Original articleMatrix Gla protein binding to hydroxyapatite is dependent on the ionic environment: calcium enhances binding affinity but phosphate and magnesium decrease affinity
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 (Ka) and the maximum bound fraction (Bmax) 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 Ka = 8.0 × 104 M−1 and Bmax = 0.58, which are close to the values of Ka and Bmax 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.
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