Interactions of amelogenin with phospholipids

Amelogenin protein has the potential to interact with other enamel matrix proteins, mineral, and cell surfaces. We investigated the interactions of recombinant amelogenin rP172 with small unilamellar vesicles as model membranes, toward the goal of understanding the mechanisms of amelogenin–cell interactions during amelogenesis. Dynamic light scattering (DLS), fluorescence spectroscopy, circular dichroism (CD), and nuclear magnetic resonance (NMR) were used. In the presence of phospholipid vesicles, a blue shift in the Trp fluorescence emission maxima of rP172 was observed (～334 nm) and the Trp residues of rP172 were inaccessible to the aqueous quencher acrylamide. DLS studies indicated complexation of rP172 and phospholipids, although the possibility of fusion of phospholipids following amelogenin addition cannot be ruled out. NMR and CD studies revealed a disorder–order transition of rP172 in a model membrane environment. Strong fluorescence resonance energy transfer from Trp in rP172 to DNS‐bound‐phospholipid was observed, and fluorescence polarization studies indicated that rP172 interacted with the hydrophobic core region of model membranes. Our data suggest that amelogenin has ability to interact with phospholipids and that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 96–108, 2015.     

Sex determination of ancient human skeletons using DNA

A method for determining the sex of human skeletons was developed using molecular genetic techniques. The amelogenin gene, found on the X and Y chromosomes, was examined using the polymerase chain reaction (PCR) and a nonradioactive dot blot procedure. DNA was analyzed from 20 modern individuals of known sex and 20 skeletons from an archaeological site in central Illinois dating to A.D. 1300. An independent assessment of the sex of each skeleton was made according to standard osteological methods. The sex of 19 ancient and 20 modern individuals was accurately determined using this molecular genetic technique. Molecular sex determination will be especially useful for juvenile and fragmentary remains when it is difficult, or impossible, to establish an individual's sex from morphological features. © 1996 Wiley-Liss, Inc.     

Sequence characteristics responsible for protein-protein interactions in the intrinsically disordered regions of caseins,amelogenins, and small heat-shock proteins

Milk caseins and dental amelogenins are intrinsically disordered proteins (IDPs) that associate with themselves and others. Paradoxically, they are also described as hydrophobic proteins, which is difficult to reconcile with a solvent-exposed conformation. We attempt to resolve this paradox. We show that caseins and amelogenins are not hydrophobic proteins but they are more hydrophobic than most IDPs. Remarkably, uncharged residues from different regions of these mature proteins have a nearly constant average hydropathy but these regions exhibit different charged residue frequencies. A novel sequence analysis method was developed to identify hydrophobic and order-promoting regions that would favor conformational collapse. We found that such regions were uncommon; most hydrophobic and order-promoting residues were adjacent to hydrophilic or disorder-promoting residues. A further reason why caseins and amelogenins do not collapse is their high proportion of disorder-promoting proline residues. We conclude that in these proteins the hydrophobic effect is not large enough to cause conformational collapse but it can contribute, along with polar interactions, to protein-protein interactions. This behaviour is similar to the interaction of the disordered N-terminal region of small heat-shock proteins with either themselves during oligomer formation or other, unfolding, proteins during chaperone action.     

A PCR-based sex-determination assay in cattle based on the bovine amelogenin locus

A method for determining the sex of bovine embryos has been established. Primers for a portion of the bovine amelogenin locus (AMX/Y) were used to amplify DNA present in either 0.1 μ1 of blood or biopsies taken from 6–7-day-old embryos. The primers amplify a 280 bp band in females and a 280 and 217bp bands in males. The method is rapid, does not require prior purification of DNA and contains an internal control which detects PCR failure.     

High‐yield recombinant bacterial expression of 13C‐, 15N‐labeled,serine‐16 phosphorylated,murine amelogenin using a modified third generation genetic code expansion protocol

Amelogenin constitutes ~90% of the enamel matrix in the secretory stage of amelogenesis, a still poorly understood process that results in the formation of the hardest and most mineralized tissue in vertebrates—enamel. Most biophysical research with amelogenin uses recombinant protein expressed in Escherichia coli. In addition to providing copious amounts of protein, recombinant expression allows ¹³C‐ and ¹⁵N‐labeling for detailed structural studies using NMR spectroscopy. However, native amelogenin is phosphorylated at one position, Ser‐16 in murine amelogenin, and there is mounting evidence that Ser‐16 phosphorylation is important. Using a modified genetic code expansion protocol we have expressed and purified uniformly ¹³C‐, ¹⁵N‐labeled murine amelogenin (pS16M179) with ~95% of the protein being correctly phosphorylated. Homogeneous phosphorylation was achieved using commercially available, enriched, ¹³C‐, ¹⁵N‐labeled media, and protein expression was induced with isopropyl β‐D‐1‐thiogalactopyranoside at 310 K. Phosphoserine incorporation was verified from one‐dimensional ³¹P NMR spectra, comparison of ¹H‐¹⁵N HSQC spectra, Phos‐tag SDS PAGE, and mass spectrometry. Phosphorus‐31 NMR spectra for pS16M179 under conditions known to trigger amelogenin self‐assembly into nanospheres confirm nanosphere models with buried N‐termini. Lambda phosphatase treatment of these nanospheres results in the dephosphorylation of pS16M179, confirming that smaller oligomers and monomers with exposed N‐termini are in equilibrium with nanospheres. Such ¹³C‐, ¹⁵N‐labeling of amelogenin with accurately encoded phosphoserine incorporation will accelerate biomineralization research to understand amelogenesis and stimulate the expanded use of genetic code expansion protocols to introduce phosphorylated amino acids into proteins.     

