Structure And Function Of Peptide And Protein Hormones

Child Health And Human Development

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Abstract

The research program is concerned with the analysis, synthesis, protein expression, and structure-function relationships of peptide and protein hormones. Current emphases are placed on the structure and function of the angiotensin II (Ang II) and gonadotropin-releasing hormone (GnRH) receptors, and related studies. Recent accomplishments include development of mass spectrometric analysis for the identification of serine and threonine phosphorylation sites by converting phospho-Ser and phospho-Thr to cysteic acid and beta-methyl cysteic acid, respectively via beta-elimination Michael addition reaction. Both cysteic acid and beta-methyl cysteic acid residues in the sequence were shown to be stable and easily identifiable in a series of y and b ions under general conditions for tandem mass spectrometric sequencing applicable to common peptides. However, the reactivity of the free hydroxyl group on serine and threonine by sodium hydroxide-induced beta-elimination has not been critically examined. Under the widely used conditions previously reported by others for beta-elimination and ethanethiol addition, two analogous phosphopeptides ( KMpSTLSYR and KMSpTLSYR) yielded additional 44 mass of ethylthio group in excess of the expected mass for the modified phosphopeptide analyzed by MALDI-TOF. ESI tandem mass spectrometric analysis confirms that the modification occurred on the hydroxyl group of Ser and Thr in addition to P-Ser and P-Thr residues. On the other hand, modification on the free hydroxyl group of Ser or Thr was not detected under the mild condition of 0.1 N NaOH/0.6 M sodium sulfite at 25?C for 24 hours. This finding suggests that temperature above 25 degree C and excessive alkalinity should be avoided in order to prevent the beta-elimination of the hydroxyl group of Ser and Thr in peptides. This is of particular concern when employing highly sensitive tandem mass spectrometric methods for the identification and localization of Ser and Thr as modification sites by the beta-elimination/Michael addition reaction. The additional modification site(s) may complicate the interpretation of data and lead to an erroneous conclusion. Previously, a 41-residue peptide with a sequence corresponding to human/rat CRH was synthesized under this program. Brief oxidation of the CRH peptide in dilute hydrogen peroxide and acetic acid solution yielded Met(O)-CRH at positions 21 and 38. These two peptides were used to develop a reverse phase HPLC system that permitted base-line separation between the two. In order to verify the production of CRH in a clonal cell line (designated as IVB) derived from rat embryonic hypothalamic tissue, its cell lysates were fractionated by this HPLC system and the aliquots were analyzed by a specific radioimmunoassy for rat CRH. The endogeneous immunoreactivity was found to co-elute with synthetic CRH. Furthermore, oxidation of the lysate converted its radioimmunoreactivity elution position to be coincident with that of the oxidized CRH separated from CRH. In addition, IVB cells exhibited high-affinity binding of CRH and shared many functional characteristics with other hypothalamic CRH cells. This new immortalized cell line may be a useful model to better understand the molecular mechanisms that are operating in the function of hypothalamic CRH neurons. Among glycoproteins, those from the pigeon egg were found to uniquely possess binding activity for uropathogenic Eschericia coli and Shigella suis. Previously, we purified and sequenced four components from pigeon egg white proteins and identified them as ovomucoid, ovotransferrin and two ovalbumins. All four proteins contain a terminal Gal-alpha 1,4-Gal sequence that is uncommon in mammals and in other avians. A major pigeon serum glycoprotein, IgG (alias IgY) was purified. N-terminal sequence analyses of the light chain and heavy chain confirmed it as IgG based on its homology to the corresponding regions of chicken, duck and human IgG. Furthermore, the heavy chain of pigeon IgG, but not the light chain, stained with GS-1 lectin and anti-P1 mAb (specific for P1 blood type), indicative of a distinctive Gal-alpha 1,4-Gal linkage on the termini of the glycan that facilitates binding to uropathogenic microbes. One glycopeptide was isolated by reverse phase HPLC from a tryptic digest of the reduced-pyridinylethylated heavy chain. Peptide sequencing of this glycopeptide before and after glycoamidase F treatment revealed an N-glycosylation site in the sequence that is homologous to that located at the CH3 domain of chicken and duck IgG. Since both avian and mammalian IgG are known to contain two N-glycosylation sites, a search of the second glycopeptide of pigeon IgG is under way.