Direct evidence that KLK4 is a hydroxyapatite-binding protein

Pérez, Vidal; Mangum, Jonathan E.; Hubbard, Michael J.

doi:10.1016/j.bbrc.2017.12.040

Cited by 5 publications

(14 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Note these values differ from classical determinations made with unstained protein ladders (Hubbard, 1995;Mangum et al, 2010) and also from later experiments done with different batches of (commercial) gels. Immunoblotting was done using optimised electrotransfer conditions (wet tank method), probing (overnight incubation in primary antibody, rapid handling thereafter), and colorimetric detection (Vectastain ABC alkaline phosphatase kit, from Vector Labs) as previously (Mangum et al, 2010;Perez et al, 2018). Standard antibody dilutions were: anti-albumin 1:2,000; anti-alpha-fetoprotein peptide, 1:500; anti-(whole alpha-fetoprotein), 1:200.…”

Section: Profiling Of Enamel Proteinsmentioning

confidence: 99%

“…Where indicated, avidin/biotin-blocking was performed in Tris-buffered saline using streptavidin (0.1 mg/ml for 15 min) then biotin (0.5 mg/ml for 60 min) before the primary-antibody step. Sample loadings were adjusted to give detection within the linear range established by imaging densitometry of serially diluted standards (Perez et al, 2018) except where indicated. Spiking with tagged recombinant proteins (albumin, alphafetoprotein) that migrated slower than native protein standards was used to establish detection sensitivity for complex specimens containing native albumin/alpha-fetoprotein (i.e., neonatal serum, opacities).…”

Section: Profiling Of Enamel Proteinsmentioning

confidence: 99%

See 1 more Smart Citation

Pathogenesis of Molar Hypomineralisation: Hypomineralised 6-Year Molars Contain Traces of Fetal Serum Albumin

et al. 2020

Self Cite

View full text Add to dashboard Cite

Molar Hypomineralisation (MH) is gaining cross-sector attention as a global health problem, making deeper enquiry into its prevention a research priority. However, causation and pathogenesis of MH remain unclear despite 100 years of investigation into "chalky" dental enamel. Contradicting aetiological dogma involving disrupted enamel-forming cells (ameloblasts), our earlier biochemical analysis of chalky enamel opacities implicated extracellular serum albumin in enamel hypomineralisation. This study sought evidence that the albumin found in chalky enamel reflected causal events during enamel development rather than later association with pre-existing enamel porosity. Hypothesising that blood-derived albumin infiltrates immature enamel and directly blocks its hardening, we developed a "molecular timestamping" method that quantifies the adult and fetal isoforms of serum albumin ratiometrically. Applying this novel approach to 6-year molars, both isoforms of albumin were detectable in 6 of 8 chalky opacities examined (corresponding to 4 of 5 cases), indicating developmental acquisition during early infancy. Addressing protein survival, in vitro analysis showed that, like adult albumin, the fetal isoform (alpha-fetoprotein) bound hydroxyapatite avidly and was resistant to kallikrein-4, the pivotal protease involved in enamel hardening. These results shift primary attention from ameloblast injury and indicate instead that an extracellular mechanism involving localised exposure of immature enamel to serum albumin constitutes the crux of MH pathogenesis. Together, our pathomechanistic findings plus the biomarker approach for onset timing open a new direction for aetiological investigations into the medical prevention of MH.

show abstract

Section: Profiling Of Enamel Proteinsmentioning

confidence: 99%

Section: Profiling Of Enamel Proteinsmentioning

confidence: 99%

Pathogenesis of Molar Hypomineralisation: Hypomineralised 6-Year Molars Contain Traces of Fetal Serum Albumin

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…Note these values differ from classical determinations made with unstained protein ladders (Mangum et al, 2010a). For immunoblotting, proteins were electrotransferred to 0.2 μm nitrocellulose membrane using a Trans-Blot Turbo system (BioRad) and immunospecific bands were detected colorimetrically (Vectastain ABC alkaline phosphatase and peroxidase kits, from Vector Labs; Mangum et al, 2010a;Perez et al, 2018). Standard antibody dilutions were: anti-albumin 1:2,000 and anti-amylase 1:1,000.…”

Section: Profiling Of Enamel Proteinsmentioning

confidence: 99%

“…Effects of dithiothreitol (DTT; from Sigma) and tris(2-carboxyethyl) phosphine (TCEP; from Pierce) were assessed by solubilising enamel proteins in SDS-PAGE sample buffer that lacked both of these reducing agents, then subsequently had reducers added alone or in combination as indicated in the figure legends. Protein bands were quantified using semi-quantitative imaging densitometry as before (Perez et al, 2018).…”

