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Control of vertebrate skeletal mineralization by polyphosphates.

Omelon S, Georgiou J, Henneman ZJ, Wise LM, Sukhu B, Hunt T, Wynnyckyj C, Holmyard D, Bielecki R, Grynpas MD - PLoS ONE (2009)

Bottom Line: Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium.The resulting reduction of both free PO(4)(3-) and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals.When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates.

View Article: PubMed Central - PubMed

Affiliation: Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Canada.

ABSTRACT

Background: Skeletons are formed in a wide variety of shapes, sizes, and compositions of organic and mineral components. Many invertebrate skeletons are constructed from carbonate or silicate minerals, whereas vertebrate skeletons are instead composed of a calcium phosphate mineral known as apatite. No one yet knows why the dynamic vertebrate skeleton, which is continually rebuilt, repaired, and resorbed during growth and normal remodeling, is composed of apatite. Nor is the control of bone and calcifying cartilage mineralization well understood, though it is thought to be associated with phosphate-cleaving proteins. Researchers have assumed that skeletal mineralization is also associated with non-crystalline, calcium- and phosphate-containing electron-dense granules that have been detected in vertebrate skeletal tissue prepared under non-aqueous conditions. Again, however, the role of these granules remains poorly understood. Here, we review bone and growth plate mineralization before showing that polymers of phosphate ions (polyphosphates: (PO(3)(-))(n)) are co-located with mineralizing cartilage and resorbing bone. We propose that the electron-dense granules contain polyphosphates, and explain how these polyphosphates may play an important role in apatite biomineralization.

Principal findings/methodology: The enzymatic formation (condensation) and destruction (hydrolytic degradation) of polyphosphates offers a simple mechanism for enzymatic control of phosphate accumulation and the relative saturation of apatite. Under circumstances in which apatite mineral formation is undesirable, such as within cartilage tissue or during bone resorption, the production of polyphosphates reduces the free orthophosphate (PO(4)(3-)) concentration while permitting the accumulation of a high total PO(4)(3-) concentration. Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium. The resulting reduction of both free PO(4)(3-) and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals. Identified in situ within resorbing bone and mineralizing cartilage by the fluorescent reporter DAPI (4',6-diamidino-2-phenylindole), polyphosphate formation prevents apatite crystal precipitation while accumulating high local concentrations of total calcium and phosphate. When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates. The hydrolytic degradation of polyphosphates in the calcium-polyphosphate complex increases orthophosphate and calcium concentrations and thereby favors apatite mineral formation. The correlation of alkaline phosphatase with this process may be explained by the destruction of polyphosphates in calcifying cartilage and areas of bone formation.

Conclusions/significance: We hypothesize that polyphosphate formation and hydrolytic degradation constitute a simple mechanism for phosphate accumulation and enzymatic control of biological apatite saturation. This enzymatic control of calcified tissue mineralization may have permitted the development of a phosphate-based, mineralized endoskeleton that can be continually remodeled.

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TNAP cleaves Pi from polyP.Separation of synthetic polyP exposed to TNAP by polyacrylamide gel electrophoresis. TNAP exposure times were 0.5, 1, 2.5, 5, 10, 15, 20, and 30 minutes. Right lane is TNAP- (control) for 30 minutes (C) and the far left lane is a pyrophosphate (P2O7)4−/Pi ladder. Synthetic polyP contains a range of polyP species, from (PO3−)3 (3 condensed Pi units) to polyP chains longer than 28 Pi units.
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pone-0005634-g007: TNAP cleaves Pi from polyP.Separation of synthetic polyP exposed to TNAP by polyacrylamide gel electrophoresis. TNAP exposure times were 0.5, 1, 2.5, 5, 10, 15, 20, and 30 minutes. Right lane is TNAP- (control) for 30 minutes (C) and the far left lane is a pyrophosphate (P2O7)4−/Pi ladder. Synthetic polyP contains a range of polyP species, from (PO3−)3 (3 condensed Pi units) to polyP chains longer than 28 Pi units.

Mentions: Figure 7 shows the result of an in vitro assay of bovine kidney ALP, a TNAP isoenzyme, with long chain polyPs. Electrophoresis of the enzymatic digestion products demonstrated that this enzyme cleaves Pi from synthetic polyPs (average chain length of 25 Pi units, with some polyP species as short as 3 Pi units). The Pi concentration increased with time in the +ALP experiment, while no notable Pi was detected in the −ALP (control) experiment, even at the longest time point (30 minutes). Densitometry measurements of the bands showed an increase in Pi and decrease in polyP concentrations over time (Supplementary Information, Figure S1).


