Matrix Gla protein binding to hydroxyapatite is dependent on the ionic environment: calcium enhances binding affinity but phosphate and magnesium decrease affinity

doi:10.1016/S8756-3282(02)00821-9

Bone

Volume 31, Issue 2, August 2002, Pages 296-302

https://doi.org/10.1016/S8756-3282(02)00821-9 Get rights and content

Abstract

Matrix Gla protein (MGP) is an inhibitor of mineralization found in bone, cartilage, developing tissues, smooth muscle, and atherosclerotic plaques. MGP interaction with hydroxyapatite (HA) has been inferred by its function, but has never been measured directly. In this study, the influence of MGP antibody (x-MGP) binding, plasmin digestion, and various ions, including calcium and phosphate, on ¹²⁵I-labeled MGP-HA binding was examined. Nonlinear regression analysis of MGP binding yielded K_a (association constant; ≈8.0 × 10⁴ M⁻¹) and B_max (maximum specific bound fraction of MGP; ≈0.53). Anti-MGP antiserum reduced K_a to less than half of control (0.33% x-MGP). Plasmin-digested MGP decreased HA binding parameters by almost a third, showing that protein binding and limited proteolysis greatly affected HA binding. The presence of free calcium ions significantly increased binding in a dose-dependent manner, with ∼1 mmol/L calcium increasing K_a by a factor of 2. Phosphate ions decreased binding significantly in a dose-dependent fashion, with ∼1 mmol/L PO₄ decreasing K_a by a third. Magnesium at ∼1 mmol/L decreased K_a significantly by half, but the effect was not dose-dependent. Carbonate, sulfate, and sodium ions had no significant effect on binding. MGP binding to HA is sensitive to protein binding, limited proteolysis, and the surrounding ionic environment.

Introduction

Matrix Gla protein (MGP), first isolated in 1983, contains calcium-binding γ-carboxyglutamic acid (Gla) residues and three phosphoserines.9, 28, 29 MGP synthesis occurs in the developing kidney, heart, and lungs, but synthesis decreases rapidly after birth.7, 43 However, MGP concentration has been shown to remain relatively constant in adult rat cartilage.21

MGP has been shown to be an inhibitor of calcification. Virally driven overexpression of MGP greatly decreased mineralization in vitro and also delayed chondrocyte maturation, suggesting that MGP is a developmentally regulated inhibitor of mineralization in cartilage, controlling the quantity of mineral made.41 Pathological calcifications have also been linked to MGP. MGP-deficient mice die within 2 months of birth due to arterial calcification and subsequent rupture of blood vessels. These mice have also displayed inappropriate calcification of cartilage.15 Gene expression for MGP has been associated with the developmental appearance of several pathologies in the tiptoe-walking Yoshimura (twy) mouse, which suffers pathological calcium deposition and osteochondral lesions.24 Keutel’s syndrome, a human genetic disorder with loss of functional MGP, is characterized by abnormal cartilage calcification.19 The expression of bovine MGP mRNA has been found to decrease as calcification of vascular smooth muscle cells increased; however, when calcification was blocked with bisphosphonates, the expression was restored to control levels.17 Rats treated with high doses of warfarin plus vitamin K suffered focal calcification of the arteries and in aortic heart valves.30 Although γ-carboxylated Gla residues are required for MGP to function as a calcification inhibitor, these residues are not necessary for its accumulation at calcification sites.31 At sites of arterial calcification, the expression of MGP was found to be downregulated in favor of proteins normally found in bone.34

The binding of a protein for hydroxyapatite (HA) offers insight into the strength of protein-mineral interactions, and the role of the protein in normal and pathological calcification. Other protein affinities have also been reported. For example, osteocalcin (Oc) has an association constant on the order of 1 × 10⁷ M⁻¹.20, 27 The affinity of HA for osteonectin,33 dentin phosphophoryn,8 and osteopontin4 has also been examined. To our knowledge, the binding of MGP to HA has never been characterized.

