Solution Synthesis of Human Midkine, a Novel Heparin‐binding Neurotrophic Factor Consisting of 121 Amino Acid Residues with Five Disulphide Bonds

Abstract

Human midkine (hMK), a novel heparin-binding neurotrophic factor consisting of 121 amino acid residues with five intramolecular disulphide bonds, was synthesized by solution procedure in order to demonstrate the usefulness of our newly developed solvent system, a mixture of dichloromethane or chloroform and trifluoroethanol. The final protected 121-residue peptide was assembled from two large fully protected intermediates, Boc-(1-59)-OH and H-(60-121)-OBzl, in CHL/TFE(3:1, v/v) using water-soluble carbodiimide in the presence of HOOBt as coupling reagents. After removal of the protecting groups by HF followed by treatment with Hg(OAc)2 in 50% acetic acid, the fully deprotected peptide was subjected to the oxidative folding reaction. The final product was confirmed to have the correct disulphide structure from its tryptic peptide mapping and to possess the same biological activities as those of the natural product. In order to clarify the active region of the hMK molecule, the N-terminal half domains [(1-59) and (60-121)] were also synthesized by the same procedure used for the hMK synthesis. The C-half domain was confirmed to show the full pattern of bioactivities except for the neuronal cell survival activity, while the N-half one showed much less activity in general.

Dimerization of Midkine by Tissue Transglutaminase and Its Functional Implication

“…Materials-The procedure for chemical synthesis of human MK and its fragments including N1/2 and C1/2 domains was described in a previous paper (42). Antibodies against the whole MK molecule, N1/2 domain, and C1/2 domain were produced in rabbits by injecting each antigen subcutaneously into animals after homogenization with Freund's complete adjuvant (Sigma).…”

“…Identification of Gln 42 , Gln 44 , and Gln 95 as Possible Amine Acceptors-The formation of multimers indicates that both murine and human MK contain both Gln and Lys residues that participate in the transglutaminase-mediated bridge formation. Both murine and human MK contain 5 Gln residues/ molecule (15,17).…”

“…N1/2 and C1/2 contain two and three Gln residues, respectively (15). Therefore, it is suggested that either Gln 42 or Gln 44 in N1/2 and one of the three Gln residues (amino acids 82, 93, and 95) in C1/2 serve as acyl-donating sites. Plasmaderived FXIIIa catalyzed the incorporation of […”

“…In the process of chemical synthesis of human MK, we synthesized 13 segments that could be coupled to form N1/2 and C1/2, and finally intact MK (42 . After the incubation, proteins in the solution were precipitated by adding 10% trichloroacetic acid and spotted onto filter paper discs, which were repeatedly washed.…”

Synthesis of Native Proteins by Chemical Ligation

“…Despite successful syntheses of the HIV-1 protease (22) and of a limited number of other proteins (30)(31)(32)(33)(34)(35), at the start of the decade of the 1990s total chemical synthesis, by the standard methods of peptide chemistry of even a small protein molecule remained a daunting task, often requiring large teams and taking years to complete, with no guarantee of success. The routine, reproducible preparation of synthetic polypeptides of defined chemical structure was limited to products of ∼50 amino acid residues (19; Figure 2).…”