Our research program focuses on the analysis, synthesis, and structure-function relationships of peptide and protein hormones. During the past year, we developed one-platform tandem mass-spectrometric analysis for the identification of serine and threonine phosphorylation and glycosylation sites in protein. We also screened for angiotensin II–mediated changes in the protein kinase expression and phosphorylation site levels in cells and performed structure-function studies of an anti–HIV nonapeptide from human lysozyme.
Mass-spectrometric analysis of O-linked glycosylation sites in protein
Chen H-C, Wang G, Wang D; in collaboration with, Backlund, Boykins
Most eukaryotic proteins are glycoproteins, and more than 50 percent of human proteins are glycosylated in one of the following forms: Asn-(N)-linked or Ser- or Thr (O)-linked. Protein glycosylation is a post-translational modification that is involved in a variety of important biological processes, including the biosynthesis, folding, solubility, stability, subcellular trafficking, turnover, and half-life of the molecules to which they are attached. While the N-linked glycosylation site can be predicted by its common occurrence on Asn in Asn-X-Ser or Asn-X-Thr sequence and identified by protein sequence analysis as Asp after specific glycosidase cleavages, similar predictions do not hold for O-linked glycosylation sites. In addition, tandem mass-spectrometric analyses of a glycopeptide are impractical due to its heterogeneities and the various chain lengths of the oligosaccharide moieties attached to the peptide, which generate highly complex spectra that are impossible to interpret. Therefore, the development of an effective O-linked glycoprotein analysis method is necessary in order to tackle many problems associated with carbohydrate-mediated biological functions.
Previously, we developed methodologies based on beta-elimination and Michael addition for the identification, by tandem mass spectrometry (MS/MS), of P-Ser and P-Thr sites in peptides. In particular, we achieved effective modification of phosphopeptide under a mild base–catalyzed beta-elimination, using barium hydroxide and the addition of propanethiol in n-propanol, which forms S-propylcysteine and beta-methyl-S-propylcysteine from P-Ser and P-Thr, respectively. We applied the same condition to modify a 3.1 kDa peptide from bovine beta-casein and achieved, for the first time, the identification of four clustered phosphorylation sites by a routine platform of electrospray ionization (ESI) ion trap MS/MS.
Barium hydroxide has long been considered ineffective for the beta-elimination of O-glycosidic Ser and Thr because the carbohydrate moieties do not form coordinate bonds with the barium ion. However, beta-elimination of O-glycosidic Ser or Thr is known to occur readily at a lower sodium hydroxide concentration than those required for P-Ser or P-Thr. Indeed, using ESI ion trap MS/MS for detection, tests of synthetic O-linked N-acetylgalactosamidic-Ser glycopeptide (KMSTLgSYR; g denotes the monosaccharide residue) under conditions normally used for phosphopeptides revealed the formation of S-propylcysteine at the O-glycosidic site. We achieved more extensive modification by increasing the barium hydroxide concentration. We then sought the optimum conditions for modifying several O-glycosidic peptides, using as a test two natural glycopeptides (Sg´TVATLEDpSPE and Pg´TSg´TPTg´TEAVE; g´ denotes complex oligosaccharides) purified from a trypsin and Glu-C digest of glycomacropeptide, also known as GMP, of bovine kappa-casein. We used ESI ion trap MS/MS to determine both phosphorylation and O-glycosylation sites in the peptides and found the optimum reaction conditions to be 50mM BaOH2, 25 percent DMF, 20 percent ETOH, 1M propanethiol, and incubation for 24 hours at room temperature.
Li W, Backlund PS, Boykins RA, Wang GY, Chen HC. 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.
