Research Paper Glycoproteome Remodeling In MLL

Theranostics 2021, Vol. 11, Issue 19R7B6 and R7B11) from different donors depleted ofCD34 stem cells. R7B11 was not further processed,whereas R7B6 was enriched in CD19 B cells, andR7B1 and R7B3 were enriched for both B- and T-cells[CD19 and TCRα/β]. To isolate normal CD19 CD10 precursor B-cells, around 2 109 of such viably frozennormal bone marrow cells from each sample wereapplied to EasySep Release Human CD19 PositiveSelection Kit (STEMCELL Technologies, Vancouver,BC, Canada, Cat#17754) and EasySep Human CD10Positive Selection Kit (STEMCELL Technologies,Cat#18358) columns.MLL samples in this study were BM0 [(11q23)relapse sample, 98% blasts], BM37 [(46XY, t(9;11)diagnosis, 98% blasts], and BM41 (60% blasts, MLLrearranged (11q23; t9;11) 46XY]. Ficoll-Plaque Plus(GE Healthcare, Cat#17-1440-02) centrifugation wasused to remove red blood cells, according tomanufacturer’s instructions, and viable cells werewashed before being frozen with DMSO. Viablyfrozen cells were thawed and washed twice withice-cold DPBS (500xg, 4 min, 4 C). Each sample,consisting of 4-6 106 total cells, was divided into twofractions,oneforproteomic/glycomic analyses and the other for RNA sequencing.Sample preparation for (glyco)proteinextractionFrozen cell pellets of normal bone marrow (n 4)and MLL-r (n 3) cells were processed according tothe same protocol: 2-3 million cells per sample werelysed in cell lysis buffer composed of Dulbecco’s PBS(Sigma, Cat#D8537) containing 1% Triton X-100(Sigma, Cat#T8787) and Protease Inhibitors (Sigma,Cat#P8340). Briefly, 400 µL of the cell lysis buffer wereadded to each sample, and tubes were kept on ice for20 min. Samples were then homogenized using a T-10ULTRA-TURRAX (IKA, Cat#0003737000) in 3 x 3sec cycles, followed by 30 sec on ice between cycles.Samples were kept on ice for 10 more minutes, afterwhich sonication was performed for 30 sec using awater-bath sonicator to shred DNA. Samples werecentrifuged for 15 min at 15,000xg, 4 C. Supernatantswere collected to new tubes and all resulting aliquotswere stored at -80 C prior use. After lysis, all sampleswere quantified in triplicates using a Pierce BCAProtein Assay Kit (Thermo Scientific, Cat#23225).Protein content extracted from similar number ofcells from samples R7B11 and BM41 was on averageapproximately 25 µg compared to 115 µg or more forthe other 5 samples (Supplementary Table 1). To ensurehigh-quality proteomics and glycomics preparations,these samples were excluded from the finalpreparations. The protein lysates of the other fivesamples were used as described in each individual9521method and as presented in Supplementary Table 1.RNA seq and RNA expression analysisRNA was extracted from frozen cell pellets usingTrizol (ThermO-Fisher 315596026) and furtherpurified using a RNeasy Mini kit (Qiagen #74104).Quality control analysis was done using BioanalyzerRNA Nano and D1000Chips. Nucleic acidconcentrations were determined by Qubit. The mRNAlibrary was prepared using an Illumina TruseqStranded mRNA High Throughput Prep kit andsamples were sequenced using an Illumina NextSeq500 Mid-Output Sequencing Reagent kit (v2, 150cycles), 132 M reads on an Illumina NextSeq 500instrument. RNA-seq results were aligned twice to thehuman genome. One analysis used the GRCh37annotation file (including around 50,000 genesincluding pseudogenes and non-coding RNA)whereas the second alignment, and of which resultswere used for proteomic comparisons presented here,made use of the genome assembly GRCh38.p13(GCA 000001405.28) genome annotation files (around19,862 mainly protein-encoding genes but notincluding IGHM). For the latter analysis, reads werequality-checked and processed using the nf-core/rnaseq analysis workflow v1.3 pipeline consisting ofNextflow v19.04.1, FastQC v0.11.8 Cutadapt v2.4,Trim Galore! v0.5.0, STAR vSTAR 2.6.1d, HISAT2v2.1.0, Picard MarkDuplicates v2.18.27, Samtools v1.9,featureCounts v1.6.4, StringTie v1.3.5, Preseq v2.0.3,deepTools, v3.2.0, RSeQC v3.0.0, MultiQCv1.7software. Raw count results were analyzed usingedgeR to determine significantly regulated genes(criteria: fold change 2; p 0.05; low