Matrix vesicles: structure, composition, formation and function in calcification
Roy E. Wuthier, Guy F. Lipscomb
Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208
TABLE OF CONTENTS
- 1. Abstract
- 2. Introduction
- 3. Morphology of matrix vesicles (MVs)
- 3.1. Conventional transmission electron microscopy
- 3.2. Cryofixation, freeze-substitution electron microscopy
- 3.3. Freeze-fracture studies
- 4. Isolation of MVs
- 4.1. Crude collagenase digestion methods
- 4.2. Non-collagenase dependent methods
- 4.3. Cell culture methods
- 4.4. Modified collagenase digestion methods
- 4.5. Other isolation methods
- 5. MV proteins
- 5.1. Early SDS-PAGE studies
- 5.2. Isolation and identification of major MV proteins 5.3. Sequential extraction, separation and characterization of major MV proteins 5.4. Proteomic characterization of MV proteins
- 6. MV-associated extracellular matrix proteins
- 6.1. Type VI collagen
- 6.2. Type X collagen
- 6.3. Proteoglycan link protein and aggrecan core protein
- 6.4. Fibrillin-1 and fibrillin-2
- 7. MV annexins - acidic phospholipid-dependent ca2+-binding proteins 7.1. Annexin A5
- 7.2. Annexin A6
- 7.3. Annexin A2
- 7.4. Annexin A1
- 7.5. Annexin A11 and Annexin A4
- 8. MV enzymes
- 8.1. Tissue-nonspecific alkaline phosphatase(TNAP)
- 8.1.1. Molecular structure
- 8.1.2. Amino acid sequence
- 8.1.3. 3-D structure
- 8.1.4. Disposition in the MV membrane
- 8.1.5. Catalytic properties
- 8.1.6. Collagen-binding properties
- 8.2. Nucleotide pyrophosphate phosphodiesterase (NPP1, PC1)
- 8.3. PHOSPHO-1 (Phosphoethanolamine/Phosphocholine phosphatase
- 8.4. Acid phosphatase
- 8.5. Proteases and peptidases
- 8.6. Lactate dehydrogenase and other glycolytic enzymes
- 8.7. Carbonic anhydrase
- 8.8. Phospholipases
- 8.8.1. Phospholipase A (PLA)
- 8.8.2. Phospholipase C (PLC)
- 8.8.3. Lysophospholipase C (LPLC)
- 8.8.4. Sphingomyelinase (Smase)
- 8.8.5. Other MV phospholipases
- 8.9. Phosphatidylserine synthases
- 8.10. Phospholipid flippases and scramblases
9. MV transporters and surface receptors
9.1. Cell-surface receptors
9.2. ATP-driven ion pumps and transporters
9.3. Channel proteins and solute-carrier transporters
9.4. Ca2+ channel proteins
- 9.4.1. L-type Ca2+ channels in growth plate chondrocytes
- 9.4.2. Annexins as Ca2+ channels
9.5. Inorganic P (Pi) transporters
- 9.5.1. Na+-dependent Pi transporters
- 9.5.2. Na+-independent Pi transporters
10. MV regulatory proteins
10.1. Syntenin
10.2. Gi Protein, alpha-2
10.3. Trp/Trp mono-oxygenase activation protein, etc.
11. MV cytoskeletal proteins
11.1. Actin
11.2. Filamen B, beta
11.3. Actinin
11.4. Tubulin
11.5. Radixin
11.6. Gelsolin and proteins with gelsolin/villin domains
11.7. Plastins
12. MV lipids
12.1. Phospholipids
- 12.1.1. Fatty acid composition
- 12.1.2. Physical form of MV phospholipids
- 12.1.3. Topological distribution of MV phospholipids
- 12.1.4. Ca2+-binding properties of MV phospholipids
12.2. Nonpolar lipids
12.3. Glycolipids
13. MV electrolytes
13.1. Chemical analyses of intracellular electrolytes in growth plate chondrocytes
13.2. Chemical analyses of electrolytes in isolated MVs
13.3. Chemical analyses of electrolytes in the extracellular fluid and blood plasma
13.4. Ultrastructural analyses of Ca2+ and P in cells and MVs
13.5. Chemical analyses of micro-electrolytes in MVs
13.6. Physico-chemical properties of MV mineral forms
13.6.1. X-ray diffraction
13.6.2. FT-IR and FT-Raman analyses
13.7. Solubility properties of nascent MV mineral
14. Formation of MVs
14.1. Electron microscopic studies
14.2. Biochemical studies
- 14.2.1. Phospholipid metabolic studies
- 14.2.2. Cell culture studies
14.3. Role of cellular Ca2+ and Pi metabolism in MV formation
- 14.3.1. Histology of cellular Ca2+ metabolism during MV formation
- 14.3.2. Confocal imaging of Ca2+ in living growth plate chondrocytes
- 14.3.3. Biochemistry of mitochondrial Ca2+ metabolism
14.4. Growth plate cellular Pi metabolism
14.4.1. Chondrocyte mitochondrial PIC Pi-transporter
14.4.2. Chondrocyte plasma membrane Pi transporters
14.4.3. Chondrocyte mitochondrial permeability transition (MPT) pore
14.5. Growth plate cellular H+ (pH) metabolism
- 14.5.1. Confocal imaging of H+ (pH) in living growth plate chondrocytes
15. Kinetics of mineral formation by MVs
15.1. Differing views of MV mineral formation
15.2. Radio-isotope analyses of the kinetics of MV Ca2+ and Pi accumulation
- 15.2.1. Stage 1 - Initial exchange
- 15.2.2. Stage 2 - Lag period
- 15.2.3. Stage 3 - Induction period