Screening of protein kinase expression and phosphorylation site mapping of proteins in angiotensin II–stimulated adrenal cells
Chen H-C; in collaboration with Chen H-D, Catt
In addition to blood pressure regulation, aldosterone secretion, and sodium balance, angiotensin II (Ang II) has potent mitogenic and pro-inflammatory activities that influence several cellular responses associated with vascular inflammation and structural remodeling. Although the primary action of Ang II is mediated by the Gq-coupled AT1 receptor and its inositol phosphate/Ca2+–mediated signaling pathways, protein kinases and phosphoproteins play pivotal roles in the control of cellular responses. In an effort to identify novel protein kinases involved in such control, we took advantage of the availability of specific antibodies against a wide variety of protein kinases and analyzed the protein kinases’ expression patterns with Western blot analysis before and after Ang II stimulation in human adrenocortical carcinoma cells known as H295R. The results indicated that Ang II stimulation increased levels of casein kinase 2, elongation factor-2 protein-serine kinase, ERK1, IKK-beta, AKT/PKB-alpha, PKC-micro, Moloney sarcoma oncogene–encoded protein-serine kinase, DAPK, FAK, and JAK1. In contrast, Ang II stimulation of H295R also resulted in decreased levels of other kinases, such as Cdk 5, Osaka thyroid oncogene protein-serine kinase, GSK3-alpha, PKC-alpha, ribosomal S6 kinase 1, CaM kinase 1, IKK-alpha, Yes-related protein–tyrosine kinase, Raf1 proto-oncogene–encoded protein–serine kinase, cGMP-dependent protein kinase, and ZIP kinase.
We also made use of anti–phospho-site antibodies to analyze the level of phosphorylation at specific sites in phosphoproteins in Ang II–stimulated H295R cells. Results indicate increases of ribosomal S6 kinases at S380/386 and T573/577, AMP-activated protein kinase-alpha at T174/172, and ERK1 at T202/Y204, GSK3beta (S9), MEK1/2 (S212/221), p38alpha MAPK (T180/Y182), p70 S6 kinase (T421/424), Raf1 (S259), and Raf1 (S259). We observed a decrease of the phosphorylation level in Cdk1 (T161/160), Cdk1 (Y15), and GSK3alpha (S21).
Our results, which concur with those of earlier studies, indicate that Ang II stimulation of H295R cells increases the expression of p38MAPK and ERK as well as phosphorylation of specific sites on the kinases. The increased levels of PKB/Akt and GSK-3-beta phosphorylation at Ser-9 are noteworthy. GSK-3-beta is known to be phosphorylated at Ser-9 by PKB/Akt after stimulation by a wide variety of agents. As a consequence, the activity of GSK-3-beta is attenuated to downregulate the downstream signaling pathway involving phosphorylation of several transcription factors. PKB/Akt is also a major kinase downstream from phosphatidylinositol 3-kinase (PI3K). Therefore, many stimuli that activate PI3K may also inhibit GSK-3-beta through PKB/Akt phosphorylation. Our results establish for the first time a linkage between Ang II stimulation and GSK-3-beta phosphorylation. Although negative regulation of GSK-3-beta causes cardiac hypertrophy, there is no report of an Ang II–induced GSK-3 effect in either cardiac or adrenal cells. It is conceivable that the cardiac hypertrophic effect of Ang II is mediated through the Gq-coupled AT1 and the PI3K-PKB/Akt to GSK-3-beta pathway. We will verify the action of the pathway by studying Ang II–induced GSK-3-beta phosphorylation in association with the rate of protein synthesis and hypertrophic gene expression in cardiac myocytes and aortic cells. Similarly, we will focus on the effect of Ang II–induced GSK3-beta expression in H295R cells followed by GSK-3-beta catalyzed phosphorylation on transcription factors, i.e., CREB, c-Jun, and NF-kappaB in search of its function.
Structural and functional modeling of HL9, a unique nonapeptide from human lysozyme with anti–HIV activity
Chen H-C; in collaboration with Lee-Huang
Lysozyme was originally described for its effects against Gram-positive bacteria. Its bactericidal property is attributable to its muramidase activity, which hydrolyzes the peptidoglycan of bacterial cell walls, resulting in cell lysis. However, a growing body of evidence suggests the existence of nonenzymic and/or nonlytic modes of action against other microbes. Even when reduced and/or partially unfolded, lysozyme still exerts broad-spectrum antimicrobial action against both Gram-negative and Gram-positive bacteria independent of muramidase catalytic activity. Moreover, lysozyme is widespread in nature and plays critical roles in host defense in both the control of microbial infection and the modulation of host immunity. Our previous report indicated that lysozyme accounts for the anti–HIV activity associated with the beta-core fraction of human chorionic gonadotropin. In search of the active sites for anti–HIV activities, we conducted peptide fragmentation and activity mapping of human lysozyme.
