Cooperativity in Calcium Ion Binding to Repetitive, Carboxylate‐serylphosphate Polypeptides and the Relationship of This Property to Dentin Mineralization

Abstract

Analogs of phosphophoryn, a calcium‐binding phosphorylated protein found in mineralized dentin, were synthesized by solid phase peptide synthesis. The dentin phosphophoryn appears to contain some sequence blocks of (Asp‐PhosphoSer)n. As this sequence is difficult to synthesize, polymers of (α‐L‐Glu‐L‐Ser) were prepared. The 30‐peptide, (α‐L‐Glu‐L‐Ser)15, was phosphorylated by reaction with orthophosphoric acid in the presence of trichloroacetonitrile in anhydrous dimethylsulfoxide. Calcium ion binding studies were carried out with both the 30‐peptide and phosphorylated 30‐peptide. The conformation of the original 30‐peptide, (Glu‐Ser)15, was examined, in the presence and absence of calcium ion, by circular dichroism measurements. Purified bovine phosphophoryn, previously studied by the same techniques, was partially dephosphorylated by alkaline phosphatase, and its calcium ion binding properties were determined. Dephosphorylation to 31% of the initial phosphorus content reduced the number of high affinity sites to ˜ 30% of the initial value. However, the stoichiometry of binding indicated that both phosphate and carboxylate groups participate in the high affinity binding and that the binding constant was decreased only slightly. Partial phosphorylation of the 30‐mer raised the calcium binding constant, Ka, from 2.1 times 102 to 3.3 × 103 M‐1 and increased the amount of binding from an electrostatic equivalent number of sites to a stoichiometric equivalent number. Concomitant with binding, there was a transition from random coil to ß‐like structure. These data suggest that the repetitive (Asp‐PhosphoSer)n regions in phosphophoryn and the (Glu‐PhosphoSer)n sequence of the synthetic polymer have special conformations which favor the unidentate binding of calcium to the carboxyl groups and phosphate groups, and which enhance the binding affinities of the carboxyl groups in such sequences in a cooperative fashion.

Specific Binding and Mineralization of Calcified Surfaces by Small Peptides

“…Proteins involved in mineralization processes in vertebrates (including DPP) often contain negatively charged, Asp- or Glu-rich domains ([ 21 ], and references within). This observation combined with computational and biochemical studies of the (DSS) n repeat region [ 2 , 3 , 13 , 15 ] indicated that this short repeated sequence governs the mineral binding and nucleation activity of DPP.…”

“…In addition, high concentrations of DPP inhibit HA crystal growth [ 4 ]. These activities have been attributed to an aspartate- and serine-rich region within the DPP sequence [ 2 , 3 , 13 ] consisting primarily of a large number of repeats of the sequence Asp-Ser-Ser [ 9 ]. This region is known to be highly flexible [ 14 ] and highly phosphorylated [ 3 ] and is thought to nucleate HA crystal formation by providing an optimal alignment of calcium ion binding sites (or, at high enough concentrations, to cap the surface of existing crystals to prevent further growth).…”

“…While some efforts have been made to explore the role of this region in biomineralization, using synthetic peptides consisting of alternating aspartate and phosphoserine residues [ 3 ] or short highly phosphorylated peptides derived directly from the DPP sequence [ 15 ], we chose to explore the nature of the fundamental repeat unit within DPP, Asp-Ser-Ser (DSS, using the single-letter amino acid symbols), in the absence of serine phosphorylation. Small peptides with varying numbers of DSS repeats have been synthesized, and their roles in the binding and deposition of calcium phosphate have been analyzed.…”

Casein Phosphopeptide–Amorphous Calcium Phosphate Nanocomplexes: A Structural Model

“…The CPP are formed in vivo by normal digestion of casein and, as they are relatively resistant to further proteolytic degradation, accumulate in the distal portion of the small intestine [17][18][19][20][21] . It has been proposed that this accumulation together with the peptides' ability to form soluble complexes with calcium phosphate are responsible for the enhanced intestinal calcium absorption that has been observed even in vitamin D deficient animals consuming dietary CPP [17][18][19][20][21] . In addition, CPP increase the calcification of in vitro cultured embryonic rat bone and again the mechanism is suggested to be associated with the peptide's ability to form soluble complexes with calcium and phosphate ions 22 .…”

Bone regeneration strategy inspired by the study of calcification behavior in deer antler

“…zones". These hole zones are rich in phosphate groups and glycoproteins, creating a local charge accumulation for calcium-rich mineral nuclei and initiation of mineral growth [49][50][51]. Therefore, the similar calcification could contribute to gain a sight in the biomineralization.…”