Limited Proteolysis of Human Growth Hormone at Low pH:  Isolation, Characterization, and Complementation of the Two Biologically Relevant Fragments 1−44 and 45−191

Abstract

The limited proteolysis approach was used to analyze the conformational features of human growth hormone (hGH) under acidic solvent conditions (A-state). Pepsin was used as the proteolytic probe because of its poor substrate specificity and its activity at low pH. Limited proteolysis of hGH in its A-state results in a selective cleavage of the Phe44-Leu45 peptide bond, leading to the production of fragments 1-44 and 45-191. The two fragments were isolated in homogeneous form for studying their conformational properties by means of spectroscopic methods. Fragment 1-44 was shown to retain little secondary and tertiary structure at neutral pH, while fragment 45-191 independently folds into a highly helical secondary structure. In particular, we have shown that the two peptic fragments are able to associate into a stable and native-like hGH complex 1-44/45-191. Our proteolysis data indicate that in acid solution hGH adopts a partly folded state characterized by a local unfolding of the first minihelix (residues 38-47) encompassing the Phe44-Leu45 peptide bond. Of interest, hGH has both insulin-like and diabetogenic effects. Two fragments of hGH occur in vivo and exert these two opposite activities, namely, fragment 1-43 showing an insulin-potentiating effect and fragment 44-191 showing a diabetogenic activity. The results of this study suggest that the conformational changes of hGH induced by an acidic pH promote the generation of the two physiologically relevant fragments by proteolytic processing of the hormone. Although pepsin cannot be the enzyme responsible for the in vivo processing of the hormone, we propose that limited proteolysis of hGH at low pH is physiologically relevant, since the hormone is exposed to an acidic environment in the cell. This study reports for the first time the analysis of the conformational features of the two individual functional domains of hGH and of their complex.

Online Electrospray Ionization Mass Spectrometric Monitoring of Protease-Catalyzed Reactions in Real Time

“…On the other hand, it was hypothesized that the pH-related conformational changes of proteins might be the reason why an enzyme can catalyze the same substrate with different catalytic mechanisms at different pH values [49][50][51]. Apparently, our results support this speculation.…”

Heme Binding by the N-Terminal Fragment 1−44 of Human Growth Hormone

“…Limited proteolysis of hGH with pepsin in 10 mM Na citrate/0.15 M NaCl, pH 4.0, at 4°C occurs selectively at the level of the peptide bond Phe44-Leu45 (39). The reaction is quite clean, and it allows the selective production of fragment 1-44 and fragment , as shown by the RP-HPLC chromatogram of an aliquot of the reaction mixture ( Figure 1).…”

“…At present, we can speculate that, if heme binding occurs, the hGH fragment adopts a folded structure that makes it rather resistant to proteolytic degradation. The properties of fragment 1-44, produced in vitro by proteolysis of the hormone (39), are expected to be very similar to those of fragment 1-43, which is circulating in vivo and shows hypoglycemic activity (33,34,38). If we accept this, it is quite unlikely that the naturally occurring fragment 1-43 circulates in vivo as a largely unfolded chain, since it can be anticipated that this random polypeptide would be degraded rapidly by proteases.…”

Characterisation of the 5 kDa growth hormone isoform

“…Proteolytic 5 kDa (purified sequences AA 1 -43 and AA 1 -44 ) and 17 kDa (a mixture of sequences AA 44 -191 and AA 45 -191 ) isoforms were obtained by limited cleavage of recombinant 22 kDa isoform in a comparable process to the described by Spolaore et al (2004), which is to be published in a separate manuscript. Thermolysin, trypsin, 1,3,5-triacryloylhexahydro-s-triazine (TAT) and 2,2,2-trifluoroethanol (TFE) were purchased from Sigma-Aldrich (Barcelona, Spain).…”

“…In this case, the stated different structure could actually be caused by the synthetic origin of the protein. This hypothesis was discarded at an antibody binding level through the use of an alternative 5 kDa protein of the same AA 1 -43 sequence originating from a specific cleavage onto the 22 kDa sequence, in a similar procedure to that described elsewhere (Spolaore et al 2004). The structure of this fragment derives from the structure of the main 22 kDa isoform and consequently, it is expected to maintain the maximum homology with the native 5 kDa isoform, also generated by proteolysis.…”