The Cell Adhesion Activity of the Joining Peptide of Proopiomelanocortin.

Proopiomelanocortin (POMC) is a precursor protein of several peptide hormones, such as ACTH and ß-endorphin. Almost all of the peptide hormones in POMC have been drastically investigated in terms of their biological activities. However, the biological activity of the joining peptide region (JP) in POMC is unknown. Therefore, to explore the biological activity of JP, sequence analyses of mammalian POMC were performed. We found an -Arg-Gly-Asp- (RGD) motif in several mammalian species, such as porcine, suggesting that JP has cell adhesion activity. To validate this hypothesis, the cell adhesion activities of the synthetic porcine JP peptides were examined using 293T cells. Cell adhesions were observed in a concentration-dependent manner of the JP peptides. In addition, the JP peptide competitively inhibited cell adhesion to the POMC-coated plates. Moreover, the cell adhesion activity of the joining peptide was inhibited by the addition of EDTA, indicating that the JP peptide mediates the cell adhesion activity via a receptor protein, integrin. Interestingly, a human JP peptide, which possesses an -Arg-Ser-Asp- (RSD) sequence in place of the RGD sequence, exhibited a higher ability in the cell adhesion activity than that of the porcine JP peptide, suggesting that the cell adhesion activity of the joining peptide is developed during the molecular evolution of POMC. In conclusion, our results reveal that the joining peptide in POMC plays an important role during cell adhesion and provide useful information related to signal transduction of nerve peptide hormones derived from POMC.

A Novel Peptide Reagent for Investigating Disulfide-Coupled Folding Intermediates of Mid-Size Proteins.

Investigations of protein folding have largely involved the use of disulfide-containing proteins, since the disulfide-coupled folding of proteins allows folding intermediates to be trapped and their conformations determined. However, studies of the folding mechanisms of mid-size proteins face several problems, one of which is that detecting folding intermediates is difficult. Therefore, to solve this issue, a novel peptide reagent, maleimidohexanoyl-Arg5-Tyr-NH2, was designed and applied to the detection of folding intermediates of model proteins. BPTI was chosen as a model small protein to estimate the ability of the novel reagent to detect folding intermediates. In addition, a precursor protein (prococoonase) of Bombyx mori cocoonase was used as a model mid-size protein. Cocoonase is classified as a serine protease and has a high homology with trypsin. We recently found that the propeptide sequence of prococoonase (proCCN) is important for the folding of cocoonase. However, it was difficult to study the folding pathway of proCCN since the folding intermediates could not be separated on a reversed-phase HPLC (RP-HPLC). Therefore, to separate the folding intermediates by RP-HPLC, the novel labeling reagent was used to accomplish this for proCCN. The results indicated that the peptide reagent allowed the intermediates to be captured, separated on SDS-PAGE, and analyzed by RP-HPLC without the occurrence of undesirable disulfide-exchange reactions during the labeling reactions. The peptide reagent reported herein is a practical tool for investigating the mechanisms of disulfide-coupled folding of mid-size proteins.

Degradation-Suppressed Cocoonase for Investigating the Propeptide-Mediated Activation Mechanism.

Cocoonase is folded in the form of a zymogen precursor protein (prococoonase) with the assistance of the propeptide region. To investigate the role of the propeptide sequence on the disulfide-coupled folding of cocoonase and prococoonase, the amino acid residues at the degradation sites during the refolding and auto-processing reactions were determined by mass spectrometric analyses and were mutated to suppress the numerous degradation reactions that occur during the reactions. In addition, the Lys8 residue at the propeptide region was also mutated to estimate whether the entire sequence is absolutely required for the activation of cocoonase. Finally, a degradation-suppressed [K8D,K63G,K131G,K133A]-proCCN protein was prepared and was found to refold readily without significant degradation. The results of an enzyme assay using casein or Bz-Arg-OEt suggested that the mutations had no significant effect on either the enzyme activity or the protein conformation. Thus, we, herein, provide the non-degradative cocoonase protein to investigate the propeptide-mediated protein folding of the molecule. We also examined the catalytic residues using the degradation-suppressed cocoonase. The point mutations at the putative catalytic residues in cocoonase resulted in the loss of catalytic activity without any secondary structural changes, indicating that the mutated residues play a role in the catalytic activity of this enzyme.

The propeptide sequence assists the correct folding required for the enzymatic activity of cocoonase.

Cocoonase, a protein that is produced by the silkworm (Bombyx mori), is thought to specifically digest the sericin protein of the cocoon and has a high homology with trypsin. Similar to trypsin, cocoonase is folded as an inactive precursor protein which is activated by releasing the propeptide moiety. However, the mechanism responsible for the activation of its catalytic structure has not yet been determined in detail. Therefore, to investigate the activation and folding mechanism of cocoonase, recombinant cocoonase (CCN) and prococoonase (proCCN) were over-expressed in E. coli cells. Both recombinant proteins (proCCN and CCN) were expressed as inclusion bodies in E. coli cells and their folding was examined under several sets of conditions. After the refolding reactions, both of the recombinant proteins were present as the oxidized soluble forms. The proCCN protein was then auto-processed to release the propeptide region for activation. Interestingly, the CCN (CCN∗) derived from the refolded proCCN showed a much stronger protease activity than the refolded CCN from the reduced CCN in a protease assay using Bz-Arg-OEt as a substrate. In addition, the secondary structure of the refolded CCN protein was similar to that of the CCN∗ protein, as evidenced by CD measurements. These results suggest that the CCN protein becomes trapped in a molten globule-like state without the assistance of the propeptide region during the folding process. We therefore conclude that the propeptide region of CCN kinetically accelerates the folding of CCN to adopt the correct conformation of cocoonase at the final step of the folding pathway.