Premium
Total chemical synthesis of human matrix Gla protein
Author(s) -
Hackeng Tilman M.,
Rosing Jan,
Spronk Henri M.H.,
Vermeer Cees
Publication year - 2001
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.44701
Subject(s) - native chemical ligation , chemistry , peptide , solubility , matrix gla protein , peptide bond , peptide synthesis , amino acid , stereochemistry , biochemistry , chemical synthesis , organic chemistry , in vitro , phosphate , hyperphosphatemia
Human matrix Gla protein (MGP) is a vitamin K–dependent extracellular matrix protein that binds Ca 2+ ions and that is involved in the prevention of vascular calcification. MGP is a 10.6‐kD protein (84 amino acids) containing five γ‐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 μg/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 Tyr 1 ‐Ala 53 was synthesized on a derivatized 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 formation of a native peptide bond between Ala 53 and Cys 54 . 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 Cys 54 ‐Cys 60 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. Ca 2+ ‐binding to MGP was confirmed by analytical HPLC, on which the retention time of MGP was reduced in the presence of CaCl 2 . Circular dichroism studies revealed a sharp increase in α‐helicity at 0.2 mM CaCl 2 that may explain the Ca 2+ ‐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.