Total chemical synthesis of human matrix Gla protein

Human matrix Gla protein (MGP) is a vitamin K-dependent extracellular matri x protein that binds Ca2+ ions and that is involved in the prevention of va scular calcification. MCP is a 10.6-kD protein (84 amino acids) containing five gamma -carboxyglutamic acid (Gla) residues and one disulfide bond. Stu dies of the mechanism by which MGP prevents calcification of the arterial m edia are hampered by the low solubility of the protein (<10 <mu>g/mL). Beca use of solubility problems, processing of a recombinantly expressed MGP-fus ion protein chimera to obtain MGP was unsuccessful. Here we describe the to tal chemical synthesis of MGP by tBoc solid-phase peptide synthesis (SPPS) and native chemical ligation. Peptide Tyr(1)-Ala(53) was synthesized on a d erivatized resin yielding a C-terminal thioester group. Peptide Cys(54)-Lys (84) was synthesized on Lys-PAM resin yielding a C-terminal carboxylic acid . Subsequent native chemical ligation of the two peptides resulted in the f ormation of a native peptide bond between Ala(53) and Cys(54). Folding of t he 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 tw o protons because of the formation of the Cys(54)-Cys(60) internal disulfid e bond. Like native MGP, synthetic MGP had the same low solubility when bro ught into aqueous buffer solutions with physiological salt concentrations, confirming its native like structure. However, the solubility of MGP marked ly increased in berate buffer at pH 7.4 in the absence of sodium chloride. Ca2+-binding to MGP was confirmed by analytical HPLC, on which the retentio n time of MGP was reduced in the presence of CaCl2. Circular dichroism stud ies revealed a sharp increase in alpha -helicity at 0.2 mM CaCl2 that may e xplain the Ca2+-dependent shift in high-pressure liquid chromatography (HPL C)-retention time of MGP. In conclusion, facile and efficient chemical synt hesis in combination with native chemical ligation yielded MGP preparations that can aid in unraveling the mechanism by which MGP prevents vascular ca lcification.