Leucine-rich amelogenin peptide induces osteogenesis by activation of the Wnt pathway

We previously showed that one of the amelogenin splicing isoforms, Leucine-rich amelogenin peptide (LRAP), induced osteogenic differentiation of mouse embryonic stem cells; however, the signaling pathway(s) activated by LRAP remained unknown. Here, we demonstrated that the canonical Wnt/β-catenin signaling is activated upon LRAP treatment, as evidenced by elevated β-catenin level and increased Wnt reporter gene activity. Furthermore, a specific Wnt inhibitor sFRP-1 completely blocks the LRAP-mediated Wnt signaling. However, exogenous recombinant Wnt3a alone was less effective at osteogenic induction of mouse ES cells in comparison to LRAP. Using a quantitative real-time PCR array, we discovered that LRAP treatment up-regulated the expression of Wnt agonists and down-regulated the expression of Wnt antagonists. We conclude that LRAP activates the canonical Wnt signaling pathway to induce osteogenic differentiation of mouse ES cells through the concerted regulation of Wnt agonists and antagonists.     

Probing the self-association, intermolecular contacts, and folding propensity of amelogenin

Amelogenins are an intrinsically disordered protein family that plays a major role in the development of tooth enamel, one of the most highly mineralized materials in nature. Monomeric porcine amelogenin possesses random coil and residual secondary structures, but it is not known which sequence regions would be conformationally attractive to potential enamel matrix targets such as other amelogenins (self-assembly), other matrix proteins, cell surfaces, or biominerals. To address this further, we investigated recombinant porcine amelogenin (rP172) using "solvent engineering" techniques to simultaneously promote native-like structure and induce amelogenin oligomerization in a manner that allows identification of intermolecular contacts between amelogenin molecules. We discovered that in the presence of 2,2,2-trifluoroethanol (TFE) significant folding transitions and stabilization occurred primarily within the N- and C-termini, while the polyproline Type II central domain was largely resistant to conformational transitions. Seven Pro residues (P2, P127, P130, P139, P154, P157, P162) exhibited conformational response to TFE, and this indicates these Pro residues act as folding enhancers in rP172. The remaining Pro residues resisted TFE perturbations and thus act as conformational stabilizers. We also noted that TFE induced rP172 self-association via the formation of intermolecular contacts involving P4-H6, V19-P33, and E40-T58 regions of the N-terminus. Collectively, these results confirm that the N- and C-termini of amelogenin are conformationally responsive and represent potential interactive sites for amelogenin-target interactions during enamel matrix mineralization. Conversely, the Pro, Gln central domain is resistant to folding and this may have important functional significance for amelogenin.     

Indigenous and foreign Y-chromosomes characterize the Lingayat and Vokkaliga populations of Southwest India

Previous studies have shown that India's vast coastal rim played an important role in the dispersal of modern humans out of Africa but the Karnataka state, which is located on the southwest coast of India, remains poorly characterized genetically. In the present study, two Dravidian populations, namely Lingayat (N = 101) and Vokkaliga (N = 102), who represent the two major communities of the Karnataka state, were examined using high-resolution analyses of Y-chromosome single nucleotide polymorphisms (Y-SNPs) and seventeen short tandem repeat (Y-STR) loci. Our results revealed that the majority of the Lingayat and Vokkaliga paternal gene pools are composed of four Y-chromosomal haplogroups (H, L, F* and R2) that are frequent in the Indian subcontinent. The high level of L1-M76 chromosomes in the Vokkaligas suggests an agricultural expansion in the region, while the predominance of R1a1a1b2-Z93 and J2a-M410 lineages in the Lingayat indicates gene flow from neighboring south Indian populations and West Asia, respectively. Lingayat (0.9981) also exhibits a relatively high haplotype diversity compared to Vokkaliga (0.9901), supporting the historical record that the Lingayat originated from multiple source populations. In addition, we detected ancient lineages such as F*-M213, H*-M69 and C*-M216 that may be indicative of genetic signatures of the earliest settlers who reached India after their migration out of Africa.