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 :
- For most of these proteins , we confirmedthe specificity of the interaction with MGL through the identificationof O-glycopeptides carrying the Tn antigen
- The glycopeptide ,mediating the interaction with MGL , could notbe identified for all MGL ligands
- 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
- 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
- Wecombined multiple strategies for data acquisition and analysis tomaximize our glycopeptide identification
- 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
- However, forthis study, we were primarily interested in identifying MGL ligands,and not necessarily the site-specific glycan assignment
- Moreover,for several peptides , we had full occupancy of all serine and threonineresidues present in the identified glycopeptide
- From the 17potential MGL-binding proteins (Table ), some could have been obtained throughan interaction with a genuine MGL ligand
- 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
- Similarly, for only one of the four LRRC8 subunits ( LRRC8D ) that we identified could a specific O-glycopeptide be found
- Because it is known that the LRRC8 subunits form heterodimers, it is conceivable that the whole LRRC8 complex was captured
- As expected, CD43 and CD45 were among the top proteins carryingthe Tn antigen in Jurkat cells
- 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
- To study proteins carrying the Tn antigen, this methodwas applied to total cell lysates of Jurkat cells
- As a result, atotal of 97 proteins harboring the Tn antigen were identified, ofwhich 27 were consistently found in all three independent biologicalreplicates
- From the total of 97 proteins , 11 were transmembrane signalingmolecules, including CD45 , Semaphorin-4D , and TNFRSF8 , which werealso identified in our study
- Some other Tn-antigen-bearing glycoproteinswere not found in our experiments
- 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
- On the contrary, it could be due to the factthat for MGL binding the presence of Tn is necessary but not sufficient
- For example, all CD45 isoforms (CD45ABC/AB/BC/B) except for one(CD45RO) bind MGL
- Unlike the others, which are highly glycosylated,CD45RO has only two O-linked glycan epitopes
- Although it isknown that MGL can have immune-modulatory activities,the specific proteins involved in the cellular response elicited by MGL are largely unknown
- 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
- 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
- For the new MGL ligands identified in this study, the outcome ofthe interaction with MGL remains to be determined
- 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
- 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
- Our data show that, at leastin tumor cells with aberrant glycosylation, Semaphorin 4D also bindsto MGL
- For several of the newly identified MGL ligands, literatureprovideslimited information about their function
- For example, although KIAA0319Lhas been demonstrated to be an essential receptor for adeno-associatedvirus infection, the cellular function, especially in immune responses,is unknown
- However, our data support previous findings that suggestedhigh levels of mucin-type O-glycosylation on KIAA0319L
- Our data also provide evidence of O-glycosylationon proteins that hitherto were unknown to be O-glycosylated, suchas EVI2B and TREML2
- 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
- Altogether, our data warrant further exploration of the functionalimplications of the interaction of MGL with the newly identified ligands
- 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
- The high level of Tn antigen is not restricted to leukemias butis frequently observed in other tumors as well
- The best characterizedligand carrying Tn or sialyl-Tn (sTn , Neu5Acα2,6-GalNAc-O-Ser/Thr)in epithelial cells is the glycoprotein MUC1
- 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
- In another study,it was demonstrated that these pathways are crucial for IL-10 production,which regulates the DC maturation phenotype
- 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
- For example, in colorectal cancer, higher levels of several GALNTshave recently been linked to the BRAFV600E mutation
- A positive correlation between this oncogenicmutation and MGL ligands on tumor cells was previously identified
- Hence, it will be interesting to study the proteinscarrying MGL ligands in these tumor cells as well
- In conclusion,here we provide an optimized method to capture MGL-binding proteins followed by glycoproteomic analysis
- The application of thisprocedure on the Jurkat cell line provides important novel insightsinto previously unknown MGL ligands
- 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
- 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):