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structure
andfunction of peptide
and proteinhormones
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Hao-Chia Chen, PhD, Head, Unit on Molecular Structure and Protein Chemistry Ja Shin
Koo, PhD, Postdoctoral
Fellow Guiyu
Wang, MD, Adjunct Investigator Sashwata Goswami, Summer Student David Wang, Summer Student |
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Our research program is concerned with the
analysis, synthesis, protein expression, and structure-function relationships
of peptide and protein hormones. In the past year, we focused on development
of mass spectrometric analysis for the identification of serine and threonine
phosphorylation sites, identification of phosphorylation sites in human
angiotensin II type 1 receptor (hAT1), overexpression of protein
corresponding to the carboxyl-terminal domain (residues 268 through 359) of
AT1, identification of an anti-HIV active site in human lysozyme, cloning of
pigeon egg white protein cDNAs, and
characterization of expressed yeast ribonuclease H2. Mass spectrometric analysis of phosphopeptides Wang,
Chen Protein phosphorylation on Ser, Thr, and Tyr
plays a major role in the regulation of eukaryotic cell function.
Approximately 30 percent of all mammalian cell proteins are phosphorylated at
any given time, and 99 percent of phosphorylation occurs on Ser and Thr
residues. Therefore, characterization of the phosphorylation sites (P-sites)
is critical to understanding how these modifications modulate the biological
activities of proteins. Despite the development of numerous other
methods to analyze intracellular protein phosphorylation, mass spectrometric
(MS) methods are the most advanced owing to their sensitivity, accuracy, and
selectivity. A problem hampering the analysis of phosphopeptides in a peptide
mixture is the inherent proton sequestration property of phosphoric acid,
resulting in poor ionization and ionization suppression in positive ion MS.
Furthermore, MS/MS identification of P-sites directly using phosphopeptides
as precursors is complicated by the loss, in most cases, of the phosphate
moiety during low-energy collision–induced dissociation (CID). To
improve the ionization of phosphopeptides for detection, the phosphate group
could be eliminated or converted into a neutral or positively charged group.
We have developed methodologies based on beta-elimination/Michael addition to
modify the P-Ser and P-Thr into CID-stable derivatives. Furthermore, we used
the same reaction to liberate O-linked glycans from glycoproteins or peptides
and to identify the exact O-glycosylation sites by tandem mass spectrometry.
Figure 4.1 illustrates the mechanism of beta-elimination/Michael addition.
However, under commonly employed conditions, i.e., the use of 0.2-0.5N NaOH for modification, nonspecific beta-elimination on
Ser and Thr could easily occur, as described by our previous studies. This
outcome is of particular concern when employing highly sensitive tandem MS
methods. The additional modification site(s) may complicate the
interpretation of data and lead to an erroneous conclusion. Therefore, we
need an optimal modification condition that can both avoid side reactions and
facilitate detection of phosphopeptides and permit accurate assignment of the
P-sites. We have systematically studied a reaction
catalyzed by the mild base barium hydroxide to eliminate O-linked phosphate
or glycans on Ser or Thr followed by the addition of propanethiol
or butanethiol as nucleophile. Under optimum
conditions (1 microM peptide, 20 mM Ba(OH)2, 30
percent n-propanol, and 0.5M alkanethiols incubated
for 24 hours at 25°C), n-propylthio and n-butylthio derivatives both P-Ser– and P-Thr–containing
peptides not only were stable during CID but also yielded at least sevenfold more positive mode ionization than the parent
molecule when analyzed by matrix-assisted laser desorption ionization (MALDI)
MS. Furthermore, we easily identified abundant y and b fragment ions under
general conditions for electrospray ionization
tandem mass spectrometry. We applied the procedure to identify the P-sites in
synthetic P-Ser and P-Thr peptides (KMpSTLSYR and KMSpTLSYR) and a 16-residue phosphopeptide
(FQpSEEQQQTEDELQDK) from bovine beta-casein.
However, the carbohydrate moiety in either synthetic O-GalNAc-Ser or -Thr
glycopeptide was only slightly eliminated under the same conditions,
indicating that barium hydroxide could catalyze beta-elimination efficiently
on O-phospho- but not on O-GalNAc-Ser and -Thr. Thus, the method permits us
to distinguish between O-phospho- and O-glycan sites based on the
beta-elimination/Michael addition reactions.
