Defense Date

1998

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biochemistry and Molecular Biology

First Advisor

Robert B. Harris

Abstract

Two biologically active peptides, gonadotropin releasing hormone (GnRH) and GnRH associated peptide (GAP) are both derived from a common prohormone precursor protein, pro-GnRH/GAP. Both peptides are cosecreted from hypothalamic neurosecretory granules and are involved in the regulation of mammalian reproduction. A calcium dependent, neutral pH serine protease discovered in this laboratory, GAP-releasing enzyme, is the most likely processing enzyme of pro-GnRH/GAP. GAP-releasing enzyme is immunologically related to PC1/3, a member of the prohormone convertase (PC) class of processing endoproteinases.

GAP-releasing enzyme recognizes the eight residue processing site within pro-GnRH/GAP, G6LRPGGKR13, and correctly cleaves the R13-D14 bond to yield bioactive GAP and a three residue extension of GnRH. We and others have postulated that the recognition site for GAP-releasing enzyme forms a defined structural element at the surface of the substrate protein and that this structural element helps mediate limited endoproteolysis.

In the work reported here, hundred mg quantities of pro-GnRH/GAP were prepared by novel methods of both chemical synthesis and bacterial expression. Large amounts of pure protein are required for enzymatic and biophysical studies of pro-GnRH/GAP, which are intended to establish whether or not the processing site within the prohormone exists as a defined structural element that plays a central role in endoproteolytic processing. Synthetic pro-GnRH/GAP was prepared in high yield but proved difficult to purify to homogeneity. Recombinant pro-GnRH/GAP was prepared in sufficient yield and purity to perform all subsequent experiments.

An immunoassay was developed against a processing site epitope within pro-GnRH/GAP. Both synthetic and recombinant pro-GnRH/GAP proteins are immunoreactive, consistent with the idea that the epitope, and, thus, the processing site, is located on the surface of the molecule. Proteolysis of synthetic or recombinant pro-GhRH/GAP by trypsin or kallikrein caused immediate loss of immunoactivity, showing that the processing site is susceptible to proteolysis and that the integrity of the processing site is required for immunoactivity. One of the kallikreih hydrolytic products was identified as GAP. Therefore, kallikrein cleaves at the R13-D14 bond.

The intrinsic fluorescence yield of the Trp residue near the processing site region of pro-GhRH/GAP is sensitive to changes in pH, but not to changes in ionic strength or calcium concentration; its fluorescence yield is maximal at neutral pH. This suggests that the processing site displays maximum structure at neutral pH. This finding is coincident with the fact that GAP-releasing enzyme is optimally active at neutral pH. However, the relative contribution of secondary structural elements, as discerned by circular dichroism, remains constant over the range of pH 5.2-10.7. Only at pH

Thermal denaturation of pro-GnRH/GAP follows a simple two-state transition at neutral pH, as assessed by differential scanning calorimetry. This shows that pro-GnRH/GAP assumes a protein—like tertiary structure at neutral pH. 1D NMR data obtained at variable pH showed changes in resonance position and spectral resolution which are consistent with pH mediated conformational change and with the assumption of organized structure at neutral pH. However, the lack of through space correlations in the 2D NOESY experiment indicates that determination of the three-dimensional structure of pro-GnRH/GAP at neutral pH may be problematic.

Comments

Scanned, with permission from the author, from the original print version, which resides in University Archives.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

2-7-2018

Included in

Biochemistry Commons

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