Conformational analysis and proteolytic processing of synthetic pre-pro-GnRH/GAP protein.

Homogeneous pre-pro-GnRH/GAP protein was recently synthesized in 100 mg quantities by solid-phase methods and surprisingly, the synthetic pre-pro-protein, which normally does not escape the endoplasmic reticulum, was found to inhibit the release of prolactin from cultured pituitary cells. This is the first demonstration of significant biological activity associated with a precursor protein and provides the rationale for its further study. We now report the results of our initial examination of the conformational properties of pre-pro-GnRH/GAP protein as a prelude to solving its solution phase conformation by homonuclear 1H-NMR protocols. Thermal and pH titration fluorescence and circular dichroism spectroscopies reveal that the protein is resistant to thermal-induced conformational changes but is particularly sensitive to pH-induced conformational changes; while Asp/Glu and Arg residues may contribute to structural stability, His and Lys residues predominate. Pre-pro-GnRH/GAP is about 30% helix in the range of 2-40 degrees C; however, even at 90 degrees C, the peptide retains nearly 50% of its helix character. There is no evidence for a cooperative transition; for this reason, differential scanning calorimetry failed to yield a defined transition thermogram. Pre-pro-GnRH/GAP apparently does not pass through a transition state as a function of temperature but appears to flex and retain a high percentage of helix structure, resulting in subtle changes in secondary structure. There is no discernible isodichroic point. On either side of the neutral pH range, however, there are dramatic changes in structure that result in nonreversible denaturation of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

GAP-releasing enzyme is a member of the pro-hormone convertase family of precursor protein processing enzymes.

The recent discovery of mammalian endoproteinases which show extensive sequence homology with the yeast Kex 2 gene product (kexin) has lead to the hypothesis that processing enzymes of pro-hormone precursor proteins belong to a family of calcium dependent, subtilisin-like serine proteinases. We previously showed that hypothalamic GAP-releasing enzyme shares these characteristics and possesses the requisite specificity to be considered as a processing enzyme of progonadotropin releasing hormone (pro-GnRH) precursor protein. Thus, GAP-releasing enzyme (and other non-related proteins) were tested for their immunological reactivity with antisera raised against pituitary pro-hormone convertase 1/3 (PC1/3) and insulinoma PC2. On the basis of indirect enzyme-linked immunosorbent (ELISA) and Western blot assays, GAP-releasing enzyme is now shown to be immunologically related to PC1/3. We can conclude that GAP-releasing enzyme is also likely to be a member of the pro-hormone convertase family and should be considered the physiologically relevant processing enzyme of pro-GnRH. It is possible that GAP-releasing enzyme represents bovine hypothalamic PC1/3.

Processing enzyme specificity is a consequence of pro-hormone precursor protein conformation.

Peptide-hormones are synthesized as higher molecular weight, precursor proteins which must initially undergo limited endoproteolysis to yield the bioactive peptide(s). The ability of two different endoproteinases, gonadotropin-associated peptide (GAP)-releasing enzyme and atrial granule serine proteinase (which are likely to be the physiologically relevant processing enzymes of bovine hypothalamic pro-gonadotropin-releasing hormone/gonadotropin-associated peptide and bovine pro-atrial natriuretic factor precursor proteins, respectively), to act at their own recognition sequences within their relevant pro-hormone proteins has now been contrasted with their ability to act at the recognition sequence for the alternate enzyme or to act at their own recognition sequence when it is placed within the protein framework of the alternate precursor protein. The results show that each enzyme acts with specificity at its own recognition sequence even when it is placed within the framework of the alternate pro-hormone. However, the enzymes fail to act (or act in a non-specific manner) at the alternate recognition sequence even if it is placed within the peptide framework of its own pro-hormone protein. Thus, despite the fact that both recognition sequences are similar in sequence and residue composition and that both contain a doublet of basic amino acids, it appears that sequence and the local conformation assumed by the processing site within the pro-hormone protein are essential for each endoproteinase to act with fidelity. As part of our continuing work, we now also report several newly determined physicochemical properties of hypothalamic GAP-releasing enzyme, the processing enzyme of pro-gonadotropin-releasing hormone/GAP protein.

Pro-gonadotropin-releasing hormone protein is processed within hypothalamic neurosecretory granules.

Peptide-hormones are synthesized as higher-molecular-weight precursor proteins which must undergo numerous posttranslational modifications to yield the bioactive peptide(s) which may include limited endoproteolysis, limited exopeptidase digestion, and C-terminal amidation. Three different enzymes which are likely to be the physiologically relevant processing enzymes of bovine pro-gonadotropin-releasing hormone (pro-GnRH) precursor protein have been colocalized to, and purified from, hypothalamic neurosecretory granules. Gonadotropin-releasing-hormone-associated-peptide-releasing enzyme initiates processing by endoproteolysis of the pro-hormone exclusively at the Arg 13-Asp 14 bond in the sequence, -Gly6-Leu-Arg-Pro-Gly 10-Gly-Lys 12-Arg 13-Asp-, which overlaps the sequence for GnRH (1-10) and GAP(14-69) within the pro-protein. Hypothalamic carboxypeptidase E then sequentially removes the -Lys12-Arg13- doublet from the newly formed peptide before peptidyl glycine alpha-amidating monooxygenase catalyzes the formation of amidated GnRH. Carboxypeptidase E activity is stimulated in vitro by cobalt ion and removes the Lys and Arg residues with equal facility. The residue which acts as the amide nitrogen donor for the alpha-amidating enzyme must be present as the free C-terminal residue of a substrate; the enzyme does not recognize peptide substrates with C-terminal extensions. Based on the mandatory ordered events for processing pro-GnRH and the relative pH profiles displayed by these enzymes, our results are consistent with the idea that endoproteolysis of the pro-hormone occurs upon formation of the secretory granule at the Golgi apparatus and that the processed peptides are the storage form within the secretory vesicles.