Graeme K. Hunter

Enamel defects and ameloblast-specific expression in Enam knock-out/lacz knock-in mice.

Enamelin is critical for proper dental enamel formation, and defects in the human enamelin gene cause autosomal dominant amelogenesis imperfecta. We used gene targeting to generate a knock-in mouse carrying a null allele of enamelin (Enam) that has a lacZ reporter gene replacing the Enam translation initiation site and gene sequences through exon 7. Correct targeting of the transgene was confirmed by Southern blotting and PCR analyses. No enamelin protein could be detected by Western blotting in the Enam-null mice. Histochemical 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) staining demonstrated ameloblast-specific expression of enamelin. The enamel of the Enam+/- mice was nearly normal in the maxillary incisors, but the mandibular incisors were discolored and tended to wear rapidly where they contacted the maxillary incisors. The Enam-/- mice showed no true enamel. Radiography, microcomputed tomography, and light and scanning electron microscopy were used to document changes in the enamel of Enam-/- mice but did not discern any perturbations of bone, dentin, or any other tissue besides the enamel layer. Although a thick layer of enamel proteins covered normal-appearing dentin of unerupted teeth, von Kossa staining revealed almost a complete absence of mineral formation in this protein layer. However, a thin, highly irregular, mineralized crust covered the dentin on erupted teeth, apparently arising from the formation and fusion of small mineralization foci (calcospherites) in the deeper part of the accumulated enamel protein layer. These results demonstrate ameloblast-specific expression of enamelin and reveal that enamelin is essential for proper enamel matrix organization and mineralization.

Binding of bone sialoprotein, osteopontin and synthetic polypeptides to hydroxyapatite.

The phosphorylated acidic glycoproteins bone sialoprotein (BSP) and osteopontin (OPN) bind to hydroxyapatite (HA) crystals and may be involved in the regulation of bone mineralization. The HA-binding properties of these proteins have been attributed to glutamic acid-rich sequences in BSP and aspartic acid-rich sequences in OPN. The present study examines the roles of these polycarboxylate sequences in the binding of BSP and OPN to HA. Porcine BSP, OPN and the synthetic polypeptides poly-L-glutamic acid [Poly(Glu)] and poly-L-aspartic acid [Poly(Asp)] were labeled with fluorescein isothiocyanate and their binding to HA determined by fluorimetry. From the binding isotherms, dissociation constants (KDs) for all the reagents tested were determined to be in the micromolar range. The saturation binding capacities of HA for Poly(Glu), Poly(Asp), BSP and OPN were similar (500–600 μg/m2). To investigate the role of glutamic acid-rich and aspartic acid-rich sequences in the binding to HA of BSP and OPN, respectively, competitive binding studies with Poly(Glu) and Poly(Asp) were performed. Poly(Glu) was able to displace a maximum of 100% of Poly(GIu), 81% of OPN, 68% of BSP and 65% of Poly(Asp). Poly(Asp) was able to displace a maximum of 100% of Poly(Glu), 99% of Poly(Asp), 95% of OPN and 89% of BSP. These results are consistent with the view that BSP and OPN bind to HA via their polycarboxylate sequences, but suggest a complex mode of interaction between polyelectrolytes and ionic crystals.

Bone sialoprotein-collagen interaction promotes hydroxyapatite nucleation.

In bone, hydroxyapatite (HA) crystals are deposited onto the type I collagen scaffold by a mechanism that has yet to be elucidated. Bone sialoprotein (BSP) is an acidic phosphoprotein that is expressed at high levels in mineralized tissues, capable of binding type I collagen, and nucleating HA. Both bone-extracted and recombinant BSP (rBSP) bind with equal affinity to collagen. The nature of the BSP-collagen interaction and its role in HA nucleation are not known. We have used a solid-phase binding assay and affinity chromatography to characterize the BSP-collagen interaction. rBSP-binding affinities of triple-helical and fibrillar type I collagen were similar (K(D) approximately 13 nM), while that of heat-denatured type I collagen was lower (K(D) approximately 44 nM), indicating the importance of triple-helical structure in binding BSP. Pepsin treatment of collagen had no effect on rBSP binding, demonstrating that the telopeptides of collagen are not involved. The majority of collagen-bound rBSP was eluted by acetonitrile, indicating that hydrophobic interactions are principally responsible for binding. Using an HA-nucleation assay, it was shown that rBSP is ten-fold more potent in reconstituted fibrillar collagen gels than in agarose gels. Nucleating potency of a non-collagen-binding, HA-nucleating peptide [rBSP(134-206)] showed no difference in the two gel systems. The work here shows that optimal binding of rBSP requires collagen to be in a native, triple-helical structure, does not require the telopeptides, and is stabilized by hydrophobic interactions. Upon binding to collagen, rBSP displays an increase in nucleation potency, implying a co-operative effect of BSP and collagen in mineral formation.

