from stone-forming patients. Interestingly, both human and rat UAP exhibited structural similarity to bikunin, a subunit of inter-a-inhibitor ( I d ) [lo, 121. Indeed, the sequence of the first 18 amino acid residues of UAP was identical to I d and bikunin. Furthermore, on Western blotting, the immuoreaction between UAP and I d antibody was positive [lo].IaI, with a molecular mass of approximately 220 kDa, is one of the plasma protease inhibitors known formerly as inter-a-trypsin inhibitor (ITI be the active part of I d involved in the inhibition...

Inter-α-trypsin Inhibitor, a Covalent Protein-Glycosaminoglycan-Protein Complex

“…One member, the urinary trypsin inhibitor (UTI), was purified much later in the 1950s (1, 2) when it was independently isolated by researchers working on mucopolysaccharides, leukemia, and urolithiasis and was given many names: acid-stable protease inhibitor, prealbumin-like protease inhibitor, urinastatin, HI30, mingin, EDC1, uronic acid-rich protein, and nephrocalin (3)(4)(5). Based on the presence of two tandem Kunitz-type protease inhibitory domains, a structure-based name, bikunin, was suggested in 1990 to avoid confusion (6).…”

Interactions between Stone-Forming Calcific Crystals and Macromolecules

“…specifically with bikunin. UAP was recently confirmed to be bikunin by western blot analysis studies using polyclonal bikunin antibody [11].…”

Acidic polyanion poly(acrylic acid) prevents calcium oxalate crystal deposition

“…4 Acidic urinary macromolecules that have been studied individually interact strongly with crystals and generally inhibit crystal formation, especially those of calcium oxalate (CaOx), in vitro. [5][6][7][8][9][10][11][12] Synthetic polyanions have similar effects on CaOx formation; however, polycations and neutral polymers are, in general, ineffective. [13][14][15][16][17] Among the synthetic polyanions, those with carboxylate side chains are more potent at inhibiting crystal formation than phosphoryl or sulfonyl, in that order.…”