For the identification of phosphorylation
sites in hAT1, we obtained tryptic fragments of solubilized proteins from
angiotensin II–stimulated human adrenal cortical carcinoma cells
(H295R). We plan to isolate phosphopeptides from the tryptic digest by
immobilized metal (Fe3+) affinity chromatography and then submit them
to the beta-elimination/propanethiol addition
described above. We will analyze the modified peptides by the combination of nanoflow HPLC on an LCQ ion-trap mass spectrometer and
data search to identify the phosphorylation sites in AT1 by the SEQUEST or
MASCOT program. 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 2003;323:94-102. Overexpression of protein
corresponding to the carboxyl-terminal domain of hAT1 receptor Koo, Chen The phosphorylation of the cytoplasmic
carboxyl-terminal sequence of G protein–coupled receptors plays a
crucial role in receptor internalization, desensitization, phosphorylation,
and recycling and has been reported to modulate interactions with numerous
proteins, including heterotrimeric G proteins, JAK2
kinase, and AT1 receptor–associated proteins. We amplified a
partial segment of the carboxyl-terminal cytoplasmic domain (CTP, residues
268 through 359) of hAT1 cDNA by PCR. We inserted the amplified DNA fragment
into a pMAL-c2X vector, which contains maltose-binding protein (MBP) cDNA,
and transfected the recombinant plasmid transfected into E . coli BL21. An MBP fusion protein containing as much as
50 percent of the total soluble protein was expressed. Using the amylose
resin method, we purified the CTP-hAT1 fusion MBP protein and cleaved off MBP
with Factor Xa. We verified that the purified
protein was the CTP by Edman degradation sequencing. Final large-scale
purification of the CTP fragment is now in progress. Identification of anti-HIV active site in
human lysozyme Chen Previously, we reported that the lysozyme
associated with the beta-core fraction of human chorionic gonadotropin (HCG)
accounts for HCG’s anti-HIV activity
(Lee-Huang S et al., Proc Natl Acad Sci USA 1999;96:2678). To
define the structural and sequence requirements for anti-HIV activity, we
identified two peptides that consist of residues 98 through 115 of human
lysozyme and its C-terminal nine-residue segment RAWVAWRNR (designated,
respectively, HL18 and HL9). Both HL18 and HL9 are potent inhibitors of HIV-1
infection and replication at a level comparable to that of intact lysozyme.
By scrambling residues 107, 113 or 115, 109, and 112 or substituting each or
in combination, we demonstrated the importance of Arg and Trp at certain
positions by the loss of anti-HIV activity. Although HL9 exists as an alpha
helix and forms a pocket with its basic residues on the surface of the
molecule, as occurs the lysozyme molecule, it does
not display the muramidase activity associated with the lysozyme because it
is located outside the muramidase catalytic site of human lysozyme. Cloning of pigeon egg white glycoproteins Chen We previously purified and sequenced four
components from pigeon egg white proteins and identified them as ovomucoid,
ovotransferrin, and two ovalbumins (high- and
low-molecular weight forms). MALDI-MS revealed a mass of 53,129.7 for the
high- and 48,551.6 for the low-molecular weight forms. Only one form
corresponding to the low-molecular weight form has been found in most avian
species despite the fact that chickens contain multiple ovalbumin genes.
Unique to pigeon egg white glycoproteins, the four proteins we have isolated
all contain a terminal Gal-alpha 1,4-Gal sequence
that is uncommon in mammals and other avians. These
proteins are capable of binding to the surface of uropathogenic
E. coli and Shigella suis. Based on our knowledge of the amino
acid sequences of fragments obtained from each of the four proteins, we
obtained full-length cDNA clones encoding the four proteins by screening a
pigeon oviduct library. The four cDNAs encode
proteins of the following lengths of amino acid sequence, with
identity/similarity to the respective chicken counterparts listed in
parentheses as percentages: ovalbumin-high (pigeon 388 versus chicken 386
residues; 58.29 percent/68.91 percent of chicken); ovalbumin-low (386 versus
386; 66.58 percent/75.65 percent); ovomucoid (209 versus 210; 70.53
percent/60.38 percent); and ovotransferrin (706 versus 705; 78.01
percent/83.40 percent). Aside from several forms of ovalbumin in pigeon,
dissimilarity in protein sequences of egg white glycoproteins between pigeon
and chicken is significant and may indicate a way to understand the evolution
of avians in general and that of related
glycoproteins in particular. Suzuki N, Khoo KH, Chen CM, Chen H-C, Lee YC. N-Glycan
structures of pigeon IgG: a major serum glycoprotein containing Gal-alpha
1,4Gal termini. J Biol Chem 2003;278:46293-46306. Characterization of yeast ribonuclease H
Rnh2Ap expressed in E. coli Chen When expressed in E. coli, Saccharomyces
cerevisiae Rnh2Ap, a homolog of bacterial ribonuclease (RNase) H2, proved
to be inactive as a single chain. To rule out that the inactivity resulted
from an error in expression or isolation of the target protein, we performed
N-terminal sequence analysis by Edman degradation method and tandem LC-MS/MS
analyses of its tryptic fragments and verified the expressed protein of
interest as Rnh2Ap. This finding led to the elucidation of the complex nature
of ribonuclease H2 of Saccharomyces cerevisiae. Two more proteins,
namely Ydr279p and Ylr154p, together with Rnh2Ap, are necessary and
sufficient for RNase H2 activity. Jeong HS, Backlund PS, Chen H-C, Karavanov AA, Crouch RJ. RNase H2 of Saccharomyces
cerevisiae is a complex of proteins. Nuclei Acid Res 2004;32:407-414. COLLABORATORS Peter S. Backlund,
PhD, Laboratory of Cellular and Molecular Biophysics, NICHD, Robert A. Boykins,
BS, Center for Biologics Evaluation and
Research, FDA, Hung-Dar Chen,
PhD, Endocrinology and Reproduction Research Branch, NICHD, Robert J. Crouch, PhD, Laboratory
of Molecular Genetics, NICHD, Yuan-Chuan
Lee, PhD, The Sylvia Lee-Huang, PhD, Wei Li, PhD, Laboratory of
Biochemical Genetics, NHLBI, Noriko
Suzuki, PhD, The For
further information, contact chenha@mail.nih.gov |
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