Identification of the Type I Collagen-binding Domain of Bone Sialoprotein and Characterization of the Mechanism of Interaction

Bone sialoprotein (BSP) is an anionic phosphorylated glycoprotein that is expressed almost exclusively in mineralized tissues and has been shown to be a potent nucleator of hydroxyapatite formation. The binding of BSP to collagen is thought to be important for the initiation of bone mineralization and in the adhesion of bone cells to the mineralized matrix. Using a solid phase assay, we have investigated the interaction between BSP and collagen. Initial studies showed that raising the ionic strength, decreasing the pH below 7, or introducing divalent cations diminishes but does not abolish the binding of BSP to collagen, indicating that the interaction is only partly electrostatic in nature. Both bone-extracted and recombinant (r)BSP exhibited similar binding affinities, indicating that post-translational modifications are not critical for binding. To identify the collagen-binding domain, recombinant peptides of BSP were studied. Peptide rBSP-(1–100) binds to type I collagen with an affinity similar to that of full-length rBSP, whereas peptides containing the sequences 99–201 or 200–301 do not bind. Further studies showed that rBSP-(1–75) competitively inhibits the binding of rBSP-(1–100), whereas rBSP-(21–100) inhibits binding to a lesser extent, and rBSP-(43–100) does not inhibit binding. These results suggest that the collagen-binding site of rat BSP is within the sequence 21–42, with residues N-terminal of this region likely also involved. This site was confirmed by the demonstration of collagen-binding activity of a synthetic peptide corresponding to residues 19–46. The collagen-binding domain, which is highly conserved among species, is enriched in hydrophobic residues and lacks acidic residues. We conclude that residues 19–46 of BSP represent a novel collagen-binding site.

Functional Analysis of Bone Sialoprotein: Identification of the Hydroxyapatite-nucleating and Cell-binding Domains by Recombinant Peptide Expression and Site-directed Mutagenesis

Mammalian bone sialoprotein (BSP) is a mineralized tissue-specific protein containing an RGD (arginine-glycine-aspartic acid) cell-attachment sequence and two distinct glutamic acid (glu)-rich regions, with each containing one contiguous glu sequence. These regions have been proposed to contribute to the attachment of bone cells to the extracellular matrix and to the nucleation of hydroxyapatite (HA), respectively. To further delineate the domains responsible for these activities, porcine BSP cDNA was used to construct expression vectors coding for two partial-length recombinant BSP peptides: P2S (residues 42-87), containing the first glutamic acid-rich domain; and P1L (residues 69-300), containing the second glutamic acid-rich region and the RGD sequence. These peptides were expressed in Escherichia coli as his-tag fusion proteins and purified by nickel affinity columns and FPLC chromatography. Digestion with trypsin released the his-tag fusion peptide, which generated P2S-TY (residues 42-87) and P1L-TY (residues 132-239). Using a steady-state agarose gel system, P2S-TY promoted HA nucleation, whereas P2S, P1L, and P1L-TY did not. This implies that the minimum requirement for nucleation of HA resides within the amino acid sequence of the first glutamic acid-rich domain, whereas the second glutamic acid-rich domain may require posttranslational modifications for activity. P1L, but not P2S, promoted RGD-mediated attachment of human gingival fibroblasts in a manner similar to that of native BSP. Deletion of the RGD domain or conversion of it to RGE (arginine-glycine-glutamic acid) abolished the cell-attachment activity of P1L. This suggests that, at least for human gingival fibroblasts, the major cell-attachment activity in the recombinant BSP peptides studied (residues 42-87 and 69-300) requires the RGD sequence located at the C-terminal domain.

The flexible polyelectrolyte hypothesis of protein−biomineral interaction

Biomineralization is characterized by a high degree of control over the location, nature, size, shape, and orientation of the crystals formed. For many years, it has been widely believed that the exquisitely precise nature of crystal formation in biological tissues is the result of stereochemically specific interactions between growing crystals and extracellular matrix proteins. That is, the ability of many mineralized tissue proteins to adsorb to particular faces of biominerals has been attributed to a steric and electrical complementarity between periodic regions of the polypeptide chain and arrays of ions on the crystal face. In recent years, however, evidence has accumulated that many mineral-associated proteins lack periodic structure even when adsorbed to crystals. It also appears that protein-crystal interactions involve a general electrostatic attraction rather than arrays of complementary charges. In the present work, we review these studies and present some relevant new findings involving the mineral-modulating phosphoprotein osteopontin. Using molecular dynamics simulations, we show that the adsorption of osteopontin peptides to hydroxyapatite crystals does not involve a unique conformation of the peptide molecule, and that the adsorbed peptides are not aligned with rows of Ca(2+) ions on the crystal face. Further, we show that the interface between osteopontin peptides and calcium oxalate monohydrate crystals consists of peptide regions of high electronegativity and crystal faces of high electropositivity. Collectively, the above-mentioned studies suggest that interactions between mineral-modulating proteins and biologically relevant crystals are primarily electrostatic in nature, and that molecular disorder assists these proteins in forming multiple bonds with cations of the crystal face.

