PMCID: PMC6399673

 

    Legend: Gene, Sites, Suger

Section : Workflow ofthe MGL Pull-Down Assay

Content :

1. To identify novelbinding partners of MGL on Jurkat T-cells, we developed a protocolwhere we used Fc-coupled MGL as a bait in pull-down assays (Figure A)
2. MGL-Fc is a chimericmolecule formed by the extracellular domains of MGL fused to the human immunoglobulin G1 Fc tail , allowing bindingto magnetic Protein G beads
3. Because the binding to the CRD of MGLis calcium-dependent, captured proteins were eluted using EDTA andsubsequently analyzed by SDS-PAGE and processed for mass-spectrometry-basedprotein identification
4. CD43 and CD45 are hitherto the only describedMGL-binding proteins on Jurkat cells
5. Therefore, to test the effectiveness of our workflow, we first determinedthe capturing of CD43 by Western blot
6. This clearly showed that CD43was captured by MGL-Fc and could be eluted with EDTA (Figure B)
7. Likewise, the additionof EDTA during the pull-down assay prevented the binding of CD43 to MGL (Figure B)

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Section : MS-Based Identification of MGL-Binding Proteins from JurkatCells

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1. Next, we performed three biologically independent pull-downexperiments with MGL-Fc and analyzed the bound proteins by LC–MS/MSfollowing trypsin digestion
2. As a negative control, pull-down assaysin the presence of EDTA were performed
3. Altogether, these experimentsresulted in the identification of 775 proteins (data not shown), ofwhich 540 were identified in at least two experiments
4. To filter for proteins specifically binding to MGL , we selected proteins that werenot observed in the negative controls and at least two times in thepull-downs with MGL or proteins that were found in all three pull-downswith MGL and at most once in a negative control
5. This resulted ina list of 85 proteins (Table S1)
6. The candidateMGL-binding proteins included intracellular, plasma membrane, andpredicted secreted proteins
7. Because we used total cell lysates forthe pull-down assays, some of these proteins may be physiologicallyless relevant
8. To filter for cell-surface proteins , which may be expectedto be in direct contact with MGL , we next selected only those proteinsthat were annotated as cell-surface proteins in the UniProtKB database ,resulting in a final list of 17 MGL-binding cell-surface proteins ( Table )
9. Importantly,these proteins were not found in the negative control samples (seealso Table S1), making them strong candidateMGL-binding proteins
10. As expected, CD45 ( PTPRC ) and CD43 ( SPN ) werethe two main MGL-binding proteins identified based on the total numberof peptide-spectrum matches (summed for the three biological replicates)for these two proteins , respectively

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Section : Characterizationof Glycopeptides

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1. MGL is known to bindterminal residues of N-acetylgalactosamine
2. To validatethe specificity of the interaction of the proteins listed in Table with MGL , we nextevaluated our data for the presence of glycopeptides carrying thismotif
3. During our LC–MS/MS analyses of the tryptic digests,we used a method that triggered additional MS/MS acquisitions oncethe MS/MS spectrum showed the presence of the characteristic HexNAcoxonium ion at m/z 204.087 ([C8H14NO5]+)
4. First of all ,for these additional MS/MS events, we used a higher number of ionsfor fragmentation , resulting in a higher signal-to-noise for the fragmentions
5. Moreover, to improve the confidence of the glycopeptide identifications,we collected Orbitrap spectra using different HCD collision energiesin addition to an ion trap CID spectrum
6. Next, we searched forglycopeptides carrying the Tn antigen following database searchesusing MaxQuant and Byonic
7. With these approaches, we could confirmthe presence of the Tn antigen on CD45 and CD43 (Table ) with peptides containing amaximum of four HexNAcs (Table )
8. However, it is known that certain regions in these twoproteins contain a high density of O-glycans, which may have beenmissed by the automatic data analysis due to limitations of the particularsoftware used, for example, due to the high number of occupied glycosylationsites in one individual peptide
9. Indeed, upon manual inspection ofour data, we observed peptides containing up to 11 HexNAcs (Figure )
10. Of note, one peptidewith an extended O-glycan on the CD45 tryptic peptide LNPTPGSNAISDVPGER(HexNAc2Hex1NeuAc1) was found using Byonic, which, considering the Cosmc mutation in Jurkat cells, could correspond to a core 6 O-glycan,a specific MGL-binder (GlcNAcβ1-6GalNAcαSer/Thr)
11. In addition to CD43 and CD45 , our database searches confirmedthepresence of the Tn antigen on another 11 proteins (Table )
12. We confirmed the Tn antigenon all of these peptides by manual interpretation of the data
13. Asan example, two glycopeptides from EVI2B and TREML2 , respectively,are shown in Figure
14. Between the peptides presented in Table , one peptide (GLFIPFSVSSVTHK)with three HexNAcs was identified from P-selectin glycoprotein ligand1 ( SELPLG ), which was not identified as a specific binder in the proteomicsdata (Table )
15. Inspectionof the data showed that, unexpectedly, it was filtered out due tothe fact that normal tryptic peptides of SELPLG were observed in onlyone of the three MGL pull-downs
16. Terminal N-acetylgalactosamines can alsobe partof N-glycans, for example, as part of the LacdiNAcepitope
17. Hence we also searched our data for the presence of N-glycopeptides using Byonic
18. Only for CD45 presented in Table we could identify N-glycopeptides (on N234 , 278, 337, 380, 421, and 470, respectively),but none of these appear to contain a terminal N-acetylgalactosamine ,as judged on the basis of the glycan com positions as well as the absenceof the LacdiNAc (GalNAcβ1-4GlcNAcβ1) marker ion at m/z 407.166 (Table S2)

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• 18. MGL, -, N234

Section : Schematic representationand validation of the experimental workflowfor the identification of MGL ligands in Jurkat cells

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1. (A) Pull-downworkflow to capture MGL-binding proteins
2. As negative control, EDTAwas added to the sample prior to incubation with MGL-Fc to preventthe binding to MGL
3. (B) Western blot analysis using monoclonal anti-CD43staining of the unbound fraction and captured proteins (elution) fromthe MGL-Fc pull-down assay in the absence (−) and presence (+,negative control) of EDTA (100 mM)
4. Fc: Fragment crystallizable region ; Prot G: protein G; TCL : total cell lysate

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Section : Highly O-glycosylated peptides of CD43 and CD45

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1. MS/MS spectraof glycopeptides from CD43 carrying 10 and 11 GalNAcs and CD45 carrying11 GalNAcs
2. Left panels: HCD MS/MS spectra
3. b and y ions representfragments without the GalNAc , unless indicated otherwise
4. The insetshows the MS spectrum of the corresponding precursor with charge stateand m/z value
5. Right panels: CIDMS/MS spectra
6. Yellow square: GalNAc

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Section : Tn-bearing O-glycosylated peptides ofEVI2B and TREML2

Content :

1. (A) Manuallyassigned HCD MS/MS spectrum of the tryptic peptide QLPSAR from EVI2Bcarrying one GalNAc
2. The inset shows the MS spectrum of the precursorion at m/z 437.735 [M + 2H]2+
3. (B) Manually assigned HCD MS/MS spectrum of the trypticpeptide LAQEAASKLTR from TREML2 carrying three GalNAcs
4. The inset shows the MS spectrum of the precursor ion at m/z 531.950 [M + 3H]3+
5. Yellow square:GalNAc
6. b and y ions represent fragments without the GalNAc , unlessotherwise indicated

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Section : 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

Content :

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

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Protein	NCBI ID	SENTENCE INDEX