expression filterrpkm 1.0). There were 3936 genes meeting thedefault criteria which were differentially expressed inthis comparison, with 1883 genes up-regulated and2053 genes down-regulated. The normalized RNAcounts were plotted using GraphPad Prism (v8.4.3).Diagnosis leukemia samples including 70 MLL-r casesand 74 normal bone marrow controls [17] from theMiLE study (GSE13159) were compared forexpression of GAG synthesis enzymes using onlinetools [18].N- and O-glycan release for glycomics analyses50 µg of (glyco)proteins were reduced by addinga volume of 500 mM of Dithiothreitol to each sample,to a final concentration of 20 mM, at 50 C for 1 hour.After cooling, samples were then subjected toalkylation using 40 mM Iodoacetamide in the dark atroom temperature. The reaction was quenched byadding another 20 mM of Dithiothreitol andincubating for 5 minutes.Proteins were precipitated using the Chloroform:http://www.thno.org

Theranostics 2021, Vol. 11, Issue 19Methanol:Water separation as described previously[19]. The resulting protein pellet was left to air dry for10 min. 5 µL of 8 M urea were added to each sample toresuspend the protein pellet using intensivevortexing. The final concentration of urea wasadjusted to 4 M by adding 5 µL of pure water(MQ-H2O).The urea dissolved proteins were dot blottedonto a PVDF membrane (Immobilon-P, 0.44 µm pore,Merck Millipore), and N- and O-glycans werereleased as described previously [20] (see alsoSupplementary Methods for details). Before massspectrometry analyses, N- and O-glycans were carboncleaned off-line using porous-graphitized carbon(PGC) material packed on top of C18 ZipTips to avoidany potential contaminants and then stored at -20 Cuntil their PGC-LC-ESI-MS/MS analyses.PGC-nanoLC-ESI-MS/MS glycomicsThe N- and O-glycome was determined usingthe PGC-nanoLC-ESI-MS/MS glycomics technologyas described previously [20-22] (see alsoSupplementary Methods for details). Previous studiesrelating to glycosylation and BCP-ALL focused on theidentification of particular glycoconjugates or glycantraits, such as the acetylation of sialic acid,9-O-acetyl-Neu5Ac [23, 24]. We note that thetechnologies used here do not allow to routinelyevaluate the level of O-acetylation of sialic acids, asthese labile modifications are lost due to the buffersused during the PGC-LC-ESI-MS/MS analyses. Inaddition, because of limited cell numbers, this studydid not examine the samples for GAGs. All details aredescribed following the respective MIRAGE(Minimum Information Required for A GlycomicsExperiment) guidelines in the supplementaryMaterial [25-28].Glycan structure determination and relativequantitationGlycan structures were determined aspreviously described [21, 22] (see also SupplementaryMethods for details). Unsupervised clustering analysisof the relative glycan abundances, and the respectiveheat map representation, were performed using thepackage pheatmap (v1.0.12) available in R studio(v1.3.1073). The relative intensities were also plottedusing GraphPad Prism (v8.4.3), and p-values werecalculated by performing an unpaired t-test. Symbolsof calculated significance (p 0.01, *) are representedwhen groups are significantly different. Allrepresented N- and O-glycan structures andmonosaccharides are depicted following the rules ofthe Symbol Nomenclature for Glycans (SNFG) [29,30].9522Cas9-CRISPR screen of glyco-enzymesKOPN8 (https://web.expasy.org/cellosaurus/CVCL 1866), an MLL-r B-cell precursor ALL cell line,was genotype-verified using STR genotyping. Cellswere stably transduced with a lentiviral vectorcontaining a blasticidin-selection marker andexpressing the Cas9 protein (AddGene #52962plasmid [31]). The sgRNA LV library was USEPR) vector [32]. Each neutral selection genecontrol [neg, LacZ, Luc and Ren] was covered by tensgRNAs each and 2 sgRNAs each were directedagainst ten essential gene controls [PCNA, POLR2D,POLR2A, PRL9, RPL2, CDK9 RPA3, RPS20, MYC,BRD4]. Cells transduced with LV expressing the lattersgRNAs would be expected to be depleted from thecell population. Target glycogenes were covered by 10sgRNAs each. The entire screen included 1082sgRNAs with 102 genes encoding