Our research program is concerned with the analysis, synthesis, protein expression, and structure-function relationships of peptide and protein hormones. We are currently focusing on the structure and function of the angiotensin II (Ang II) and gonadotropin-releasing hormone (GnRH) receptors and related studies. Our major accomplishments over the past year include the development of mass spectrometric analysis for the identification of serine and threonine phosphorylation sites, verification of corticotropin-releasing hormone (CRH) production in an immortalized rat hypothalamic cell line, and determination of a glycosylation site of pigeon IgG.

Mass spectrometric analysis of phosphopeptides

Li, Wang, H-C. Chen

Protein phosphorylation is considered the most important reaction in the regulation of eukaryotic cell function. Of the known proteins phosphorylated, 99 percent are phosphorylated at serine or threonine. We have developed a chemical method that converts phospho-Ser and phospho-Thr to cysteic acid and beta-methyl cysteic acid, respectively, for direct sequencing of the Ser and Thr phosphorylation sites by electrospray ionization (ESI) tandem mass spectrometry. The conversion can be carried out at 1 microM concentration of the peptide. We showed that the cysteic acid and beta-methyl cysteic acid residues in the sequence are both 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 an ethylthio group with a mass 44 in excess of that expected 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. On the other hand, we could not detect modification on the free hydroxyl group of Ser or Thr when conditions were mild, viz, 0.1 N NaOH/0.6 M sodium sulte at 25° C for 24 hours. This finding suggests that temperatures above 25ºC and excessive alkalinity should be avoided in order to prevent the beta-elimination of the hydroxyl group of Ser and Thr in peptides. The outcome 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.

Li W, Backlund PS, Boykins RA, Wang GY, Chen H-C. Susceptibility of the hydroxyl group in serine

and threonine to beta-elimination/Michael addition under commonly used moderately high temperature conditions. Anal Biochem; in press.

Li W, Boykins RA, Backlund PS, Wang G, Chen H-C. Identification of phospho-serine and phospho-

threonine as cysteic acid and beta-methylcysteic acid residues in peptides by tandem mass spectroscopic sequencing. Anal Chem 2002;74:5701-5710.
 

Verification of CRH production in an immortalized rat hypothalamic cell line

Morell, H-C. Chen

Previously, we chemically synthesized a 41-residue peptide with a sequence corresponding to human/rat CRH. Brief oxidation of the CRH peptide in dilute hydrogen peroxide and acetic acid solution yielded Met(O)-CRH at positions 21 and 38. We used the two peptides to develop a reverse-phase HPLC system that permitted base-line separation between the two. To verify the production of CRH in a clonal cell line (designated as IVB) derived from rat embryonic hypothalamic tissue, we fractionated cell lysates by the HPLC system and analyzed the aliquots by a specific radioimmunoassay for rat CRH. The endogeneous immunoreactivity coeluted with synthetic CRH. Furthermore, oxidation of the lysate converted its radioimmunoreactivity elution position to one 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 for understanding the molecular mechanisms that operate in hypothalamic CRH neurons.

Kasckow J, Mulchahey JJ, Aguilera G, Pisarska M, Nikodemova M, Chen H-C, Herman JP, Murphy

EK, Liu Y, Rizvi TA, Dautzenberg FM, Sheriff S. Corticotropin-releasing hormone (CRF) expression and protein kinase A mediated CRH receptor signaling in an immortalized hypothalamic cell line. J Neuroendocrinol 2003;15:521-529.

Identification of pigeon IgG and its N-glycopeptide

H-D. Chen, H-C. Chen

Among glycoproteins, those from the pigeon egg were found uniquely to 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. We purified a major pigeon serum glycoprotein, IgG (alias IgY). 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. Using reverse-phase HPLC, we isolated one glycopeptide 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. Given that 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. 

Suzuki N, Khoo KH, Chen CM, Chen HC, Lee YC. N-glycan structures of pigeon IgG: a major serum

glycoprotein containing Gal-alpha 1,4Gal termini. J Biol Chem; in press.

COLLABORATORS

Greti Aguilera, MD, Laboratory of Developmental Endocrinology, NICHD, Bethesda MD 
Peter S. Backlund, PhD, Laboratory of Cellular and Molecular Biophysics, NICHD, Bethesda MD 
Robert A. Boykins, BS, Center for Biologics Evaluation and Research, FDA, Bethesda MD 
John Kasckow, MD, University of Cincinnati School of Medicine, Cincinnati OH

Yuan-Chuan Lee, PhD, The Johns Hopkins University, Baltimore MD

Wei Li, PhD, Laboratory of Biochemical Genetics, NHLBI, Bethesda MD

Noriko Suzuki, PhD, The Johns Hopkins University, Baltimore MD

For further information, contact chen@helix.nih.gov