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Mass Spectrometric Analysis of Ehrlichia Chaffeensis Tandem Repeat Proteins Reveals Evidence of Phosphorylation and Absence of Glycosylation

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Mass Spectrometric Analysis of Ehrlichia Chaffeensis Tandem Repeat Proteins Reveals Evidence of Phosphorylation and Absence of Glycosylation CORE Metadata, citation and similar papers at core.ac.uk Provided by PubMed Central Mass Spectrometric Analysis of Ehrlichia chaffeensis Tandem Repeat Proteins Reveals Evidence of Phosphorylation and Absence of Glycosylation Abdul Wakeel1, Xiaofeng Zhang1, Jere W. McBride1,2,3,4,5* 1 Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America, 2 Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, United States of America, 3 Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas, United States of America, 4 Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, Texas, United States of America, 5 Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, Texas, United States of America Abstract Background: Ehrlichia chaffeensis has a small subset of immunoreactive secreted, acidic (pI ,4), tandem repeat (TR)- containing proteins (TRPs), which exhibit abnormally large electrophoretic masses that have been associated with glycosylation of the TR domain. Methodology/Principal Findings: In this study, we examined the extent and nature of posttranslational modifications on the native TRP47 and TRP32 using mass spectrometry. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) demonstrated that the mass of native TRP47 (33,104.5 Da) and TRP32 (22,736.8 Da) were slightly larger (179- and 288-Da, respectively) than their predicted masses. The anomalous migration of native and recombinant TRP47, and the recombinant TR domain (C-terminal region) were normalized by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) modification of negatively charged carboxylates to neutral amides. Exhaustive tandem mass spectrometric analysis (92% coverage) performed on trypsin and Asp-N digested native TRP47 identified peptides consistent with their predicted masses. Two TRP47 peptides not identified were located in the normally migrating amino (N)-terminal region of TRP47 and contained predicted phosphorylation sites (tyrosine and serine residues). Moreover, native TRP47 was immunoprecipitated from E. chaffeensis-infected cell lysate with anti-phosphotyrosine (anti-pTyr) antibody. Conclusions/Significance: TRP47 and TRP32 are not modified by glycans and the substantial net negative charge of the ehrlichial TRPs, and particularly the highly acidic TRs present within the ehrlichial TRPs, is responsible for larger-than- predicted masses. Furthermore, this study provides evidence that the N-terminal region of the TRP47 is tyrosine phosphorylated. Citation: Wakeel A, Zhang X, McBride JW (2010) Mass Spectrometric Analysis of Ehrlichia chaffeensis Tandem Repeat Proteins Reveals Evidence of Phosphorylation and Absence of Glycosylation. PLoS ONE 5(3): e9552. doi:10.1371/journal.pone.0009552 Editor: Deb Fox, The Research Institute for Children at Children’s Hospital New Orleans, United States of America Received November 11, 2009; Accepted February 12, 2010; Published March 4, 2010 Copyright: ß 2010 Wakeel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institute of Allergy and Infectious Diseases (AI 071145). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction Various functions have been associated with TRPs in pathogenic bacteria, including immune evasion, adhesion, actin nucleation, and Human monocytotropic ehrlichiosis (HME) is an emerging other host-pathogen interactions [11–18]. Similarly, TRPs identi- life-threatening tick-borne zoonosis caused by the obligately fied in E. chaffeensis and E. ruminantium and closely related Anaplasma intracellular Gram-negative bacterium Ehrlichia chaffeensis. E. marginale appear to play a role in cell adhesion [19–23], but the chaffeensis exhibits tropism for mononuclear phagocytes, and function of several immunoreactive TRPs in A. phagocytophilum is still survives by evading the innate host defenses [1–3]. A small subset unknown [24]. A more recent study has demonstrated that E. of E. chaffeensis proteins react strongly with antibodies in sera from chaffeensis TRP47 interacts with a network of host cell proteins infected humans or dogs [4–7], and the molecularly characterized involved in signaling, modulation of gene expression, and immunoreactive proteins of E. chaffeensis include tandem repeat intracellular vesicle trafficking [25]. E. chaffeensis TRP47 is acidic protein (TRP) 47, TRP120, and TRP32 (variable-length