Phosphorylated osteopontin peptides suppress crystallization by inhibitingthe growth of calcium oxalate crystals

Background. Osteopontin isolated from human urine [uropontin (uOPN)] is a p otent inhibitor of calcium oxalate (CaOx) monohydrate (COM) crystallization . However. specific structural features responsible for its effects on CaOx crystallization were not previously known. The present studies were design ed to define molecular features responsible for interactions of uOPN with C OM crystals and the inhibition of crystallization. Methods. Peptides and phosphopeptides with sequences corresponding to poten tial crystal binding domains within the protein sequence of osteopontin wer e synthesized. Then the effects of these peptides on COM crystal growth and crystal aggregation were investigated and their secondary structures analy zed. Results. Growth of COM crystals was inhibited by similar to 50% at 1000 nmo l/L concentrations by the two unmodified peptides with the closest clusteri ng of aspartic acid residues. Growth was not inhibited by the other two unm odified peptides, with aspartic residues more evenly distributed within the ir sequences. Phosphorylation markedly increased inhibition of COM crystal growth, so that each of the four phosphorylated peptides inhibited growth b y at least 50% at concentrations of less than or equal to 200 nmol/L. Phosp horylation of these peptides did not cause changes in secondary structure t hat would favor interaction with COM crystal surfaces. Conclusions. These studies of synthetic peptides identify molecular feature s within the osteopontin molecule that contribute to the inhibition of one aspect of COM crystallization. The inhibition of crystal growth induced by phosphorylation appears to result from altered local patterns of charge den sity, since conformational changes favoring interaction with crystals were not caused by phosphorylation.