Semisynthesis of human somatotropin analogs.

Abstract

Complementation of the natural NHrterminal 134amino acid fragment of the reduced, carbamoylmethylated human somatotropin with synthetic analogs of COOH-terminal fragments of 57, 42, or 38 amino acids of reduced, carbamoylmethylated human somatotropin has been investigated. It was found that synthetic fragments of 57 and 42 amino acids gave recombinants with full growth-promoting activity, whereas attempts to obtain a recombinant with the synthetic 38-amino acid fragment were unsuccessful. The synthesis of two analogs of the COOH-terminal fragment of human somatotropin, human [Nlel70, Alal65,i82 1891 somatotropin(150-191) and human [Nlel7O, Ala's65'82,, is herein described.It has been shown that the two plasmic fragments (1) of human somatotropin (HGH) (see Fig. 1 give a recombinant protein with the full biological activities (2, 3) of the natural hormone. Subsequently, it was found that complementation of the natural NH2-terminal fragment with the synthetic COOH-terminal fragment or its synthetic analogs gave rise to fully active recombinants (4, 5). This paper reports complementation reactions of the natural NH2-terminal 134-amino acid (AA) fragment with synthetic COGH-terminal fragment analogs of different chain lengths to obtain full growth-promoting activity. MATERIALS AND METHODSHuman somatotropin was isolated from fresh-frozen pituitary glands as described (6). Plasmic digests of HGH and the isolation of the NH2-terminal 134-AA and COOH-terminal 51-AA fragments were obtained as described (1) except that the NH2-terminal fragment was further purified by gel filtration on Sephadex G-100 in 0.01 M NH4HCO3 at pH 8.2 (7). Analogs of the COGH-terminal fragment were synthesized by the solid-phase method (8). The synthesis of one of the analogs, [Lysl35,136,138 7Glul37,139, Nle170, Alal65,182,, has already been described (9). The complementation reactions were carried out by published procedures (2, 10). Briefly, 3.0 mg (0.20 ,umol) of the NH2-terminal fragment in 1.5 ml of 0.1 M Tris-HCl, pH 8.4/5% (vol/vol) n-butanol was added to 1.3 mg (0.21 ,mol) of COGH-terminal fragment or synthetic fragment in 0.1 ml of 1 M NH40H. Both solutions were clear before mixing and became turbid after mixing. The mixtures were stored at 20C for 10 days. After removal of the insoluble material, the clear supernatants were purified by exclusion chromatography on a Sephadex G-100 column (1.5 X 60 cm) in 0.01 M NH4HCO3 (pH 8.2) at 22°C. The growth-promoting activity was estimated by the rat tibia test (11).Peptide Synthesis. The peptides [Nle170, Alal65'82,189]- and [Nle'70, were synthesized as described for the corresponding analogs and HGH-(145-191) (9). t-Butoxycarbonylphenylalanine (Boc-Phe) resin was alternately deblocked with 55% trifluoroacetic acid/methylene chloride, neutralized with N-methylmorpholine or diisopropylethylamine, and coupled with the symmetrical anhydride (12) of the Boc-amino acid. N-Methylmorpholine was used for the bulk of the neutralization for the aspartyl-containing peptide to minimize base-cat...

Two contiguous thrombin fragments of human somatotropin form a functionally active recombinant, but the two homologous fragments from sheep hormone do not

“…Plasmin and thrombin were shown to split the same Arg134-Thr135 peptide bond of HGH, thus allowing the isolation of the same 134-residue N-terminal fragment from either digest after reduction and carbamidomethylation of the Cys53-Cys165 disulfide bond of the hormone (Li & Graf, 1974;Graf et al, 1976). In the present study this N-terminal fragment has been shown to interact noncovalently with the 57-residue thrombin fragment of HGH, just like with the 51-residue C-terminal plasmin fragment of HGH (Li & Bewley, 1976) and several 42-57-residue synthetic analogues of this plasmin fragment (Li et al, 1977(Li et al, , 1981Li & Blake, 1979). It may be noted, however, that the recombinant reported in this paper is the first one which contains the full primary structure of HGH with carbamidomethyl groups on the four cysteine residues.…”

“…The lack of apparent recombination between the HGH and SGH fragments cannot be due to the presence of the blocking groups on Cys164, Cys181, and Cys189 of the SGH fragment, since even the free cysteine-containing derivative was unable to interact with the N-terminal HGH fragment. In addition, the substitution of the same cysteines with alanines in some synthetic analogues of the coresponding HGH fragment did not seem to affect the complementation reaction between the two protions of HGH (Li et al, 1978;Li & Blake, 1979). It is also known from the previous complementation studies that neither the omission of the first 15 N-terminal residues from HGH-(135-191) (Li & Blake, 1979) or the last 4 C-terminal residues from (Li et al, 1981) nor substitutions at positions 153 and 179 of the segment (Li, 1978)] nor the replacement of Met170 with Nle in several synthetic analogues (Li et al, 1978(Li et al, ,1981Li & Blake, 1979) prevented the recombination of these synthetic and natural analogues of the C-terminal plasmin fragment .…”

“…(1) This method is essentially the same routinely used in this laboratory (Li & Bewley, 1976;Li & Blake, 1979).…”

“…In the case of HGH the two natural fragments (residues 1-134 and 141-191) of the complementing system were derived by plasmin digestion and reduction-carbamidomethylation of the hormone (Li & Graf, 1974). The structural requirements of this recombination reaction have been extensively studied by complementing the large N-terminal plasmin fragment of HGH (residues 1-134) with different synthetic analogues of the C-terminal plasmin fragment (Li et al, 1977(Li et al, , 1978; Li & Blake, 1979), and it has been concluded that even considerable structural changes of the Cterminal fragment, like its shortening to residues (Li & Blake, 1979), cause no significant change in the complementation reaction. On the other hand, no recombinant molecule containing the full primary structure of native HGH has been prepared so far.…”

Complementation of Human Growth Hormone (GH) Peptide 1–134 with C-Terminal Fragments of Human GH Produced by Digestion with Bromelain*

“…This finding is compatible with the experiments of Li and co-workers (4, 5) using synthetic analogs GLUCOSE OXIDATION BY ADIPOSE TISSUE l80r (6) of the C-terminal third of the hGH molecule, in which methionine 170 was substituted with norleucine and the half-cystine residues at positions 165, 182, and 189 with alanine. They found that such synthetic, substituted analogs of sequences 145-191 and 150-191 would complement with S-carbamidomethylated peptide 1-134 derived from plasmin-digested hGH, whereas an analog of the sequence 154-191 would not (4). Whether natural fragments shorter than peptide 146-191 can complement noncovalently with peptide 1-134 remains to be established.…”