Phosphorylation of osteopontin is required for inhibition of calcium oxalate crystallization.
Under near-physiological pH, temperature, and ionic strength, a kinetics constant composition (CC) method was used to examine the roles of phosphorylation of a 14 amino acid segment (DDVDDTDDSHQSDE) corresponding to potential crystal binding domains within the osteopontin (OPN) sequence. The phosphorylated 14-mer OPN peptide segment significantly inhibits both the nucleation and growth of calcium oxalate monohydrate (COM), inhibiting nucleation by markedly increasing induction times and delaying subsequent growth by at least 50% at concentrations less than 44 nM. Molecular modeling predicts that the doubly phosphorylated peptide binds much more strongly to both (-101) and (010) faces of COM. The estimated binding energies are, in part, consistent with the CC experimental observations. Circular dichroism spectroscopy indicates that phosphorylation does not result in conformational changes in the secondary peptide structure, suggesting that the local binding of negatively charged phosphate side chains to crystal faces controls growth inhibition. These in vitro results reveal that the interactions between phosphorylated peptide and COM crystal faces are predominantly electrostatic, further supporting the importance of macromolecules rich in anionic side chains in the inhibition of kidney stone formation. In addition, the phosphorylation-deficient form of this segment fails to inhibit COM crystal growth up to concentrations of 1450 nM. However, at sufficiently high concentrations, this nonphosphorylated segment promotes COM nucleation. Dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) results confirm that aggregation of the nonphosphorylated peptide segment takes place in solution above 900 nM when the aggregated peptide particles may exceed a well-defined minimum size to be effective crystallization promoters.
Wang
Lijun
L
Department of Chemistry, The State University of New York at Buffalo, Amherst, New York 14260, USA.
Guan
Xiangying
X
Tang
Ruikang
R
Hoyer
John R
JR
Wierzbicki
Andrzej
A
De Yoreo
James J
JJ
Nancollas
George H
GH
D000595:Amino Acid Sequence; D002129:Calcium Oxalate / Q000737:chemistry*; D002942:Circular Dichroism; D003460:Crystallization; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009994:Osmolar Concentration; D053495:Osteopontin / Q000737:chemistry* / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D053838:Scattering, Small Angle; D013696:Temperature; D014961:X-Ray Diffraction
D010455:Peptides; D053495:Osteopontin; D002129:Calcium Oxalate