Identification of structural domains in inter-α-trypsin inhibitor involved in calcium oxalate crystallization

Abstract

The urinary glycoprotein that inhibits calcium oxalate (CaOx) crystallization in vitro shows a structural similarity to urinary trypsin inhibitor (UTI; recently termed bikunin), the light chain of inter-α- trypsin inhibitor (IαI). The functional domains of IαI involved in its inhibitory activity of CaOx crystallization have been investigated using isolated intact domains of IαI produced from controlled proteolytic digests of the protein. The fragments investigated include the heavy chains of IαI, UTI, chondroitinase AC-treated UTI, and the carboxyl-terminal domain of UTI (termed HI-8). The effects of IαI and its fragments on the inhibitory activity of CaOx crystallization were evaluated in vitro using CaOx crystal aggregation and growth assays, and seeded crystal generation assay as well as using crystal matrix protein generation assay. UTI, but not the heavy chains of IαI, had a discernible effect on CaOx crystallization inhibitory activity. Less requirement of the carbohydrate moiety of UTI is implicated by the observation that chondroitinase AC-treated UTI fragment was also found to inhibit CaOx crystallization with almost the same activity as UTI. HI-8 also efficiently inhibited CaOx crystallization, while IαI showed a weak inhibitory activity. The results are almost consistent with a seed crystal generation assay and a crystal adsorption inhibition assay, in which IαI or its derivatives inhibits prothrombin fragment 1 (F1) adsorption to CaOx crystals. In conclusion, these results suggest that the part of the IαI protein responsible for inhibition of CaOx crystallization is the carboxyl- terminal domain of UTI.