Section: Profiling Of Enamel Proteinsmentioning

confidence: 99%

“…Although causation and pathogenesis of MH remain unclear after 100 years of investigation into chalky enamel, recent biochemical investigations have opened an enticing new direction for aetiological research (Gottlieb, 1920;Suckling et al, 1976;Mangum et al, 2010a;Perez et al, 2018;Williams et al, 2020). Demarcated opacities have long been thought to arise from systemic injuries to enamel-forming cells (ameloblasts) during the hardening (maturation) stage of enamel formation (Suckling, 1989;Suga, 1989;Jalevik et al, 2001;Weerheijm, 2003;Alaluusua, 2010;Babajko et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Pathogenesis of Molar Hypomineralisation: Aged Albumin Demarcates Chalky Regions of Hypomineralised Enamel

2020

Self Cite

View full text Add to dashboard Cite

Molar hypomineralisation (MH) is becoming globally recognised as a significant public health problem linked to childhood tooth decay. However, with causation and pathogenesis unclear after 100 years of investigation, better pathological understanding is needed if MH is to become preventable. Our studies have implicated serum albumin in an extracellular pathomechanism for chalky enamel, opposing longheld dogma about systemic injury to enamel-forming cells. Hypothesising that chalky enamel arises through developmental exposure to serum albumin, this study used biochemical approaches to characterise demarcated opacities from 6-year molars. Addressing contradictory literature, normal enamel was found to completely lack albumin subject to removal of surface contamination. Querying surface permeability, intact opacities were found to lack salivary amylase, indicating that "enamel albumin" had become entrapped before tooth eruption. Thirdly, comparative profiling of chalky and hard-white enamel supported a dose-response relationship between albumin and clinical hardness of opacities. Moreover, albumin abundance delineated chalky enamel from white transitional enamel at opacity borders. Finally, addressing the corollary that enamel albumin had been entrapped for several years, clear signs of molecular ageing (oxidative aggregation and fragmentation) were identified. By establishing aged albumin as a biomarker for chalky enamel, these findings hold methodological, clinical, and aetiological significance. Foremost, direct inhibition of enamel-crystal growth by albumin (here termed "mineralisation poisoning") at last provides a cogent explanation for the clinical presentation of demarcated opacities. Together, these findings justify pursuit of an extracellular paradigm for the pathogenesis of MH and offer exciting new prospects for alleviating childhood tooth decay through medical prevention of MH.

show abstract

Deficiency of G protein‐coupled receptor Gpr111/Adgrf2 causes enamel hypomineralization in mice by alteration of the expression of kallikrein‐related peptidase 4 (Klk4) during pH cycling process

et al. 2023

View full text Add to dashboard Cite

Enamel is formed by the repetitive secretion of a tooth‐specific extracellular matrix and its decomposition. Calcification of the enamel matrix via hydroxyapatite (HAP) maturation requires pH cycling to be tightly regulated through the neutralization of protons released during HAP synthesis. We found that Gpr115, which responds to changes in extracellular pH, plays an important role in enamel formation. Gpr115‐deficient mice show partial enamel hypomineralization, suggesting that other pH‐responsive molecules may be involved. In this study, we focused on the role of Gpr111/Adgrf2, a duplicate gene of Gpr115, in tooth development. Gpr111 was highly expressed in mature ameloblasts. Gpr111‐KO mice showed enamel hypomineralization. Dysplasia of enamel rods and high carbon content seen in Gpr111‐deficient mice suggested the presence of residual enamel matrices in enamel. Depletion of Gpr111 in dental epithelial cells induced the expression of ameloblast‐specific protease, kallikrein‐related peptidase 4 (Klk4), suggesting that Gpr111 may act as a suppressor of Klk4 expression. Moreover, reduction of extracellular pH to 6.8 suppressed the expression of Gpr111, while the converse increased Klk4 expression. Such induction of Klk4 was synergistically enhanced by Gpr111 knockdown, suggesting that proper enamel mineralization may be linked to the modulation of Klk4 expression by Gpr111. Furthermore, our in vitro suppression of Gpr111 and Gpr115 expression indicated that their suppressive effect on calcification was additive. These results suggest that both Gpr111 and Gpr115 respond to extracellular pH, contribute to the expression of proteolytic enzymes, and regulate the pH cycle, thereby playing important roles in enamel formation.

show abstract

Direct evidence that KLK4 is a hydroxyapatite-binding protein

Cited by 5 publications

References 16 publications

Pathogenesis of Molar Hypomineralisation: Hypomineralised 6-Year Molars Contain Traces of Fetal Serum Albumin

Pathogenesis of Molar Hypomineralisation: Hypomineralised 6-Year Molars Contain Traces of Fetal Serum Albumin

Pathogenesis of Molar Hypomineralisation: Aged Albumin Demarcates Chalky Regions of Hypomineralised Enamel

Deficiency of G protein‐coupled receptor Gpr111/Adgrf2 causes enamel hypomineralization in mice by alteration of the expression of kallikrein‐related peptidase 4 (Klk4) during pH cycling process

Contact Info

Product

Resources

About