We identified two fragments, denoted HL18 and HL9, that possess anti–HIV activity comparable to the intact molecule. Structure-function studies of synthetic analogues of HL9 revealed that anti–HIV activity is sequence-specific, as scrambling the sequence abolishes the activity. Anti–HIV activity is also dependent on charge and hydrophobicity, as substitution of positively charged arginine residues 107, 113, and 115 or hydrophobic tryptophan residues 109 and 112 results in the loss of anti–HIV activity. The three-dimensional structure of lysozyme indicates that HL9 (RAWVAWRNR; residues 107 through 115) constitutes helix 4. This sequence is unique and found only in mammalian lysozymes. In native lysozyme, HL9 exists as an alpha-helix in a region of the protein that is distinct from the muramidase catalytic site. Thus, muramidase activity and antiviral activity are distinct features of intact human lysozyme.
With different glycan substrates, lysozyme is known to exist in diverse folding conformations around its active site. A helix-loop-helix motif in hen egg white lysozyme and human lysozyme has been reported to confer antimicrobial activity with membrane permeation. Energy-minimizing simulation predicts that HL9 can adopt an alpha-helical conformation essentially identical to that found in the native molecule, even without any flanking sequences. Circular dichroism (CD) studies show that HL9 adopts alpha-helical conformations mainly in a solution containing helicogenic trifluoroethanol (TFE) but otherwise assumes a random-coil confirmation structure in aqueous buffer. Modeling and CD studies indicate that helical propensity alone is not sufficient for and does not correlate with antiviral activity. Thus, inactive peptides with scrambled sequence or amino acid substitutions formed identical CD spectra indicative of alpha-helix and random coil in TFE and aqueous buffer, respectively. Neither its basicity (pI 12.3) nor its secondary structure alone can fully explain peptide HL9’s anti–HIV activity; other peptides with identical pIs and alpha-helical structure, such as the HL9 scrambled sequence and the HL9 RÆK mutants, exhibit no anti–HIV activity. We therefore conclude that anti–HIV activity requires a unique amino acid sequence, specific structural features, and specific folding topologies.
HL9 has direct effects on HIV-1 viral entrance as well as on replication, with several mechanisms potentially responsible for these effects. As proposed for other cationic peptides, HL9 may disrupt the viral particle or prevent its binding and entry into target cells. Alternatively, HL9 may signal the cell in a receptor-dependent manner, resulting in an intracellular antiviral state that affects a post-entry step(s) in the viral life cycle. The cDNA microarray studies on the effect of HL9 on HIV-1–infected target cells provide a sensitive profiling of the host response during viral infection and following antiviral treatment. Our findings suggest that the effects of HL9 on signaling pathways involved in survival, stress, TGF-beta, p53, NFkappaB, protein kinase C, and hedgehog signaling may reflect changes in host cells that affect susceptibility to infection.
In summary, our work represents the first identification, structure/activity mapping, and modeling of small anti–HIV mimetics from human lysozyme. HL9 peptide can be easily synthesized and is readily available. The main advantage of host peptides as effectors of innate immunity is that they can function without either high specificity or memory. Structural and functional characterization of natural antimicrobial peptides generated from physiological precursors is of growing interest because of potential therapeutic applications. Our findings that HL18 and HL9 are active against HIV-1 may lead to new strategies for the treatment of HIV-1 and other viral infections.
Lee-Huang S, Maiorov V, Huang PL, Ng A, Lee HC, Chang YT, Kallenbach N, Huang PL, Chen HC. Structural and functional modeling of human lysozyme reveals a unique nonapeptide, HL9, with anti-HIV activity. Biochemistry 2005;44:4648-4655.
COLLABORATORS
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
Kevin Catt, MD, PhD, Endocrinology and Reproduction Research Branch, NICHD, Bethesda, MD
Hung-Dar Chen, PhD, Endocrinology and Reproduction Research Branch, NICHD, Bethesda, MD
Sylvia Lee-Huang, PhD, New York University School of Medicine, New York, NY
For further information, contact chenha@mail.nih.gov.