Total chemical synthesis of human matrix Gla protein

Abstract

Human matrix Gla protein (MGP) is a vitamin K-dependent extracellular matrix protein that binds Ca2+ ions and that is involved in the prevention of vascular calcification. MGP is a 10.6-kD protein (84 amino acids) containing five gamma-carboxyglutamic acid (Gla) residues and one disulfide bond. Studies of the mechanism by which MGP prevents calcification of the arterial media are hampered by the low solubility of the protein (<10 microg/mL). Because of solubility problems, processing of a recombinantly expressed MGP-fusion protein chimera to obtain MGP was unsuccessful. Here we describe the total chemical synthesis of MGP by tBoc solid-phase peptide synthesis (SPPS) and native chemical ligation. Peptide Tyr1-Ala53 was synthesized on a derivatized resin yielding a C-terminal thioester group. Peptide Cys54-Lys84 was synthesized on Lys-PAM resin yielding a C-terminal carboxylic acid. Subsequent native chemical ligation of the two peptides resulted in the formation of a native peptide bond between Ala53 and Cys54. Folding of the 1-84-polypeptide chain in 3 M guanidine (pH 8) resulted in a decrease of molecular mass from 10,605 to 10,603 (ESI-MS), representing the loss of two protons because of the formation of the Cys54-Cys60 internal disulfide bond. Like native MGP, synthetic MGP had the same low solubility when brought into aqueous buffer solutions with physiological salt concentrations, confirming its native like structure. However, the solubility of MGP markedly increased in borate buffer at pH 7.4 in the absence of sodium chloride. Ca2+-binding to MGP was confirmed by analytical HPLC, on which the retention time of MGP was reduced in the presence of CaCl2. Circular dichroism studies revealed a sharp increase in alpha-helicity at 0.2 mM CaCl2 that may explain the Ca2+-dependent shift in high-pressure liquid chromatography (HPLC)-retention time of MGP. In conclusion, facile and efficient chemical synthesis in combination with native chemical ligation yielded MGP preparations that can aid in unraveling the mechanism by which MGP prevents vascular calcification.

Fig. 1.

Amino acid sequence of matrix Gla protein. The gray residues at position 53–54 represent the alanine-cysteine native chemical ligation site. The solid line represents the disulfide-bond. γ denotes γ-carboxyglutamic acid. Sequence from nucleotide analysis (Kiefer et al. 1988; Chen et al. 1990).

Fig. 2.

Native chemical ligation strategy for the total chemical synthesis of human matrix Gla protein. The 53-residue N-terminal peptide-thioester (COSR) fragment and the 30-residue C-terminal fragment of MGP were synthesized by stepwise SPPS techniques using Boc-chemistry protocols. The two fragments are initially joined by thioester formation (not observed as a discrete intermediate), and a subsequent spontaneous, rapid rearrangement results in the formation of a native peptide bond at the site of ligation.

Fig. 3.

Total synthesis of human matrix Gla protein by native chemical ligation. Synthesis of MGP fragments: (A) HPLC chromatogram (C18: 22.5%–41% acetonitrile, 1.23%/min) of the starting synthetic peptide segments MGP 54–84 and MGP 1–53. (B) ESI-MS spectrum of MGP 1–53 shows the m/z ratios (fourth through ninth ionized states) with an calculated mass of 7282.3 ± 0.3 D (theoretical average mass of 7282.6). (C) ESI-MS spectrum of MGP 54–84 shows the m/z ratios (second through sixth ionized states) with an calculated mass of 3872.8 ± 0.7 D (theoretical average mass: 3873.4 D). Native chemical ligation of MGP fragments: (D) HPLC chromatograms of the ligation reaction, started by the addition of 2% (v/v) thiophenol and 2% benzylmercaptan to the peptide mixture MGP 1–53 and MGP 54–84. At t = 1 h, ligated product (1–84) is shown as well as the C-terminal segment starting material. The unreacted N-terminal material can be accounted for as multiple intermediates eluting between 7 and 13 min (see text). At t = 24 h, ligation was almost complete. (E) ESI-MS spectrum shows the m/z pattern of the ligated material (fifth through fifteenth ionized states) with a calculated mass of 10,604.9 ± 0.7 D (theoretical average mass of the reduced 84-residue MGP: 10,604.8 D). Folding and disulfide formation of MGP: (F) HPLC chromatograms of the purified, reduced MGP polypeptide (t = 0), the crude, folded material (t = 24 h), and the purified final product (MGP). (G) ESI-MS spectrum shows the m/z pattern (fifth through fourteenth ionized state) with a calculated molecular mass of 10,602.9 ± 1.0 D (theoretical average mass of MGP containing one disulfide: 10,602.8 D).

Fig. 4.

Ca²⁺ binding to synthetic MGP. HPLC chromatograms (C18: 22.5%–41% acetonitrile, 1.23%/min) of synthetic MGP in the absence or in the presence of 5 mM CaCl₂.

Fig. 5.

Circular dichroism spectrum of synthetic matrix Gla protein. (A) The spectrum for matrix Gla protein at 25°C in boric acid shows 21% α-helical structure as can be estimated from the mean residual weight (MRW) ellipticity at 222 nm. (B) The increase (25%) of mean residual weight ellipticity of MGP at 222 nm as a function of CaCl₂ concentration.

Fig. 6.

SDS-PAGE of MGP. Synthetic MGP (lane 1), purified MGP from human bone (lane 2), MGP peptide 1–53-COSR (lane 3), and crude MGP from human bone (lane 4) were applied to 12.5% SDS-PAGE, Western blotting, and detection with a monoclonal antibody against peptide 3–15 from human MGP.