Epitaxial deposition of calcium oxalate on uric acid rich stone matrix is induced by a 29 kDa protein.
BACKGROUND
Association of macromolecules particularly the role of proteins in urolithiasis has been studied for last few centuries, but still a complete profile of stone matrix proteins that mediate co-precipitation of uric acid and calcium oxalate has not been characterized. We isolated and characterize proteins from uric acid rich stone matrix, which have oxalate binding activity.


METHODS
Matrix proteins were isolated from uric acid rich stone matrix using EDTA as a demineralizing agent. The radiolabelled solubilized proteins were fractionated with increasing ionic concentration by DEAE cellulose column chromatography to identify the oxalate binding protein. It was purified using Sephadex G-200 column chromatography. Amino acid composition was determined and monoclonal antibody was produced against the oxalate binding uric acid rich stone matrix protein. Urinary uric acid binding proteins were isolated from stone formers urine, their oxalate binding activity assayed and cross reactivity with the produced monoclonal antibody were checked using ELISA and Western blotting.


RESULTS
Matrix on DEAE column chromatography elution yielded 3 protein peaks and they were named as fraction I, II and III among which fraction I had higher oxalate binding activity which was further purified with Sephadex G-200 column which yielded 2 protein peaks designated as Ia and Ib. Fraction Ib with molecular weight 29 kDa exhibited the maximum oxalate binding activity. Forty percent of this 29 kDa protein is comprised of basic amino acids. Monoclonal antibody (IgG1) was produced against the 29 kDa stone matrix protein. Urinary uric acid binding proteins were isolated from stone formers, 4 protein peaks were obtained named as fraction I to IV. Among them, fraction IV having molecular weight of approximately 29 kDa cross reacted up to 85.6% with 29 kDa stone matrix protein. Moreover, urinary 29 kDa protein exhibited oxalate binding activity of 94.16 +/- 6.08 pmol/mg protein at pH 5.5.


CONCLUSIONS
The 29 kDa protein isolated from uric acid rich stone matrix and urine are one and the same, thereby insinuating that 29 kDa protein might play a major role in epitaxial deposition of calcium oxalate over uric acid core, consequently favoring the lithogenic events like uric acid and calcium oxalate nucleation, aggregation and retention.
Srinivasan
S
S
Department of Medical Biochemistry, Dr. A.L. Mudaliar, Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai 600 113, India. srini@musc.edu
Kalaiselvi
P
P
Varalakshmi
P
P
D000818:Animals; D015153:Blotting, Western; D002129:Calcium Oxalate / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007669:Kidney Calculi / Q000737:chemistry / Q000378:metabolism* / Q000652:urine; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D029543:Nucleocytoplasmic Transport Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D014527:Uric Acid / Q000737:chemistry / Q000378:metabolism*
D002352:Carrier Proteins; D029543:Nucleocytoplasmic Transport Proteins; D011506:Proteins; C477940:oxalate binding protein, human; D002129:Calcium Oxalate; D014527:Uric Acid