Determination of the Hydroxyapatite-Nucleating Region of Bone Sialoprotein

Bone sialoprotein (BSP) was shown to be a potent nucleator of hydroxyapatite (HA) in a steady-state agarose gel system (Hunter and Goldberg, 1993, PNAS 90: 8562). Nucleation of HA was also demonstrated with the homopolymer poly-glutamic acid but not with poly-aspartic acid or osteopontin. Since BSP contains contiguous sequences of glutamic acid, it is reasonable to suggest that the HA-nucleating activity of BSP resides within these regions. Purified porcine BSP was treated with trypsin and digests fractionated by gel filtration. In addition to small peptides (P3-5), two peptides of 38 kDa (P1) and 25 kDA (P2) were recovered, and after characterization assigned to the regions within BSP encompassing residues 133-272 (P1) and 42-125 (P2). Each of these peptides contained one of the two glutamic acid-rich regions of porcine BSP. In the steady-state agarose gel system, BSP, P1 and P2 induced HA formation, whereas the pooled small BSP-derived peptides (P3-5) did not. Analysis by circular dichroism spectroscopy revealed that the homopolymer poly-L-glutamic acid assumes a helical structure, while poly-L-aspartic acid does not. These findings suggest that the nucleating activity does not require intact molecules, that the nucleation of HA and BSP appears to require glutamic acid-rich sequences in a helical conformation and that there are two domains in porcine BSP that are each capable of nucleating HA.

Role of osteopontin in modulation of hydroxyapatite formation.

The presence of osteopontin (OPN) at high levels in both mineralized tissues such as bone and ectopic calcifications such as atherosclerotic plaque presents a conundrum: is OPN a promoter or inhibitor of hydroxyapatite (HA) formation? In vitro studies show that OPN adsorbs tightly to HA and is a potent inhibitor of crystal growth. Although the mechanism of the OPN–HA interaction is not fully understood, it is probably electrostatic in nature. Phosphorylation enhances OPN’s ability to adsorb to and inhibit the growth of HA crystals, although other anionic groups also contribute to these properties. Recent findings suggest that OPN is an intrinsically unordered protein and that its lack of folded structure facilitates the protein’s adsorption by allowing multiple binding geometries and the sequential formation of ionic bonds with Ca2+ ions of the crystal surface. By analogy with other biominerals, it is likely that adsorption of OPN to HA results in “pinning” of growth steps. The abundance of OPN at sites of ectopic calcification reflects upregulation of the protein in response to crystal formation or even in response to elevated phosphate levels. Therefore, it appears that OPN is one of a group of proteins that function to prevent crystal formation in soft tissues. The role of OPN in bone mineralization, if any, is less clear. However, it is possible that it modulates HA formation, either by preventing crystal growth in “inappropriate” areas such as the osteoid seam or by regulating crystal growth habit (size and shape).

Matrix gla protein inhibits ectopic calcification by a direct interaction with hydroxyapatite crystals

Mice lacking the gene encoding matrix gla protein (MGP) exhibit massive mineral deposition in blood vessels and die soon after birth. We hypothesize that MGP prevents arterial calcification by adsorbing to growing hydroxyapatite (HA) crystals. To test this, we have used a combined experimental-computational approach. We synthesized peptides covering the entire sequence of human MGP, which contains three sites of serine phosphorylation and five sites of γ-carboxylation, and studied their effects on HA crystal growth using a constant-composition autotitration assay. In parallel studies, the interactions of these sequences with the {100} and {001} faces of HA were analyzed using atomistic molecular dynamics (MD) simulations. YGlapS (amino acids 1-14 of human MGP) and SK-Gla (MGP43-56) adsorbed rapidly to the {100} and {001} faces and strongly inhibited HA growth (IC(50) = 2.96 μg/mL and 4.96 μg/mL, respectively). QR-Gla (MGP29-42) adsorbed more slowly and was a moderate growth inhibitor, while the remaining three (nonpost-translationally modified) peptides had little or no effect in either analysis. Substitution of gla with glutamic acid reduced the adsorption and inhibition activities of SK-Gla and (to a lesser extent) QR-Gla but not YGlapS; substitution of phosphoserine with serine reduced the inhibitory potency of YGlapS. These studies suggest that MGP prevents arterial calcification by a direct interaction with HA crystals that involves both phosphate groups and gla residues of the protein. The strong correlation between simulated adsorption and measured growth inhibition indicates that MD provides a powerful tool to predict the effects of proteins and peptides on crystal formation.

Interfacial aspects of biomineralization

Abstract The identification of crystal planes that can specifically interact with mineralized tissue proteins, together with the detailed biochemical analysis of these proteins, has shown how the organic matrix can direct the formation of biological crystals. Recent studies have identified protein sequences and conformations associated with the nucleation of calcium carbonate and calcium phosphate crystals, determined how nucleation from a specific crystal face can cause alignment of crystallographic axes with matrix structures, and demonstrated several mechanisms by which the growth habits of biogenic crystals may be controlled by components of the organic matrix.