glycan-remodelingenzymes, 4 neutral genes and 11 genes of whichablation is expected to reduce cell growth andviability. On d0 biological duplicates of around 20x106KOPN8/Cas9 cells in 1640 medium were transducedusing 10 µg/mL polybrene at a low MOI to ensurethat most cells would be transduced with one or nosgRNAs. After 24 h, puromycin selection at 4 µg/mLwas applied for 9 days, and 3 µg/mL puromycin wasused from d10-d32. On d20 of selection almost all cellscontained an ipUSEPR construct based on FACS forthe RFP marker. Cells were harvested for DNAisolation on d28. After d32 cells were also plated on anOP9 stromal feeder layer and grown for an additionalperiod under puromycin selection. Cells in the culturesupernatant were harvested on d48 and also used forDNA isolation to obtain two biological replicates.Each isolate from 1-5 106 cells contained sufficientDNA to yield an approximately 1000x coverage.DNAs were sequenced on an Illumina NextSeq 550.Results were analyzed using MAGeCK [Model-basedAnalysis of Genome-wide CRISPR-Cas9 Knockout[33] https://hpc.nih.gov/apps/MAGeCK.html. GiniIndex values for numbers of sgRNAs with 0 readcounts varied between 0.09 and 0.36. The mediannormalized read counts and the distribution of readcounts were comparable across samples.High-pH fractionation and C18-nanoLCESI-MS/MS analyses of peptides50 µg of protein (samples R7B1, R7B3, R7B6, BM0and BM37) and 10 µg (samples R7B11 and BM41)were reduced, alkylated and precipitated withChloroform-Methanol as described above. Theprotein pellet was air-dried for 10 minutes before 100µL of 25 mM of ammonium bicarbonate (Sigma,Cat#09830) were added to the pellets. Trypsin washttp://www.thno.org

Theranostics 2021, Vol. 11, Issue 19added at a ratio 1:25 (enzyme:protein ratio) andsamples were incubated for 18 h at 37 C. Afterdigestion, trypsin was heat-inactivated at 95 C for 10min, and samples were dried under vacuum. 1000 Uof PNGase F (2 µL) prepared in 50 µL of H218O (Sigma,Cat#329878) were added, and samples wereincubated at 37 C for 3 h to deglycosylate N-linkedglycopeptides before drying under vacuum.Peptides were resuspended in 300 µL of 0.1%trifluoroacetic acid (TFA) and fractionated using aPierce HighpHReversed-PhasePeptideFractionation Kit (Sigma, Cat#84868) following themanufacturer’s instructions. Briefly, the resuspendedpeptides were loaded to the pre-conditioned suppliedspin columns, and washed (3,000xg, 2 min) once usingwater. Increasing concentrations of acetonitrile (ACN)(5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, and 50%) in0.1% triethylamine (TEA) buffer were used to elute(3,000 g, 2 min) the bound peptides into eight distinctfractions. All the resulting fractions were dried undervacuum and kept at -20 C until analysis. Sampleswere resuspended in 0.1% TFA and peptide amountswere quantified using a Thermo Scientific NanoDrop One/OneC Microvolume UV-VisSpectrophotometer.The off-line fractionated peptides were identifiedusing an Orbitrap Fusion Tribrid MassSpectrometer coupled to an UltiMate 3000 UHPLCnanoLC (both Thermo Scientific ) (SupplementaryMethods for details on the columns and methodsused). Based on the NanoDrop quantitation, a volumecorresponding to 600 ng of peptides were injected ofeach fraction.Proteomics data analysesAll files were analyzed using the Andromedasearch engine integrated into the MaxQuant suit(v6.10.43) [34]. For high pH fractionated sampleanalyses using MaxQuant, the 8 fractions werecombined according to their respective sample. Astwo injections were made for each fraction, thisresulted in two combinations of 8 fractions, namelyinjection 1 and injection 2 for each sample. TheHCD-MS/MS spectra were searched against in silicotryptic digest of Homo sapiens proteins from theUniProt sequence database (v10; May 2020)containing 20,359 protein sequences (Swiss-Prot IDs).All MS/MS spectra were searched with the pre-setMaxQuant parameters, and the following modifications were used: cysteine carbamidomethylation wasset as a fixed modification; methionine oxidation,acetylation of protein N-terminus, and asparaginedeamidation and 18O deamidation were allowed asvariable modifications. False discovery