Identification of structural domains in inter-alpha-trypsin involved in calcium oxalate crystallization.
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-alpha-trypsin inhibitor (I alpha I). The functional domains of I alpha I involved in its inhibitory activity of CaOx crystallization have been investigated using isolated intact domains of I alpha I produced from controlled proteolytic digests of the protein. The fragments investigated include the heavy chains of I alpha I, UTI, chondroitinase AC-treated UTI, and the carboxyl-terminal domain of UTI (termed HI-8). The effects of I alpha I 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 I alpha I, 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 I alpha I showed a weak inhibitory activity. The results are almost consistent with a seed crystal generation assay and a crystal adsorption inhibition assay, in which I alpha I or its derivatives inhibits prothrombin fragment 1 (F1) adsorption to CaOx crystals. In conclusion, these results suggest that the part of the I alpha I protein responsible for inhibition of CaOx crystallization is the carboxyl-terminal domain of UTI.
Kobayashi
H
H
Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Shizuoka, Japan.
Shibata
K
K
Fujie
M
M
Sugino
D
D
Terao
T
T
D000510:Alpha-Globulins / Q000737:chemistry* / Q000494:pharmacology*; D002129:Calcium Oxalate / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000652:urine; D002808:Chondroitin Lyases / Q000494:pharmacology; D003460:Crystallization; D006023:Glycoproteins / Q000187:drug effects / Q000494:pharmacology; D006801:Humans; D008562:Membrane Glycoproteins; D009994:Osmolar Concentration; D011506:Proteins / Q000032:analysis; D011516:Prothrombin / Q000378:metabolism; D014360:Trypsin Inhibitor, Kunitz Soybean
D000510:Alpha-Globulins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D011506:Proteins; C109148:SPINT2 protein, human; C083359:crystal matrix protein, human; D002129:Calcium Oxalate; C011877:inter-alpha-inhibitor; D011516:Prothrombin; D014360:Trypsin Inhibitor, Kunitz Soybean; D002808:Chondroitin Lyases; C028665:urinastatin