Several HA binding studies have examined the influence of the ionic environment. Calcium (Ca²⁺), one of the main components of bone, affects the binding of osteocalcin to HA.11, 26 Calcium increases the HA binding affinity of osteocalcin as well as its α-helical content.12, 32 Magnesium (Mg²⁺), an inhibitor of mineralization in conditions of variable ionic strength,1, 16, 25, 36 has been shown to decrease OC-HA binding,40 despite inducing a somewhat α-helical structure in osteocalcin.10, 12 Cadmium (Cd²⁺) inhibits crystal growth and incorporates into HA crystals.3, 25 Cadmium also causes osteocalcin to change its conformation, mimicking the effects of calcium.32

The function of MGP as an inhibitor of HA mineralization necessitates the characterization of its interaction with HA. The purpose of the present study was to determine the influence of the ionic environment on MGP-HA binding in a well-defined environment. To determine how physiologically relevant ions affect HA binding, the association constant (K_a) and the maximum bound fraction (B_max) were calculated in the presence of different ions, including calcium and phosphate. Finally, the effects of x-MGP binding and plasmin digestion of MGP on MGP-HA binding were examined to establish the effect of MGP-protein binding and the requirement of intact MGP, respectively.

Section snippets

Materials

PIPES [piperazine-N,N"-bis(2-ethanesulfonic acid)], hydroxyapatite, and bovine serum albumin (BSA, fraction V) were obtained from Sigma (St. Louis, MO). Potassium hydroxide, concentrated hydrochloric acid, potassium chloride, potassium phosphate dibasic, calcium chloride dihydrate, and sodium bicarbonate were obtained from Mallinckrodt, Inc. (Paris, KY). Magnesium chloride hexahydrate was obtained from Fluka (Buchs, Switzerland). Potassium fluoride hydrate and cadmium chloride anhydrous were

Results

The results of three experiments performed on consecutive days are shown in Figure 1, as a plot of bound fraction vs. [HA]. The inset shows the x axis on a log scale. Each individual nonlinear regression curve had an excellent fit (r² > 0.98), as did the composite curve (r² > 0.95). The composite binding parameters for the data shown are K_a = 8.0 × 10⁴ M⁻¹ and B_max = 0.58, which are close to the values of K_a and B_max obtained from eight different experiments (8.0 × 10⁴ M⁻¹ and 0.53,

Discussion

Matrix Gla protein binds to increasing concentrations of HA with a first order relationship. After subtracting nonspecific binding, the binding curves were rectangular hyperbolas, indicating one HA binding site on MGP. Analyses with a two-binding-site model5 did not fit the data. Additional assays with 1/10 and 10 times the concentration of MGP used in these experiments yielded the same percentage bound (over 95%) at the highest [HA]. MGP-HA binding had therefore reached completion and was at

Acknowledgements

This project was funded by the American Heart Association Southeast (Grant-in-Aid 995771V). M.E.R. was also supported by the Herbert Herff Foundation and the National Institutes of Health (AR 45297). The authors acknowledge the support of Dr. Jae-Young Rho at the University of Memphis, for his support and input during this research.

References (43)