Control of vertebrate skeletal mineralization by polyphosphates.

Omelon S, Georgiou J, Henneman ZJ, Wise LM, Sukhu B, Hunt T, Wynnyckyj C, Holmyard D, Bielecki R, Grynpas MD - PLoS ONE (2009)

TNAP cleaves Pi from polyP.Separation of synthetic polyP exposed to TNAP by polyacrylamide gel electrophoresis. TNAP exposure times were 0.5, 1, 2.5, 5, 10, 15, 20, and 30 minutes. Right lane is TNAP- (control) for 30 minutes (C) and the far left lane is a pyrophosphate (P2O7)4−/Pi ladder. Synthetic polyP contains a range of polyP species, from (PO3−)3 (3 condensed Pi units) to polyP chains longer than 28 Pi units.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2683572&req=5

pone-0005634-g007: TNAP cleaves Pi from polyP.Separation of synthetic polyP exposed to TNAP by polyacrylamide gel electrophoresis. TNAP exposure times were 0.5, 1, 2.5, 5, 10, 15, 20, and 30 minutes. Right lane is TNAP- (control) for 30 minutes (C) and the far left lane is a pyrophosphate (P2O7)4−/Pi ladder. Synthetic polyP contains a range of polyP species, from (PO3−)3 (3 condensed Pi units) to polyP chains longer than 28 Pi units.
Mentions: Figure 7 shows the result of an in vitro assay of bovine kidney ALP, a TNAP isoenzyme, with long chain polyPs. Electrophoresis of the enzymatic digestion products demonstrated that this enzyme cleaves Pi from synthetic polyPs (average chain length of 25 Pi units, with some polyP species as short as 3 Pi units). The Pi concentration increased with time in the +ALP experiment, while no notable Pi was detected in the −ALP (control) experiment, even at the longest time point (30 minutes). Densitometry measurements of the bands showed an increase in Pi and decrease in polyP concentrations over time (Supplementary Information, Figure S1).

Bottom Line: Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium.The resulting reduction of both free PO(4)(3-) and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals.When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates.

View Article: PubMed Central - PubMed

Affiliation: Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Canada.

ABSTRACT

Background: Skeletons are formed in a wide variety of shapes, sizes, and compositions of organic and mineral components. Many invertebrate skeletons are constructed from carbonate or silicate minerals, whereas vertebrate skeletons are instead composed of a calcium phosphate mineral known as apatite. No one yet knows why the dynamic vertebrate skeleton, which is continually rebuilt, repaired, and resorbed during growth and normal remodeling, is composed of apatite. Nor is the control of bone and calcifying cartilage mineralization well understood, though it is thought to be associated with phosphate-cleaving proteins. Researchers have assumed that skeletal mineralization is also associated with non-crystalline, calcium- and phosphate-containing electron-dense granules that have been detected in vertebrate skeletal tissue prepared under non-aqueous conditions. Again, however, the role of these granules remains poorly understood. Here, we review bone and growth plate mineralization before showing that polymers of phosphate ions (polyphosphates: (PO(3)(-))(n)) are co-located with mineralizing cartilage and resorbing bone. We propose that the electron-dense granules contain polyphosphates, and explain how these polyphosphates may play an important role in apatite biomineralization.

Principal findings/methodology: The enzymatic formation (condensation) and destruction (hydrolytic degradation) of polyphosphates offers a simple mechanism for enzymatic control of phosphate accumulation and the relative saturation of apatite. Under circumstances in which apatite mineral formation is undesirable, such as within cartilage tissue or during bone resorption, the production of polyphosphates reduces the free orthophosphate (PO(4)(3-)) concentration while permitting the accumulation of a high total PO(4)(3-) concentration. Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium. The resulting reduction of both free PO(4)(3-) and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals. Identified in situ within resorbing bone and mineralizing cartilage by the fluorescent reporter DAPI (4',6-diamidino-2-phenylindole), polyphosphate formation prevents apatite crystal precipitation while accumulating high local concentrations of total calcium and phosphate. When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates. The hydrolytic degradation of polyphosphates in the calcium-polyphosphate complex increases orthophosphate and calcium concentrations and thereby favors apatite mineral formation. The correlation of alkaline phosphatase with this process may be explained by the destruction of polyphosphates in calcifying cartilage and areas of bone formation.

Conclusions/significance: We hypothesize that polyphosphate formation and hydrolytic degradation constitute a simple mechanism for phosphate accumulation and enzymatic control of biological apatite saturation. This enzymatic control of calcified tissue mineralization may have permitted the development of a phosphate-based, mineralized endoskeleton that can be continually remodeled.

Show MeSH
Related in: MedlinePlus