- 15.2.4. Stage 4 - Rapid acquisition period
- 15.2.5. Stage 5 - Plateau period
16. The nucleation core: The driving force in MV Ca2+ and Pi accumulation
16.1. Discovery of the nucleation core (NC)
16.2. Isolation of the NC
16.3. Chemical characterization of the NC
16.3.1. Electrophoretic analysis of proteins
16.3.2. Chromatographic analysis of lipids
16.4. Physical characterization of the NC
- 16.4.1. Transmission electron microscopy (TEM)
- 16.4.2. FT-IR analysis
- 16.4.3. 31P-NMR analysis
- 16.4.4. pH sensitivity
16.5. Summary of NC structure and composition
17. Reconstitution of the NC: role of PS-Ca2+-Pi complex (PS-CPLX) in MV crystal nucleation
17.1. Reconstitution of the nucleational core
17.2. Mathematical analysis of the kinetics of mineral formation
17.3. The physical and nucleational properties of pure PS-CPLX
17.4. Requirements for PS-CPLX formation: pH tolerance
17.5. Molecular rearrangements during PS-CPLX formation
17.6. Effect of Mg2+ incorporation on nucleation activity of PS-CPLX
17.7. Role of AnxA5 in MV crystal nucleation
17.8. Mechanism of MV nucleation of OCP
18. Modeling of MV using synthetic unilamellar liposomes
18.1. Effects of Ca2+ ionophores on Ca2+ uptake
18.2. Minimal effects of AnxA5 on 45Ca2+ uptake
18.3. Effects of detergents and phospholipase A2
18.4. Proteoliposomal MV models by other groups
19. Regulation of MV Calcification
19.1. Factors that primarily effect nucleation
19.2. Effects of phospholipid composition
19.3. Stimulatory effects of AnxA5 on MV nucleation
19.4. Effect of non-apatitic electrolytes
- 19.4.1. Effects of Mg2+
- 19.4.2. Effects of Zn2+
- 19.4.3. Effects of PPi
19.5. Inhibitory effects of proteoglycans
19.6. Stimulatory effects of type II and X collagens
19.7. Effects of non-collagenous bone-related proteins
20. Further evaluation of the roles of key MV proteins
20.1. Tissue-nonspecific alkaline phosphatase (TNAP)
- 20.1.1. Role of TNAP in PPi hydrolysis
- 20.1.2. Additional roles of TNAP
- 20.1.3. Improperly assigned roles of TNAP
20.2. Annexin A5 (AnxA5)
- 20.2.1. Questionable role in Ca2+ entrance into MVs
- 20.2.2. Activation of the nucleational core
- 20.2.3. Does AnxA5 have a physiological function?
- 20.2.4. Effects of double knockout of AnxA5 and AnxA6 on expression of other genes
- 20.2.5. Possible substitution of PS-receptor (Ptdsr) for AnxA5
- 20.2.6. What would be the effects of expression of defective AnxA5 mutants?
- 20.2.7. Is the need for AnxA5 stress-dependent?
20.3. PS Synthase
- 20.3.1. PS synthesis is not high-energy dependent, but requires Ca2+
- 20.3.2. PSS1 and PSS2 gene knockouts
20.4. Phospholipase A and C activities
- 20.4.1. Membrane lysis and Ca2+ access to nucleational core
- 20.4.2. Role in the egress of mineral from the vesicle lumen
- 20.4.3. Salvage of phospholipid P for mineral formation
20.5. PHOSPHO-1
- 20.5.1. Salvage of Pi and choline+ from phospholipid breakdown
- 20.5.2. Inhibition of PHOSPHO1 activity
- 20.5.3. PHOSPHO1 gene knockout
- 20.5.4. Co-modulation of TNAP and PHOSPHO1 gene expression
- 20.5.5. Effect on MV mineral formation
21. Perspectives
22. Acknowledgements
23. References
1. ABSTRACT
Matrix vesicles (MVs) induce calcification during endochondral bone formation. Experimental methods for structural, compositional, and functional analysis of MVs are reviewed. MV proteins, enzymes, receptors, transporters, regulators, lipids and electrolytes are detailed. MV formation is considered from both structural and biochemical perspectives. Confocal imaging of Ca2+ and H+ were used to depict how living chondrocytes form MVs. Biochemical studies revealed that coordinated mitochondrial Ca2+ and Pi metabolism produce MVs containing a nucleational complex (NC) of amorphous calcium phosphate, phosphatidylserine and annexin A5 - all critical to the mechanism of mineral nucleation. Reconstitution of the NC and modeling with unilamellar vesicles reveal how the NC transforms into octacalcium phosphate, regulated by Mg2+, Zn2+ and annexin A5. Extravasation of intravesicular mineral is mediated by phospholipases and tissue-nonspecific alkaline phosphatase (TNAP). In the extravesicular matrix, hydroxyapatite crystal propagation is enhanced by cartilage collagens and TNAP, which destroys inhibitory PPi, and by metalloproteases that degrade proteoglycans. Other proteins also modulate mineral formation. Recent findings from single and multiple gene knockouts of TNAP, NPP1, ANK, PHOSPHO1, and Annexin A5 are reviewed.