F. Barrere et al.
Biomimetic calcium phosphate coatings on Ti6AI4VA crystal growth study of octacalcium phosphate and inhibition by Mg²⁺ and HCO₃⁻
Bone
(1999)
E. Bertoni et al.
Nanocrystals of magnesium and fluoride substituted hydroxyapatite
J Inorg Biochem
(1998)
A.L. Boskey et al.
Osteopontin-hydroxyapatite interactions in vitroInhibition of hydroxyapatite formation and growth in a gelatin-gel
Bone Miner
(1993)
E. Busch et al.
Calcium affinity, cooperativity, and domain interactions of extracellular EF-hands present in BM-40
J Biol Chem
(2000)
J.D. Fraser et al.
Lung, heart, and kidney express high levels of mRNA for the vitamin K-dependent matrix Gla proteinImplications for the possible functions of matrix Gla protein and for the tissue distribution of the γ-carboxylase
J Biol Chem
(1988)
K. Mori et al.
Expression of matrix Gla protein (MGP) in an in vitro model of vascular calcification
Fed Eur Bone Soc Lett
(1998)
S.K. Nishimoto et al.
The vitamin K-dependent bone protein is accumulated within cultured osteosarcoma cells in the presence of the vitamin K antagonist warfarin
J Biol Chem
(1985)
S.K. Nishimoto et al.
Effect of aging and dietary restriction on matrix Gla protein and other components of rat tracheal cartilage
Matrix
(1993)
P.A. Price et al.
Matrix Gla protein, a new γ-carboxyglutamic acid-containing protein which is associated with the organic matrix of bone
Biochem Biophys Res Commun
(1983)
R.W. Romberg et al.
Isolation and characterization of native adult osteonectin
J Biol Chem
(1985)

D.T.H. Wassell et al.

Adsorption of bovine serum albumin onto hydroxyapatite

Biomaterials

(1995)

A. Yasukawa et al.

Preparation and characterization of barium-strontium hydroxyapatites

J Colloid Interf Sci

(1997)

J. Zhao et al.

Matrix Gla protein gene expression is elevated during postnatal development

Matrix Biol

(1996)

N.C. Blumenthal et al.

The effect of cadmium on the formation and properties of hydroxyapatite in vitro and its relation to cadmium toxicity in the skeletal system

Calcif Tissue Int

(1995)

J.P. Devogelaer et al.

Fluoride therapy of type I osteoporosis

Clin Rheumatol

(1995)

R. Fujisawa et al.

Changes in interaction of bovine dentin phosphophoryn with calcium and hydroxyapatite by chemical modifications

Calcif Tissue Int

(1986)

C.M. Gundberg et al.

Vitamin K-dependent proteins of bone and cartilage

G. Gundlach et al.

Conformational changes of γ-carboxyglutamic acid-containing protein from bovine bone by binding of alkaline earth ions

Hoppe Seyler Z Physiol Chem

(1983)

P.V. Hauschka et al.

Purification and calcium-binding properties of osteocalcin, the γ-carboxyglutamate-containing protein of bone

P.V. Hauschka et al.

Calcium-dependent α-helical structure in osteocalcin

Biochemistry

(1982)

D.I. Hay et al.

Differential adsorption and chemical affinities of proteins for apatitic surfaces

J Dent Res

(1979)

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Original articleMatrix Gla protein binding to hydroxyapatite is dependent on the ionic environment: calcium enhances binding affinity but phosphate and magnesium decrease affinity

Abstract

Introduction

Section snippets

Materials

Results

Discussion

Acknowledgements

Bone

J Inorg Biochem

Bone Miner

J Biol Chem

J Biol Chem

Fed Eur Bone Soc Lett

J Biol Chem

Matrix

Biochem Biophys Res Commun

J Biol Chem

Biomaterials

J Colloid Interf Sci

Matrix Biol

The effect of cadmium on the formation and properties of hydroxyapatite in vitro and its relation to cadmium toxicity in the skeletal system

Calcif Tissue Int

Fluoride therapy of type I osteoporosis

Clin Rheumatol

Changes in interaction of bovine dentin phosphophoryn with calcium and hydroxyapatite by chemical modifications

Calcif Tissue Int

Vitamin K-dependent proteins of bone and cartilage

Conformational changes of γ-carboxyglutamic acid-containing protein from bovine bone by binding of alkaline earth ions

Hoppe Seyler Z Physiol Chem

Purification and calcium-binding properties of osteocalcin, the γ-carboxyglutamate-containing protein of bone

Calcium-dependent α-helical structure in osteocalcin

Biochemistry

Differential adsorption and chemical affinities of proteins for apatitic surfaces

J Dent Res

Original article
Matrix Gla protein binding to hydroxyapatite is dependent on the ionic environment: calcium enhances binding affinity but phosphate and magnesium decrease affinity