PCR (pI 4.2), contains seven 19-mer TRs (pI 2.9) in the C-terminal target) [8–10]. The TR domains of the TRPs are acidic, exhibit domain, and has a predicted molecular mass of 33 kDa, but exhibits high serine/threonine content, have predicted sites for posttrans- an electrophoretic mass of ,47 kDa. The TRP47 C-terminal TR lational modifications (glycosylation and/or phosphorylation), domain is homologous to renin receptor, DNA polymerase III exhibit larger-than-predicted molecular masses during electro- subunits gamma and tau-conserved domain, and ribonuclease E. E. phoresis, and contain major continuous immunodeterminants chaffeensis TRP32 is acidic (pI, 4.1), contains four TRs, and also [8–10]. migrates at a larger (32 kDa) than predicted (22.5 kDa) mass. PLoS ONE | www.plosone.org 1 March 2010 | Volume 5 | Issue 3 | e9552 E. chaffeensis TRP47-MALDI-TOF Glycoproteins have been identified in many bacteria including predicted masses demonstrating that these polypeptides were not Borrelia, Chlamydia, Escherichia, Neisseria, and Pseudomonas [26,27], modified (Table 1). and many of the characterized glycoproteins appear to be involved in host-pathogen interactions [20,26–30]. Moreover, carbohydrate MALDI-MS and Tandem (MS/MS) Mass Spectrometric has been detected on Ehrlichia and Anaplasma outer membrane Analysis of Trypsin-Digested TRP47 proteins and TRPs [8,20,28,31–35]. Glycosyltransferases have MALDI-MS performed on trypsin-digested TRP47 peptides been identified in the genomes of many bacteria that have exhibited high relative intensity of several abundant ions (m/z). glycoproteins; however, glycosyltransferases have not been iden- Those abundant ions with high relative intensity were selected for tified in Ehrlichia spp. genomes [36–38], suggesting that additional further MS/MS analysis. For protein/peptide identification, the studies to define the mass of these proteins in order to understand bacteria NCBI endopeptidase taxonomy searched in the NCBI non the extent and nature of the glycans (composition, structure and redundant database identified three peptides with significant protein/ attachment sites) on the native and recombinant proteins are peptide match, based on both the peptide mass fingerprint (PMF) and needed. the MS/MS data from several precursor ions with low expectation The objective of this study was to examine the native and values and high protein score. A total of 12% sequence coverage was recombinant E. chaffeensis TRP47 and TRP32 using mass achieved with trypsin digested TRP47, which included three of the spectrometry (MALDI-TOF and MS/MS), in order to define nine peptides that were generated from typsin-digestion (Table 2). the posttranslational modifications. We determined by mass The identified peptides were NNGHVISDFR, GVQAENFVFDIK, spectrometry that the native TRP47 and TRP32 were nearly and DSLLNEEDMAAQFGNR with molecular masses of 1158.2, identical to the predicted mass. Furthermore, we demonstrate that 1366.5, and 1809.9 Da, respectively, and consistent with the the highly acidic TRs present within the ehrlichial TRPs are predicted molecular masses indicating they were not posttranslation- responsible for the anomalous electrophoretic behavior of these ally modified. The remaining six peptides were not identified by MS/ proteins and not glycosylation. Moreover, we provide mass MS analysis of TRP47 tryptic digests. spectrometry and immunoprecipitation evidence that TRP47 is tyrosine phosphorylated. MALDI-MS and Tandem Mass Spectrometric Analysis of Asp-N Digested TRP47 Results To obtain more sequence coverage, TRP47 was digested with Analysis of E. chaffeensis Secreted Proteins by Single and Asp-N endopeptidase resulting in 23 peptide fragments. Twenty- one peptides with molecular masses were identified extending the Two-Dimensional Gel Electrophoresis (2-DE) and Western total TRP47 sequence coverage to 90%, and these peptides all had Immunoblotting masses that matched their predicted molecular masses (Table 3). Examination of the E. chaffeensis-secreted proteome by Western The acidic TRs of TRP47, which exhibited abnormal electropho- immunoblotting using dog anti–E. chaffeensis identified several major retic mobility and contained predicted sites of posttranslational immunoreactive proteins (Figure 1A). The highly acidic TRPs modifications (phosphorylation/glycosylation) were also identified. proteins, including TRP120 (pI 4.1), TRP47 (pI 4.2), and TRP32 Their molecular masses were consistent with their predicted (pI 4.1), which were distinctly separated and resolved during 2-DE, molecular masses demonstrating absence of
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