Comparative calcium binding of leucine‐rich amelogenin peptide and full‐length amelogenin

Abstract

Leucine-rich amelogenin peptide (LRAP) is an alternately spliced amelogenin. LRAP is known to bind to hydroxyapatite, and has been shown to signal mesenchymal cells to proliferate, but its function in enamel formation is unclear. The purpose of this study was to determine the calcium-binding properties and structure of recombinant human LRAP (rLRAP) compared with full-length amelogenin (rH174). rLRAP and rH174 were synthesized in Escherichia coli and purified by affinity chromatography and reverse-phase high-performance liquid chromatography. Calcium binding was measured by isothermal titration calorimetry (ITC) at pH 7.5 and 25 degrees C, and raw data were analyzed by origin 7.0 software. The structure of rLRAP was analyzed by nuclear magnetic resonance (NMR) and circular dichroism (CD) in the absence or presence of Ca2+, pH 7.5 and 4.0, at 25 degrees C. Thermodynamic values showed that rLRAP had a Ca2+-binding affinity approximately 6.4-times greater than rH174. NMR and CD data revealed that rLRAP was randomly coiled, and that this structure was not altered by Ca2+, which bound to rLRAP and rH174 via ionic interactions. Unlike r174 (beta-spiral), rLRAP had a random-coiled structure. The calcium binding and structural differences between rLRAP and rH174 suggest that these proteins have different functions in enamel biomineralization.

Amelogenin as a promoter of nucleation and crystal growth of apatite

“…Previously reported results based on using atomic absorption spectroscopy similarly indicated drops in Ca 2 + levels in rH174 sols but relatively constant phosphate levels therein during a set of constant composition experiments[13]. The initial drop in Ca 2 + levels corresponds well to the results of a study by Le et al[24], who reported sequestration of calcium ions by rH174 monomers, resulting in approximately 6 Ca 2 + ions bound to a single rH174 molecule.Hence 0.12 mM of Ca 2 + would be required to fully saturate rH174 molecules in concentration of 0.4 mg/ml rH174. This model explains the immediate drop from [Ca 2 + ]¼1.60 mM to [Ca 2 + ]¼1.47 mM following mixing of the given amount of CaCl 2 with 0.4 mg/ml rH174.…”

Gene Duplication and the Evolution of Vertebrate Skeletal Mineralization

“…However, experimental analyses do not always support the idea that the large middle portion of AMEL consists of a disordered structure, but instead suggest that this portion contains extended polyproline II and/or ␤ -spiral structures [Matsushima et al, 1998;Le et al, 2006;Margolis et al, 2006;Robinson, 2006]. Among milk/saliva/ enamel SCPPs, AMEL is especially rich in Pro and Gln, and has Pro-Xaa-Yaa repeats (Xaa and Yaa represent any amino acids but Pro and Gln appear frequently) within the C-terminal half.…”

NMR studies of biomineralisation

“…This enables them to regulate the shape and organisation of the calcium phosphate crystals during enamel mineralisation [184]. Combined with CD measurements, NMR analysis ( 1 H-15 N correlation spectroscopy) revealed a random coil structure for LRAP in aqueous solution at pH 4.0 and pH 7.5 [183]. Furthermore, CD spectroscopy and isothermal titration calorimetry showed that Ca 2+ ions interact with LRAP without inducing conformational changes of the protein.…”

“…Recently, another hydroxyapatite-binding biomolecule, leucinerich amelogenin peptide (LRAP), has been studied by NMR spectroscopy [183]. LRAP (5 kDa) is one of the amelogenin isoforms.…”