This 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. Accomplishments of these projects include: (1) A rhodamine B conjugate of Ang II at its amino-terminus and a sulforhodamine 101 (Texas Red) conjugate of [D-Lys-6, des Gly-10amide]GnRH-ethylamide at epsilon-amino group of D-Lys-6 were synthesized. Both conjugates are biologically active as revealed on confocal microscopy by their ability to bind to their receptors and to internalize in the same manner as the natural ligands. (2) A method for converting phospho-Ser and phospho-Thr to cysteic acid and beta-methyl cysteic acid, respectively was developed. The stability of the sulfo-moiety over the phospho-moiety for MS/MS peptide sequence analysis during low energy collision in electron spray ionization was shown to simplify the identification of phosphorylation sites in peptides. (3) By a yeast two-hybrid technique using the C-terminal fragment (res. 301-359) of the human Ang II receptor type 1 (hAT1), in which potential phosphorylation sites (Thr-332, -336 and Ser-335) were mutated to Asp to mimic phosphorylated residues as bait, one clone known as CD74 (MHC class II-associated invariant chain, Ii) was obtained from screening of the human kidney cDNA library. Association of hAT1 and Ii in vivo was demonstrated by yeast mating experiments and by co-localization at the plasma membrane of human embryonic kidney (HEK-293) cells on confocal microscopy. HEK cells were transfected with both AT1-green fluorescent protein and Ii-red fluorescent protein fusion proteins. (4) Among egg glycoproteins, those from the pigeon uniquely possess binding activity for uropathogenic Eschericia coli and Shigella suis. A preparative reverse-phase HPLC system was devised to isolate four components from pigeon egg white proteins that were characterized by sequencing and SwissProt homology search, allowing identification of them as ovomucoid, ovotransferrin and two ovalbumins. All four proteins contain a terminal Gal-alpha1-4Gal sequence that is uncommon in mammals and other avians but is known to facilitate binding to the microbes. Matrix-assisted laser desorption/ionization mass spectrometric analysis revealed pigeon ovomucoid and a high molecular weight albumin to be larger than their chicken counterparts. These differences are attributable to the number and length of their oligosaccharide chains.
