Solution synthesis and biological activity of human pleiotrophin, a novel heparin-binding neurotrophic factor consisting of 136 amino acid residues with five disulfide bonds.
Human pleiotrophin (hPTN), a novel heparin-binding neurotrophic factor consisting of 136 amino acid residues with five intramolecular disulfide bonds, was synthesized by solution procedure in order to demonstrate the utility of our strategy using our newly developed solvent system, a mixture of trifluoroethanol (TFE) and dichloromethane (DCM) or chloroform (CHL). The final protected peptide was synthesized by coupling two larger protected intermediates, Boc-(1-64)-OH and H-(65-136)-OBzl, in CHL/TFE (3:1; v/v) using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) in the presence of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt). After removal of all protecting groups using the HF procedure followed by treatment with Hg(OAc)2, the fully deprotected peptide was subjected to an oxidative folding reaction. The product was confirmed as having the correct disulfide structure by examining the cystine peptides obtained by enzymatic digestions, and as possessing the same biological activities as those of the natural product. The N- and C-terminal half domains (1-64 and 65-136) were also synthesized, and measurement of their biological activities indicated that the C-terminal half domain displays almost all the activities of the full-length molecule, whereas the N-terminal half domain shows almost no activity. From these results, we were able to confirm that the C-terminal half domain is responsible for the expression of biological activities in the same manner as human midkine (hMK), another heparin-binding neurotrophic growth factor.
Inui
T
T
Peptide Institute Inc., Protein Research Foundation, Osaka, Japan.
Nakao
M
M
Nishio
H
H
Nishiuchi
Y
Y
Kojima
S
S
Muramatsu
T
T
Kimura
T
T
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001921:Brain / Q000196:embryology; D002352:Carrier Proteins / Q000096:biosynthesis / Q000138:chemical synthesis* / Q000737:chemistry*; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis / Q000138:chemical synthesis* / Q000737:chemistry*; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D006801:Humans; D051379:Mice; D000078792:Midkine; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000138:chemical synthesis / Q000737:chemistry; D010452:Peptide Biosynthesis; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis / Q000737:chemistry; D010960:Plasminogen Activators / Q000096:biosynthesis / Q000737:chemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D000596:Amino Acids; D002352:Carrier Proteins; D016207:Cytokines; D004220:Disulfides; D009414:Nerve Growth Factors; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; C066845:pleiotrophin; D000078792:Midkine; D010960:Plasminogen Activators