rate (FDR) ofthe peptide spectral matches (PSMs), protein, and site9523were set to 1% based on Andromeda score. Matchbetween runs (MBR) algorithm was activated to allowmatching MS features between the different samplefractions and improve quantification [34].LFQ-Analyst was used for the label-freequantitation (LFQ) of the MaxQuant pre-processedproteomic datasets [35]. Two main “Conditions” weredefined as “MLL-r” and “Normal BM”, and eachinjection was used as an independent replicate. In theAdvanced Options setting, the “Adjusted p-valuecut-off” was defined to 0.01 (q-value, FDR 1%),whilst the “Log2 fold change cut-off” (log2FC) wasdefined to 2.The log2 and p-values calculated by LFQ-Analystwere used to generate a volcano plot representationusing the package EnhancedVolcano (v1.7.16) in Rstudio (v1.3.1073). GraphPad Prism (v8.4.3) was usedfor the RNA-protein integrative analyses, by plottingthe calculated magnitude (log2) differences derivedfrom our proteomics and RNA-seq analyses, targetingsolely the differentially expressed proteins.ResultsMLL-r patient cells undergo a distinct proteinO-glycome transformationThe initiating step of protein O-glycosylation istightly controlled by 20 distinct GALNTs enzymesthat post-translationally transfer a N-acetylgalactosamine (GalNAc) on folded glycoproteins [36].Of these, GALNT1, 2 and 3 are considered to be themost widely expressed and responsible forglycosylating the bulk of glycoprotein acceptorsubstrates [37]. Each GALNT has specific preferencesfor the protein sequence/structure motifs that it canglycosylate, and the activity of some GALNTs canalso depend on the previous action of other GALNTs.Because these enzymes are highly regulated in a cell-,tissue- and protein-specific manner [37, 38],O-glycosylation is a major regulator of cell function[16]. RNA-seq analyses identified ten GALNT genetranscripts of which six showed expression leveldifferences between MLL-r and control cells (Fig. 1A,Supplementary Table 2). On a protein level, expressionof GALNT1, GALNT2 and GALNT7 was confirmed,which also revealed significantly increased GALNT7in MLL-r cells (Fig. 1B and Supplementary Table 3).The activity of GALNTs is the rate-limiting firststep in O-glycan biosynthesis, but further O-glycanextension is regulated by the concerted andcompetitive action of various glycosyltransferases(GTs). The mRNA expression levels of GTsresponsible for extending the 3-position of the initialGalNAc residue, such as C1GALT1 (encoded byC1GALT1), were unchanged. In contrast, expressionhttp://www.thno.org

Theranostics 2021, Vol. 11, Issue 19levels of its essential molecular chaperone Cosmc(C1GALT1C1) [39, 40] doubled in MLL-r cells (Fig. 1C).mRNA expression was three-fold increased forGCNT1, the transferase responsible for initiating Core2 type O-glycan synthesis (Fig. 1C), whereasexpression of ST3GAL1, the sialyltransferase knownto add a sialic acid on the core 1 galactose, remainedunchanged (Fig. 1C). In addition, expression ofST6GALNAC1, the sialyltransferase competing withGCNT1 and thus preventing Core 2 typeO-glycosylation, was very low in MLL-r cells (Fig. 1C).These transcriptomics data suggest a majorremodeling of the MLL-r O-glycome.9524O-glycomics by LC MS/MS confirmed thatMLL-r BCP-ALL cells exhibited a significantly alteredO-glycome, shifting towards Core 2-type O-glycans,while Core 1-types were the major forms in normalcontrol BCP cells. Overall, we identified 21 distinctO-glycans, including five Core 1, thirteen Core 2 andtwo O-fucose type glycans next to a sialylated hexosedisaccharide (Fig. 2A and 2B; Supplementary Table 4). InMLL-r cells, 51% of all glycans were Core 2 O-glycanscompared to 26% in normal BCP cells. The level ofCore 1 type O-glycans was almost halved in MLL-r(37% versus 64%, Fig. 2B and C).Figure 1. MLL-r cells have altered expression of key enzymes involved in O-glycan synthesis. RNA-seq (A, C) or proteomic (B) results for the indicated enzymes.Symbols in panel (B) each represent one sample. Panel (C) also shows a schematic structure of two glycans and the enzymes responsible for their synthesis. *p 0.05; **p 0.01;***p,0.001; ****p 0.0001. Results are represented as median with 95% CI.http://www.thno.org

