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

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 FindingsIn 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/SignificanceTRP47 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.

Tyrosine-Phosphorylated Ehrlichia chaffeensis and Ehrlichia canis Tandem Repeat Orthologs Contain a Major Continuous Cross-Reactive Antibody Epitope in Lysine-Rich Repeats

“…A tyrosine-rich C-terminal domain conserved between TRP75 and TRP95 had three predicted tyrosine phosphorylation sites, suggesting that this region is modified by phosphate. This finding is consistent with our previous reports demonstrating that TRP47 is also tyrosine phosphorylated (28). The TRP47 interacts with the host cell tyrosine kinase Fyn, and it appears that TRP75 may also be secreted to a location where it interacts with host cell tyrosine kinase(s).…”

“…Consistent with other TRPs, the native TRP orthologs in this report migrated electrophoretically with larger than the predicted masses. Furthermore, the recombinant TR and C-terminal regions also migrated electrophoretically with larger than predicted masses, We have previously reported the relationship between acidic TRPs and abnormal migration (28) and demonstrated that these charged regions are associated with larger electrophoretic masses, a characteristic associated with incomplete SDS binding (6,28). We observed abnormal migration of recombinant protein fragments representing the TR region (pI 10) and the acidic (pI 4) C-terminal region, indicating that each of these regions contributes to the abnormal electrophoretic mobility.…”

“…We observed that the native and recombinant TRPs migrate larger than predicted by SDS-gel electrophoresis. We have previously determined that the larger than predicted masses associated with the acidic TR regions of ehrlichial TRPs are associated with the acidic nature of the proteins and the lack of SDS binding (28). We performed MALDI-TOF on the E. chaffeensis and E. canis TRP recombinant fragments that exhibited abnormal electrophoretic migration.…”

“…Mass spectrometry was performed using a matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometer (Voyager-DE STR; Applied Biosystems) at the University of Texas Medical Branch Mass Spectrometry Core Laboratory as previously described (28).…”

“…Lysates were collected by centrifugation at 12,000 ϫ g for 10 min at 4°C. Tyrosine phosphorylation on the native TRPs was detected by Western immunoblot using anti-phosphotyrosine antibody (PY99; Santa Cruz Biotechnology, Santa Cruz, CA) as previously described (28). Immunoprecipitations were performed as previously described (28) with the following modifications.…”