PMID: PMC6399673-2

 

    Legend: Gene, Sites

Title : We developed a workflow for the identificationof MGL ligands,which resulted in the identification of 17 cell-surface proteins ofJurkat cells that bind to MGL

Abstract :
  1. For most of these proteins , we confirmedthe specificity of the interaction with MGL through the identificationof O-glycopeptides carrying the Tn antigen
  2. The glycopeptide ,mediating the interaction with MGL , could notbe identified for all MGL ligands
  3. These peptides may have been misseddue to insufficient sensitivity or low MS/MS spectrum quality, whichwould hamper identification by both search algorithms as well as manualinspection
  4. Although in the past years, we and others have made considerableprogress in the mass-spectrometry-based identification of glycopeptides , several issues related to their fragmentation behavior still oftenimpede straightforward (data) analysis, even though the Tn antigenrepresents a relatively simple post-translational modification
  5. Wecombined multiple strategies for data acquisition and analysis tomaximize our glycopeptide identification
  6. Other methods, such as electron-transferdissociation (ETD), have also been valuable tools for the identificationof glycopeptides , especially in caseswhere the localization of the GalNAc(s) was ambiguous
  7. However, forthis study, we were primarily interested in identifying MGL ligands,and not necessarily the site-specific glycan assignment
  8. Moreover,for several peptides , we had full occupancy of all serine and threonineresidues present in the identified glycopeptide
  9. From the 17potential MGL-binding proteins (Table ), some could have been obtained throughan interaction with a genuine MGL ligand
  10. For example, protein tyrosinephosphatase receptor type C-associated protein ( PTPRCAP ), for whichwe could not find the corresponding glycopeptide , was captured duringthe MGL pull-down probably because it is a binding partner of CD45
  11. Similarly, for only one of the four LRRC8 subunits ( LRRC8D ) that we identified could a specific O-glycopeptide be found
  12. Because it is known that the LRRC8 subunits form heterodimers, it is conceivable that the whole LRRC8 complex was captured
  13. As expected, CD43 and CD45 were among the top proteins carryingthe Tn antigen in Jurkat cells
  14. A recent study presented a workflowwhere a combination of enzymatic and chemical methods was used toselectively tag terminal GalNAc, and not GlcNAc residues , allowing the enrichment of the correspondingglycopeptides
  15. To study proteins carrying the Tn antigen, this methodwas applied to total cell lysates of Jurkat cells
  16. As a result, atotal of 97 proteins harboring the Tn antigen were identified, ofwhich 27 were consistently found in all three independent biologicalreplicates
  17. From the total of 97 proteins , 11 were transmembrane signalingmolecules, including CD45 , Semaphorin-4D , and TNFRSF8 , which werealso identified in our study
  18. Some other Tn-antigen-bearing glycoproteinswere not found in our experiments
  19. The observed differences may relateto the different experimental approaches because several Tn-bearingglycoproteins were clearly identified in our experiments, such as, CD43 , EVI2B , and TREML2 , but not in the above-mentioned study
  20. On the contrary, it could be due to the factthat for MGL binding the presence of Tn is necessary but not sufficient
  21. For example, all CD45 isoforms (CD45ABC/AB/BC/B) except for one(CD45RO) bind MGL
  22. Unlike the others, which are highly glycosylated,CD45RO has only two O-linked glycan epitopes
  23. Although it isknown that MGL can have immune-modulatory activities,the specific proteins involved in the cellular response elicited by MGL are largely unknown
  24. Tolerogenic antigen presenting cells ( APCs )express high levels of MGL on the cell surface, and the binding of MGL to CD45 suppresses TCR-mediated T-cell activation
  25. This resultsin an anti-inflammatory response characterized by the reduced productionof pro-inflammatory cytokines and lower proliferation of T-cells andinduction of cell death in Jurkat
  26. For the new MGL ligands identified in this study, the outcome ofthe interaction with MGL remains to be determined