Theranostics 2021, Vol. 11, Issue 199525Figure 2. The O-glycome differs between MLL-r and normal BCP cells. (A) Heat map showing the z-score of the detected relative abundance of the O-glycanstructures indicated with a number to the right (see Supplementary Table 4 for corresponding structures). Sample identifiers are indicated below the image. Numbers refer totechnical replicates. Glycans are categorized as indicated in the key to the right. (B) Relative intensity (as percentage of the total) of the major categories of O-glycans found inMLL-r and normal BCP cells. (C) Representation of the five most abundant and important sialylated O-glycan structures. Monosaccharide symbols are represented according toSNFG guidelines [29, 30]. ****p 0.0001.More than 1300 human proteins are reported tobe O-glycosylated [41-43]. We found that mRNAs foraround 70% of these were expressed inMLL-r/control cells, with 40% exhibiting significantlydifferent expression in MLL-r samples (159 higher,197 lower, Supplementary Table 2) compared to normalprecursor B cell controls. Proteomics confirmed thepresence of 241 previously reported O-glycoproteins,of which 33 were differentially expressed (with 9upregulated in MLL-r cells; Supplementary Table 3).Together, these glycomic, transcriptomic andproteomic data provide clear evidence for theextensive remodelling in the O-glycoprotein andO-glycan components of the MLL-r cell glycocalyx.Sialylation and Lewis X fucosylation areincreased on N-glycans of MLL-r cellsProtein N-glycosylation is critical for correctprotein folding, cell-cell recognition and cellularinteractions [44]. In both MLL-r and control cells,oligomannose and complex type N-glycans were themain forms of N-glycosylation (Fig. 3A). MLL-r cellsshowed increased complex type N-glycan levelscompared to controls (52.4% versus 42.6%), while thelevels of paucimannose (10.9% and 11.9%) and hybridtype ( 5%) N-glycans were similar. The complex typeN-glycans were mainly biantennary. Only 5% of allN-glycans showed features consistent with tri- ortetra-antennary structures, but these could not behttp://www.thno.org

Theranostics 2021, Vol. 11, Issue 19further characterized as a consequence of their lowabundance levels.The overall N-glycan sialylation levels wereincreased in MLL-r cells, with N-glycan structurescarrying α2-6, both α2-3 and α2-6, or exclusively α2-3linked sialic acids (Fig. 3B). These glycomic findingscorrelated well with the increased ST3GAL3 mRNAlevels (Fig. 3C), which is likely to be the cause of theobserved increased sialylation of tri- and tetraantennary N-glycans in MLL-r cells. For ST6GAL1,however, a correlation between the decreased mRNA9526and protein levels in MLL-r cells and a decrease inα2-6 sialylation could not be found, as the overall α2-6sialylation levels remained unchanged (Fig. 3B-D).The functional behavior of glycoproteins andcells is also well-known to be influenced byfucosylation [45, 46]. The human genome contains 13functionally-distinct fucosyltransferase genes (FUTs),of which 5 were detected at the transcript level (Fig.3E). Our glycomics analyses showed that corefucosylation, a product of FUT8 activity, was themajor fucose modification present on about one-thirdFigure 3. Differential sialylation and fucosylation of N-glycans in MLL-r B-ALL samples. (A) Relative intensities of total N-glycans indicated in the graph, as detailedin Supplementary Table 5. (B) Relative intensity of different sialic acid linkages on N-glycans. MLL-r cells contained higher levels of tri-antennary, fully sialylated N-glycans(Supplementary Table 5, structures 43a and b) while incompletely sialylated, tri-antennary and also tetra-antennary N-glycans (41b and 42a, respectively) were the most abundantforms of these branched N-glycans found on the controls. (C) Normalized mRNA expression of the indicated sialyltransferases, and their re

The mRNA library was prepared using an Illumina Truseq Stranded mRNA High Throughput Prep kit and samples were sequenced using an Illumina NextSeq 500 Mid-Output Sequencing Reagent kit (v2, 150 cycles), 132 M reads on an Illumina NextSeq 5