  27. Semaphorin 4D ,also known as CD100 , is a homodimeric transmembrane protein of 150kDa that is highly expressed in secondary lymphoid organs and constitutivelyon naïve T-cells
  28. It binds its ligand CD72 , a C-typelectin expressed on the surface of APCs , such as B cells and DCs, but also macrophages and some subpopulationof T-cells
  29. Our data show that, at leastin tumor cells with aberrant glycosylation, Semaphorin 4D also bindsto MGL
  30. For several of the newly identified MGL ligands, literatureprovideslimited information about their function
  31. For example, although KIAA0319Lhas been demonstrated to be an essential receptor for adeno-associatedvirus infection, the cellular function, especially in immune responses,is unknown
  32. However, our data support previous findings that suggestedhigh levels of mucin-type O-glycosylation on KIAA0319L
  33. Our data also provide evidence of O-glycosylationon proteins that hitherto were unknown to be O-glycosylated, suchas EVI2B and TREML2
  34. Although the cellular function of these proteinsis largely unknown, it has been demonstrated that EVI2B is the targetfor the transcription factor CCAAT/enhancer-binding protein alpha(C/EBPα ), and TREML2 binding byCD276 leads to enhanced T-cell responses
  35. Altogether, our data warrant further exploration of the functionalimplications of the interaction of MGL with the newly identified ligands
  36. Notwithstanding the importance of this, some responses elicited by MGL might also be the result of the interaction of MGL with glycolipids, which was not the topic of our study
  37. The high level of Tn antigen is not restricted to leukemias butis frequently observed in other tumors as well
  38. The best characterizedligand carrying Tn or sialyl-Tn (sTn , Neu5Acα2,6-GalNAc-O-Ser/Thr)in epithelial cells is the glycoprotein MUC1
  39. MUC1-derived glycopeptidesbind to MGL on DCs and induce the activation of the extracellularsignal-regulated kinases 1 and 2 (ERK1 ,2) and the nuclear factor-κB (NF-κB) pathways
  40. In another study,it was demonstrated that these pathways are crucial for IL-10 production,which regulates the DC maturation phenotype
  41. The higher level of Tn in other cancers is not necessarilydueto Cosmc mutations, as in the Jurkat T-cell usedin our study, but may also be the result of other genetic alterations
  42. For example, in colorectal cancer, higher levels of several GALNTshave recently been linked to the BRAFV600E mutation
  43. A positive correlation between this oncogenicmutation and MGL ligands on tumor cells was previously identified
  44. Hence, it will be interesting to study the proteinscarrying MGL ligands in these tumor cells as well
  45. In conclusion,here we provide an optimized method to capture MGL-binding proteins followed by glycoproteomic analysis
  46. The application of thisprocedure on the Jurkat cell line provides important novel insightsinto previously unknown MGL ligands
  47. However, further investigationsshould evaluate the functional immune responses triggered by MGL-specificrecognition of those proteins , as has been previously reported forthe already known interaction partner CD45 by van Vliet et al
  48. This will provide a deeper understanding ofthe MGL involvement in cancer progression and the glycan-specificimmune responses mediated by this lectin
Output (sent_index, trigger, protein, sugar, site):
  • 1. O-glycopeptides, , -, -, O-glycopeptides
  • 10. glycopeptide, , -, -, glycopeptide
  • 11. O-glycopeptide, , -, -, O-glycopeptide
  • 14. correspondingglycopeptides, , -, -, correspondingglycopeptides
  • 19. Tn-bearingglycoproteins, , Tn-bearingglycoproteins, -, -
  • 2. glycopeptide, , -, -, glycopeptide
  • 32. O-glycosylation, , KIAA0319L, -, -
  • 33. O-glycosylated, , proteins, -, -
  • 38. glycoprotein, , glycoprotein, -, -
  • 4. glycopeptides, , -, -, glycopeptides
  • 5. glycopeptide, , -, -, glycopeptide
  • 6. glycopeptides, , -, -, glycopeptides
  • 8. glycopeptide, , -, -, glycopeptide
  • 8. occupancy, , -, -, serine and threonineresidues
Output(Part-Of) (sent_index, protein, site):
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX