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Mycobacterium tuberculosis Infection Manipulates the Glycosylation Machinery and the N-Glycoproteome of Human Macrophages and their Microparticles Nathan J. Hare, Ling Y. Lee, Ian Loke, Warwick J. Britton, Bernadette M. Saunders, and Morten Thaysen-Andersen J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00685 • Publication Date (Web): 19 Oct 2016 Downloaded from http://pubs.acs.org on October 23, 2016

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Journal of Proteome Research

Mycobacterium tuberculosis Infection Manipulates the Glycosylation Machinery and the N-Glycoproteome of Human Macrophages and their Microparticles Nathan J. Hare1,^, Ling Y. Lee2,^, Ian Loke2, Warwick J. Britton1, Bernadette M. Saunders1,3,‡, and Morten Thaysen-Andersen2,‡,* 1

Tuberculosis Research Program, Centenary Institute, Discipline of Medicine, Infectious Diseases

and Immunology, Sydney Medical School, The University of Sydney, Newtown, NSW, Australia 2

Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW 2109,

Australia 3

School of Life Science, University of Technology Sydney, NSW 2007, Australia

^,‡

These authors contributed equally

Running title: M. tuberculosis infection manipulates protein N-glycosylation of human macrophages

Keywords: Mycobacterium tuberculosis, tuberculosis, microparticle, macrophage, N-glycosylation, LC-MS/MS, exoglycosidase, glycoproteome, proteomics, glycoprofiling, glycomics

*Corresponding author: Dr. Morten Thaysen-Andersen Department of Chemistry and Biomolecular Sciences Macquarie University Sydney, NSW 2109 Australia Phone / Fax: (+61) 2 9850 7487 / (+61) 2 9850 6192 E-mail: [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT Tuberculosis (TB) remains a prevalent and lethal infectious disease. The glycobiology associated with Mycobacterium tuberculosis infection of frontline alveolar macrophages is still unresolved. Herein, we investigated the regulation of protein N-glycosylation in human macrophages and their secreted microparticles (MPs) used for intercellular communication upon M. tb infection. LC-MS/MS-based proteomics and glycomics were performed to monitor the regulation of glycosylation enzymes and receptors and the N-glycome in in vitro-differentiated macrophages and in isolated MPs upon M. tb infection. Infection promoted a dramatic regulation of the macrophage proteome. Most notably, significant infection-dependent down-regulation (4-26 fold) of 11 lysosomal exoglycosidases e.g. βgalactosidase, β-hexosaminidases and α-/β-mannosidases was observed. Relative weak infectiondriven transcriptional regulation of these exoglycosidases and a stronger augmentation of the extracellular hexosaminidase activity demonstrated that the lysosome-centric changes may originate predominantly from infection-induced secretion of the lysosomal content. The macrophages showed heterogeneous N-glycan profiles and displayed significant up-regulation of complex-type glycosylation

and

concomitant

down-regulation

of

paucimannosylation

upon

infection.

Complementary intact N-glycopeptide analysis supported a subcellular-specific manipulation of the glycosylation machinery and altered glycosylation patterns of lysosomal N-glycoproteins within infected macrophages. Interestingly, the corresponding macrophage-derived MPs displayed unique Nglycome and proteome signatures supporting a preferential packaging from plasma membranes. The MPs were devoid of infection-dependent N-glycosylation signatures, but interestingly displayed increased levels of the glyco-initiating oligosaccharyltransferase complex and associated αglucosidases that correlated with increased formation, N-glycan precursor levels and N-glycan density of infected MPs. In conclusion, this system-wide study provides new insight into the host- and pathogen-driven N-glycoproteome manipulation of macrophages in TB.


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INTRODUCTION With more than a million annual deaths and over nine million new infections each year, tuberculosis (TB), which is caused by the pathogen Mycobacterium tuberculosis (M. tb), remains one of the most serious threats to human health (1). Major challenges associated with controlling this prevalent infectious disease include the low efficacy of the available TB vaccine, the rapid spread of drugresistant sub-types of M. tb and the paucity of new drugs to treat TB (2). Through thousands of years of co-evolution, M. tb has adapted to, and learnt to manipulate and survive within, host alveolar macrophages that perform constant surveillance of the respiratory tract and which paradoxically form the natural habitat of the pathogen in humans (3, 4). It is now understood that the bacilli, at least in part, gain entry into the resident alveolar macrophages through mannose-based recognition mechanisms that are receiving increasing attention in the context of host-pathogen interaction and host immune communication (5). The heavily mannosylated surface of M. tb that comprises a range of lipomannans, manno-proteins and the mannose-capped lipoarabinomannans (6-9) is recognized by various macrophage surface C-type lectins including members of the mannose receptor (MR) family (10) and dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) (11). These glycoepitope:receptor interactions facilitate an effective route-of-entry into the host macrophages. Glycosylation of specific lectin receptors has even been shown to be important for macrophage uptake (12). Other established survival strategies displayed by M. tb including the inhibition of the lysosome-phagosome fusion and strain-specific virulence as well as a functional macrophage response have also been shown to involve advanced carbohydrate mechanisms (9, 1315). These features contribute to the increased M. tb survival, replication rate and an accelerated macrophage cell death through necrosis that ultimately allow the bacterium to successfully infect and transmit between fully immune-competent individuals (16). However, the extremely complex nature of TB pathogenesis that involves key elements of both the innate and adaptive immune system has still not been mapped to sufficient resolution at the cellular and molecular level to enable the rational design of efficacious therapeutics that may prevent, or cure, the disease. In addition, reliable and



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cheap biomarkers for the accurate and sensitive detection of latent and active forms of TB are urgently required to minimize bacilli spread and allow early treatment of infected individuals. Understanding the cell-cell communication of the immune system in general, and in macrophages in particular, is of high importance to dissect the infection strategies and host response mechanisms associated with TB. We have previously shown that microparticles (MPs), which are a type of plasma membrane-derived irregular-shaped extracellular vesicles (EVs) ranging from 100-1,000 nm used for inter-cell communication under normal and altered physiology (17), play an important role in macrophage biology in the context of TB pathogenesis (18). Specifically, we showed using in vitro and in vivo experiments that macrophages infected with M. tb secrete MPs that have the capacity to stimulate and activate naïve macrophages with higher potency than MPs derived from non-infected macrophages. Very recently, we investigated the proteome of MPs derived from M. tb infected and uninfected macrophages and identified an infection-specific proinflammatory type-1 interferon inducible protein signature (19). The MPs from infected macrophages expressed increased ubiquitinlike protein ISG15 and interferon-induced protein with tetratricopeptide repeats 1-3 (IFIT1-3), which may explain their proinflammatory nature. Several important questions stemmed from this recent study, including whether the cellular (glyco)proteome is manipulated within infected macrophages and, if so, whether this regulation reflects the infection-dependent protein alterations observed in the MPs. In addition, since TB pathogenesis is strongly associated with mechanisms involving protein glycosylation we speculated whether the glycosylation machinery is altered in infected macrophages and whether any such changes are responsible for generating altered glycosylation signatures in infected macrophages and in their secreted MPs. To address these glycobiology-related research questions that are critical to advance our understanding of the pathogenesis of TB, we set out to map the N-glycoproteome of human macrophages and their secreted MPs upon M. tb infection and to study the underlying mechanisms associated with the manipulations driven by the enzymatic glycosylation machinery. This study provides system-based insight into the host- and/or pathogen-driven glycoproteome changes that seemingly appear relatively quickly in macrophages during TB infection.


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EXPERIMENTAL SECTION 2.1 Host cell culture, bacterial culture and bacilli infection Human myelo-monocytic THP-1 cells (TIB-202, ATCC) were cultured in RPMI (Life Technologies) with 10% fetal calf serum and differentiated by the addition of 100 nM phorbol 12-myristate 13acetate (PMA) (Sigma-Aldrich) to the culture medium (CM) for 48 h at 37°C. M. tb H37Rv was cultured at 37°C in supplemented Middlebrook 7H9 broth (Sigma-Aldrich) to mid-logarithmic phase (18). Bacterial counts were determined by culturing samples for three weeks on Middlebrook 7H11supplemented agar (18). The differentiated THP-1 (macrophage-like) cells were cultured to a level of 2 × 107 cells per flask for the N-glycome and proteome analyses of the isolated microparticles (MPs) and the cell lysate (CL) fractions. Macrophage cultures used for qPCR were grown to 5 × 105 cells per well. Macrophages were infected (I) in biological triplicates with M. tb H37Rv (multiplicity of infection ratio, 1:1) for 4 h or left uninfected (UI). Cells were then washed twice to remove extracellular bacteria and cultured for a further 24 h, 48 h and 72 h for qPCR experiments using the CLs and for hexosaminidase activity experiments using the collected CM, or cultured for the entire 72 h before isolation of the secreted MPs and the paired CLs that were used for the proteomics and glycomics experiments, see Fig. 1 for experimental design and nomenclature.

2.2 MP isolation Isolation of the macrophage-derived MPs were performed and size distribution measured as previously described (18). In short, culture supernatants were collected and centrifuged at 524 × g for 10 min at 4°C to remove cellular debris. Supernatants were collected and centrifuged at 3,270 × g for 30 min at 4°C to remove free bacteria, before being further centrifuged at 27,000 × g for 2 h at 4°C with low brake to pellet MPs. The MP pellet was washed with triple-filtered PBS (twice 0.2 µm, once 0.1 µm) and pelleted by centrifugation at 27,000 × g for 2 h at 4°C. MPs isolated in this manner were previously shown to be of high purity and less than 750 nm in size (19).

2.3 Quantitative real-time polymerase chain reaction (qPCR)



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The gene expression of targeted glycosylation enzymes was determined at the indicated time points using qPCR. Messenger RNA was purified using the Trisure reagent (Bioline) according to the manufacturer’s instructions. Samples were DNase I (Roche) treated, and cDNA synthesized using the Tetro cDNA synthesis kit (Bioline) according to the manufacturer’s instructions. The qPCR measurements were performed using SensiFAST SYBR green (Bioline) on a 7900HT (Applied Biosystems) using the following protocol: 95°C for 5 min equilibration followed by 95°C for 10 s, 60°C for 30 s repeated for 40 cycles using specific primers for the infection control gene i.e. ISG15, and the exoglycosidase enzymes i.e. HEXA, HEXB, GLB1, MAN2B1, and MANBA, Supplementary Table S1. The total cDNA abundances were normalized using 18S rRNA (20). Statistical evaluation of the mRNA levels (mean ± SD) was performed using one-tailed T-tests, type 2, n = 3.

2.4 Isolation and concentration of proteins from macrophage CLs and MPs MPs were lyzed in a RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% w/v SDS, 0.5% w/v sodium-deoxycholate, 1% v/v Triton X-100) supplemented with complete protease inhibitor cocktail (Roche) and 1 mM phenylmethane sulfonyl fluoride by incubation for 30 min at 4°C, centrifuged at 18,000 x g for 30 min at 4°C and stored at -20°C. The sterility of the MP protein samples were confirmed by plating 1-2 μl of the resuspended protein pellet onto 7H11 agar and incubation for 21 days at 37°C. Macrophages were lyzed in the same RIPA buffer supplemented with the same protease inhibitors (5 x 107 cells/ml) for 30 min at 4°C. The protein complement was crudely isolated by centrifugation (18,000 x g) for 30 min at 4°C and stored at -20°C. Protein concentrations were determined using a BCA assay. Separate fractions of the prepared protein isolates from the MP and macrophage lysates were used for the downstream N-glycomics and proteomics experiments.

2.5 Sample preparation of MP and CL proteins for N-glycomics N-glycans were released from 20 µg isolated proteins of the CL and MP fractions as previously described (21, 22). Briefly, the isolated protein fractions were reduced (5 mM dithiothreitol) for 10 min at 70°C and alkylated (10 mM iodoacetamide, both final concentrations) for 30 min at 25°C in 6 ACS Paragon Plus Environment

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the dark, then immobilized on a methanol-activated PVDF membrane (Millipore). After overnight drying, the membrane-bound proteins were incubated with 2.5 U peptide:N-glycosidase F in a total volume of 10 µl H2O per well for 16 h at 37°C to ensure complete release of the conjugated Nglycans. Released N-glycans were incubated with 100 mM ammonium acetate (pH 5) for 1 h at room temperature and subsequently dried by vacuum centrifugation. Reduction of N-glycans was performed with 20 µl 1 M sodium borohydride in 50 mM potassium hydroxide for 3 h at 50°C. Reduced samples were quenched with 2 µl glacial acetic acid and desalted on columns of AG 50W X8 cation exchange resin packed on top of ZipTip C18 columns (Millipore) as described. The desalted N-glycans were dried by vacuum centrifugation and residual borate was removed by repeated cycles of methanol addition and evaporation in a vacuum centrifuge. Further desalting was performed on small homemade columns packed with porous graphitized carbon (PGC) resin (Grace) on top of ZipTip C18 columns. The purified N-glycans were eluted with 40% (v/v) acetonitrile (ACN) containing 0.1% (v/v) trifluoroacetic acid, dried and stored at -80°C if not analyzed immediately.

2.6 Sample preparation of MP and CL proteins for proteomics and intact glycopeptide analysis Protein isolates (~50 µg) of the CL and MP fractions were reduced and alkylated as described above and loaded on a 4-12% Bis-Tris SDS polyacrylamide gel for electrophoresis at 200 V for 20 min. The gel was fixed in 40% (v/v) ethanol and 10% (v/v) acetic acid for 1 h, stained overnight with Coomassie Blue G250 and then destained in water. A gel approach was chosen since the amount of infection-driven protein regulation and the sample-to-sample proteome variation could quickly and visually be estimated from the protein band pattern prior to the downstream LC-MS/MS and in order to support that the identified peptides of lysosomal nature were indeed belonging to intact or only weakly degraded/truncated proteins. In-gel trypsin digestions of all samples were performed by cutting each of the lanes into five equal sized gel fractions (see Supplementary Fig. S-1 for representative gel images), which were further sliced into 1 mm pieces and placed in Eppendorf tubes. The five gel fractions from each sample were handled and analyzed individually. The gel pieces were destained with 50% (v/v) ACN in 50 mM ammonium bicarbonate, then dehydrated in 100% ACN and



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dried. Porcine trypsin (sequence grade, Promega) was added at a weight ratio of 1:30 to digest the proteins overnight at 37C. The tryptic peptides were extracted, dried and stored at -80C until used for LC-MS/MS analysis.

2.7 LC-MS/MS of N-glycans The purified N-glycan alditols were taken up in water, transferred to high recovery vials (Agilent) and injected onto a PGC LC column (Hypercarb KAPPA, 5 µm particle size, 100 mm length x 200 µm inner diameter, 250 Å pore size, Thermo Scientific) on a 1260 Infinity LC system (Agilent) connected directly to an ESI-MS/MS 3D ion trap mass spectrometer (LC/MSD Trap XCT Plus Series 1100, Agilent Technologies). Separation was performed for 85 min over a linear gradient of 0-45% (v/v) ACN/10 mM ammonium bicarbonate at a constant flow rate of 2 µl/min. Data were acquired in negative ion polarity mode with three scan events at a scan speed of 8,100 m/z/s. MS full scans (m/z 400-2,200) were followed by data-dependent MS/MS after isolation and resonance activation CID fragmentation of the top two most intense precursor ions with an absolute intensity threshold >30,000 and a relative intensity threshold >5% relative to the base peak. Multi-point m/z calibration of the mass spectrometer was performed using a tune mix (Agilent) prior to acquisition. N-glycans released in parallel from bovine fetuin (Sigma) served as a control for the sample preparation and the LCMS/MS performance. Differences between experimental and theoretical precursor and fragment masses were generally better than 0.2 Da.

2.8 LC-MS/MS of peptides and intact N-glycopeptides The isolated proteins were taken up in 0.1% (v/v) formic acid, transferred to high recovery vials (Agilent) and analyzed using an Easy-nLC (Thermo Scientific) connected directly to the nano-ESI source of a Q-Exactive Plus Orbitrap mass spectrometer (Thermo Scientific). Peptides were separated on a custom-made reversed-phase LC column packed with Halo C18 resin (2.7 µm particle size, 100 mm length, 75 µm inner diameter, 160 Å pore size, Advance Materials Technology, Wilmington, DE). The LC was equipped with an LC trapping column (35 mm length, 100 µm inner diameter)


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made from the same resin. The flow rate was kept constant at 300 nL/min and the column was equilibrated in solvent A consisting of 0.1% (v/v) aqueous formic acid prior to sample injection. Peptides were separated over 60 min where a linear gradient increasing from 0-50% of solvent B consisting of 100% (v/v) ACN were applied over the first 50 min, then a linear increase to 85% solvent B over 2 min and then solvent B was maintained at 85% for 8 min. MS full scans were acquired with a resolution of 35,000 (at m/z 200) in positive polarity ion mode over a m/z 350–2,000 range and an automatic gain control (AGC) target value of 1×106. The top 10 most intense precursor ions were selected for MS/MS in each full scan, isolated, and fragmented using higher energy collisional dissociation (HCD) fragmentation at 17,500 resolution (at m/z 200) with the following settings: collision energy: 30%; AGC target: 2×105; isolation window: m/z 3.0; and dynamic exclusion enabled. Precursors with unassigned charge states or singly charged ions were ignored for the MS/MS selection. Please note that the mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (23) partner repository with the data set identifier PXD005169 and 10.6019/PXD005169.

2.9 Analysis of N-glycome data LC-MS/MS raw data files of the N-glycome were viewed and manually analyzed using Data Analysis v4.0 (Bruker Daltonics). Monoisotopic masses were derived after deconvolution and searched for possible N-glycan monosaccharide compositions using GlycoMod (http://web.expasy.org/glycomod/). The monosaccharide compositions were subsequently verified manually by de novo sequencing of the corresponding CID-MS/MS spectra and by the absolute and relative PGC-LC retention time according to the established elution order of N-glycans (24). The identified monosaccharide compositions were all assumed to have a trimannosylchitobiose core i.e. two N-acetylglucosamine (GlcNAc) and three mannose (Man) residues unless they were truncated to paucimannosidic or chitobiose core type structures. Core fucosylated N-glycans were distinguished from antenna fucosylated structures by the presence of the diagnostic ions (m/z 350.1/368.1) corresponding to the glycosidic fragments comprising an α1,6-linked fucose (Fuc) attached to the reducing end GlcNAc



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residue. Diagnostic fragment ions (m/z 290.1/655.3) were used to determine sialylated N-glycans. The 2,3- or 2,6-sialic acid isomers linked to the penultimate galactose (Gal) residues were identified based on differential PGC-LC retention. Bisecting GlcNAc-containing structures were differentiated from the other isomeric tri-antennary structures based on the observation of particularly abundant diagnostic fragment ions i.e. D-ion and D-221 Da and their weaker retention on PGC-LC. The distribution of the observed N-glycans was calculated using their relative abundances as measured by the relative peak areas of their corresponding extracted ion chromatograms (EICs) of all the observed N-glycan charge states as a percentage (mean ± SD, n = 3) out of the total glycome. The N-glycans were grouped according to their glycan type and other features and their relative abundances statistically evaluated using student’s T-tests across samples.

2.10 Analysis of the proteome and intact glycopeptide data The analysis of the LC-MS/MS-based proteomic data was performed largely as described (19). Briefly, peak lists were extracted from the Q-Exactive raw data by Mascot Daemon/extract_msn (Matrix Science, Thermo), using the default parameters. All MS/MS data were submitted to an inhouse Mascot server (Matrix Science, London, UK; v2.4.0) and X! Tandem (The GPM, thegpm.org; version CYCLONE (2010.12.01.1)) and searched against the human proteome database (Homo sapiens, 20,203 reviewed entries in UniProtKB, released March 2015), by selecting trypsin as the proteolytic enzyme and a single missed cleavage for Mascot or two missed cleavages for X! Tandem. Separate searches against M. tb and “All species” proteome databases were also performed, but showed no sign of bacterial proteins. Mass tolerances of 0.020 Da and 20 ppm were selected for product and precursor ions, respectively. For both database searches, deamidation of N-terminal Gln, oxidation of Met, carbamidomethylation and propionamide of Cys were set as variable modifications. Additional variable modifications specified in X! Tandem included Glu→pyro-Glu of the N-terminus, ammonia-loss of the N-terminus and Gln→pyroGlu of the N-terminus. Scaffold (version Scaffold_4.4.1, Proteome Software Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identification. Peptide identifications were accepted if they exceeded specific database


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search engine thresholds. Mascot identification required that ion scores were greater than both the associated identity scores and 30, 40, and 50 for, doubly, triply, and quadruply charged peptides, respectively. X! Tandem identifications required at least –Log (expect scores) scores of greater than 4.0. The protein identifications were only accepted if they contained at least two identified nonredundant peptides. Homologous proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. Proteins were annotated with GO terms from gene_association.goa_human (downloaded July, 2013). Relative label-free quantitation analysis was performed to determine differentially expressed proteins based on spectral counting and significance testing (25, 26). The spectral count of each identified protein in a sample was normalized on the total sum of spectral counts of all identified proteins in the sample. The normalized values were used for calculating fold change between M. tb-infected and uninfected samples, with P values calculated by Fisher’s exact test and adjusted after Benjamini-Hochberg multiple testing correction. Protein expressions were considered to be significantly altered if the fold change was ≥ ±2 and P < 0.05. The spectral counts per protein were generally reasonably high and the SDs were generally reasonably low e.g. for the approximately 1,100 proteins identified from the I-MP sample, the average spectral count per protein was ~30 and the SD was ~7.5 (calculations not shown). Functional pathway analyses were performed using publicly-available DAVID Functional Annotation Tool (v6.8) (http://david.abcc.ncifcrf.gov/) (27) and aided by the use of Ingenuity Pathway Analysis and the KEGG pathway analysis tools (www.genome.jp/kegg/pathway). For the intact glycopeptide analysis, raw data files from the Q-Exactive Orbitrap analyses were converted to mgf using Proteome Discoverer v1.4 (Thermo Fisher) and the glycopeptide-containing HCD-MS/MS spectra were extracted using an in-house filtering tool isolating specifically oxonium (GlcNAc) ion-containing fragment spectra (m/z 204.08). The extracted data were searched for Nglycopeptides using ByonicTM v2.6 (Protein Metrics Inc.). Specifically, the searches against the entire human proteome (Homo sapiens, 20,198 reviewed entries in UniProtKB, release September 2015) and against the common mammalian N-glycome (309 monosaccharide compositions, default N-glycome database in Byonic). The following search criteria were used: mass tolerances of 10 and 20 ppm for 11 ACS Paragon Plus Environment


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the precursor and product ions, respectively. A maximum of two missed cleavages were allowed. Carbamidomethylation was chosen as a fixed Cys modification; Met oxidation and Asn/Gln deamidation were variable modifications. Only confidently identified glycopeptides were accepted by using a Byonic score >100, which has recently been evaluated to be an appropriate threshold (28, 29). The glycopeptide-based quantitation of the glycan types (expressed as a mean ± SD, n = 3) was performed using spectral counting.

2.11 β-hexosaminidase activity assay Total β-hexosaminidase (A: αβ and B: ββ) and β-hexosaminidase A (αβ) activity were measured in the culture media (CM) at the indicated time points. Equal volumes of the CM from the individual samples were incubated in a phosphate citrate buffer (pH 4.4) with 4-methylumbelliferyl-2acetamido-2-deoxy-β-D-glucopyranoside

and

4-methylumbelliferyl-6-sulfo-N-acetyl-β-D-

glucosaminide (Merck Millipore) in separate experiments for 30 min at 37°C. Reactions were quenched with 0.25 M glycine carbonate buffer (pH 10.0). The release of 4-methylumbelliferone (4MU) was then quantified using a fluorometer plate reader (BMG technologies) with excitation at 360 nm and emission at 450 nm. The hexosaminidase activities were determined by comparison to a premade standard curve performed using known amounts of 4-MU standards (Sigma-Aldrich) after subtraction of background hexosaminidase activity originating from the fetal calf serum within the CM. These data points are expressed as a mean ± SEM.

RESULTS AND DISCUSSION Cell lysates (CLs) of macrophages and their secreted microparticles (MPs) were studied in vitro in paired biological triplicate experiments 72 h after infection with virulent M. tb H37Rv (fractions called I-CL and I-MP, respectively) and without infection (called UI-CL and UI-MP, respectively). Gel-separated proteins and N-glycans of non-separated proteins of these cellular fractions of human macrophages were profiled using LC-MS/MS-based label-free quantitative proteomics and N-


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glycomics, respectively, Fig. 1. The regulation of intact N-glycopeptides derived from macrophage CL proteins was also estimated upon infection using the proteomics data. In addition, the mRNA levels of exoglycosidases that were regulated at the proteome level were monitored from 4-72 h postinfection to investigate time-dependent regulation at the transcriptional level. Finally, the culture media (CM) were also monitored for hexosaminidase activity within this period. We have previously utilized this TB model system demonstrating both high macrophage cell viability over the 72 h time course and high purity isolation of released MPs (100-750 nm in size) (18, 19). In agreement with these past reports, M. tb infection induced significant up-regulation of a number of proinflammatory markers at the protein and transcript level, including the interferon-induced proteins i.e. IFIT1-3 and ISG15 (data not shown).

M. tb infection alters the macrophage N-glycoproteome by manipulation of lysosomal exoglycosidases Conventional LC-MS/MS-based proteomics of gel-separated proteins was used to monitor the regulation of the usually lower-abundant glycosylation enzymes and receptors in macrophages upon M. tb infection. In total, 1,495 human proteins (UI-CL: 136 unique proteins, I-CL: 233 unique proteins and 1,126 shared proteins) were confidently identified from the macrophage lysates, Supplementary Fig. S-1A-B and see also Supplementary Table S-2 and Supplementary Table S-3 for more information of the identified peptides/proteins. Since only very few M. tb proteins were identified in the infected samples (data not shown), we decided not to investigate the low-abundant bacterial proteome further. The quantitative analysis, which was performed by spectral counting and significance testing (25, 26), identified 207 (13.8%) up- and 228 (15.3%) down-regulated proteins from infected macrophages relative to uninfected macrophages, Supplementary Fig. S-1C. Functional pathway analysis indicated that amongst other well-known phagocyte, immune and metabolism features of the macrophages, the usually abundant glycan degradation processes central to the lysosome (P = 1.4 x 10-3) and other general lysosomal processes (P = 2.4 x 10-5) were attenuated upon M. tb infection.



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In total, 25 glycosylation enzymes and nine glycan receptors (lectins) were detected and quantified within the macrophage proteome, Table 1. Amongst the lectins, significant infection-dependent down-regulation (5.3x ↓) of the C-type mannose receptor 2 was observed. This macrophage cell surface lectin receptor, together with other related members of the MR family (10) and other C-type lectins, including DC-SIGN and the Mincle receptor, contributes to the uptake of M. tb (30). Another interesting lectin altered by M. tb infection was sialoadhesin (also known as Siglec-1 or CD169) (6.0x ↑). Siglec-1 is an inflammatory receptor on the cell surface of macrophages that mediates sialic aciddependent antigen presentation to both T and B cells and NK cells (31). Further research is required to understand the involvement of these infection-induced lectin responses in the pathogenesis of, and response to, TB infection. One of the most key survival features of M. tb is its ability to modulate the host macrophage to prevent the M. tb carrying phagosome from fusing with lysosomes, thereby avoiding degradation by the potent lysosomal hydrolytic enzymes (4). The phagosome-lysosome fusion event is a complex and only partially understood process involving key lysosomal glycoproteins, including synaptotagmin (32, 33). We therefore focused our investigation on the glycoproteome regulation of the lysosomal compartment. Significant down-regulation of all 11 lysosomal exoglycosidases was observed upon M. tb infection, these included β-galactosidase (18.7x ↓), β-hexosaminidase subunit α and β (20.0x ↓ and 25.6x ↓, respectively), α-mannosidase (4.1x ↓) and β-mannosidase (10.7x ↓), Table 1. These hydrolases are linkage-specific exoglycosidases that facilitate sequential truncation of non-reducing end monosaccharide residues of N-glycoproteins in the relative acidic lysosomal environment (pH 4.5-5), Fig. 2A. Surprisingly, the lysosomal neuraminidase (also known as sialidase-1, UniProtKB: Q99519) was not identified in this near-complete list of N-glycan glycosidases although this sialic acid-degrading enzyme has been reported to be expressed in a lysosomal complex with βgalactosidase and thus expected to be present at similar levels (34). Exoglycosidases responsible for glycolipid e.g. α-galactosidase A and glycosaminoglycan e.g. β-glucuronidase degradation/truncation were also observed, but not investigated further here since our focus was specifically on the Nglycoproteome. The β subunit forming part of the heterodimeric (Hex A, αβ) or the homodimeric (Hex B, ββ) β-hexosaminidase was the most down-regulated glycosidase. Interestingly, the β14 ACS Paragon Plus Environment

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hexosaminidases have been shown to be critical for limiting infection of mycobacteria (35) and other bacteria (36) in mammalian cells and in lower model organisms. As indicated by these studies, the enzyme acts by digesting the alternating β1,4-GlcNAc residues of the bacterial peptidoglycan making up the stabilizing cell wall of most bacteria. We then investigated using qPCR if the alterations of the lysosomal glycosidases were being regulated at the transcriptional level. These experiments demonstrated that expression of the genes coding for exoglycosidases responsible for lysosomal N-glycoprotein truncation were only weakly attenuated after 72 h infection i.e. GLB1 (β-galactosidase), HEXA (β-hexosaminidase subunit α), HEXB (β-hexosaminidase subunit β), MAN2B1 (α-mannosidase) and MANBA (β-mannosidase) (all P < 0.05 except HEXB), Fig. 2B. These data indicate that the dramatic manipulation of the lysosomal exoglycosidases observed at the proteome level may only partially be explained by transcriptional regulation. The exact mechanisms controlling the gene expression of the diverse glycosylation enzymes involved in the complex glycosylation machinery remain largely undefined. Indeed our understanding of how these complicated pathways function in both pathologies and in normal physiology is still developing (37). Post-transcriptional regulation of gene expression by microRNA (miRNA) has also been suggested to play an important role in TB (38), but any miRNA-based control over the glycosylation machinery remains undescribed in a TB context. Reduced protein trafficking to the lysosome is another mechanism that may contribute to a lysosomal regulation. This was supported by three observations: 1) the cation-independent mannose-6-phosphate receptor that is partially responsible for sequestering N-glycoproteins from the cis-Golgi network to the lysosome compartment (39) was found to be down-regulated (~2 fold, P < 0.0001) upon infection (see Table 1), 2) pathway analysis showed that infection increased lysosomal protein signatures in the secreted MPs (see below) suggesting a re-routing of some of the proteins originally destined to the lysosome, and 3) a major subset of the proteins identified to be of lysosomal origin as classified based on gene ontology (34 of 59 proteins, 57.6%) were significantly down-regulated. These proteins were additionally classified as being involved in processes associated with degradation. However, a subset of lysosomal proteins (9 of 59 proteins, 15.3%) were significantly up-regulated upon M. tb infection and several other proteins remained unchanged, including the cation-dependent mannose-6-phosphate 15 ACS Paragon Plus Environment


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receptor, another lectin responsible for lysosomal trafficking (39), thus indicating that not the entire lysosomal sequestering pathway is attenuated following M. tb infection. A more significant contributor to the dramatic lysosomal proteome changes may be the selective secretion of the lysosomal content including the abundant exoglycosidases into the extracellular environment via exocytosis/degranulation mechanisms upon microbial infection. This hypothesis is supported by the observation that soluble and fully functional β-hexosaminidase and α-glucuronidase enzymes are released by mycobacteria, furthermore β-hexosaminidase deficient macrophages permitted enhanced mycobacterial growth (35, 36, 40). Using enzymatic assays to assess the hexosaminidase activities within the culture media, we confirmed that M. tb infection induces the release of significant Hex A (αβ) (P = 4.4 x 10-4), though the total hexosaminidase activity (capturing both Hex A (αβ) and Hex B (ββ) activity) was not significantly different, Fig. 2C-D. Together these data indicate a constitutive secretion of Hex B (and possibly Hex A) and a time-dependent infectioninduced release of Hex A from macrophages. Further studies are required to detail the exact mechanism(s) underpinning these intriguing observations. The effect of the infection-driven alterations of the lysosomal exoglycosidases on macrophage Nglycosylation was then investigated using established glycomics technology on a PGC-LC-MS/MS platform (41). This N-glycome profiling showed that macrophage proteins carry heterogeneous Nglycosylation comprising 42 N-glycan structures spanning 30 monosaccharide compositions including the abundant high mannosidic (Man5-9GlcNAc2, ~35-40%) and paucimannosidic (Man23GlcNAc2Fuc0-1,

~40-50%) structures, Table 2. Some hybrid and complex-type N-glycans were also

identified in macrophage lysates, covering predominantly bi- and tri-antennary type structures with partial α2,3- and α2,6-NeuAc-type sialylation and α1,6-(core) type fucosylation. The observed Nglycans were biosynthetically related species, Fig. 3. Although these observations are largely in agreement with a recent glycome study of uninfected THP-1 cells (42), the abundance of paucimannosylation was not reported, possibly due to an analytical focus on larger N-glycans and/or differences in analytical acquisition styles. We recently showed that paucimannosylation is an abundant yet under-reported type of N-glycosylation of azurophilic granule proteins of human neutrophils (43). We showed that these unusual glyco-epitopes appear on intact and bioactive 16 ACS Paragon Plus Environment

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neutrophil proteins (44). In fact, these paucimannosylated azurophil granule proteins constitute the major mycobactericidal proteins in neutrophils, endocytosed by macrophages to enhance mycobacterial killing (45). Although we previously indicated the presence of paucimannosylation in differentiated THP-1 cells using immune-fluorescence (46), it remains to be investigated if these structures are present on intact glycoproteins or are hydrolytic degradation products within the lysosome of macrophages. In support of the former, the glycoprotein appeared to not undergo excessive proteolytic cleavage as evaluated by SDS-PAGE (Supplementary Fig. S-1A) and the observation that intact human glycopeptides displayed these structures (see below). Although M. tb was reported to carry protein glycosylation including O-mannosylation (9, 47), to the best of our knowledge, no mycobacteria N-glycosylation has yet been reported. Together with the lack of M. tb proteins in our proteomics data, we conclude that the profiled N-glycome is of human cell origin. When grouped according to glycan types, significant up-regulation of the complex-type glycosylation (P = 0.002) and a concomitant down-regulation of paucimannosylation (P = 0.023, both two-tailed Ttests) were observed upon infection, Fig. 4A. Many of the complex glycans in the macrophage lysates were increased upon M. tb infection as demonstrated by the significant up-regulation of 9 of the 10 regulated complex type structures in the I-CL relative to the UI-CL samples. However, it was observed that the short GlcNAc-capped complex glycans (e.g. glycan #9, 1.5 percentage point and 4.6 fold increase) contributed relatively more to this increase, see Supplementary Table S-2 for details. This regulation of the N-glycome showing an infection-dependent decrease of N-glycan truncation, and of individual glycan species (i.e. glycan #4, #7a/b and #9), agrees well with the reduction of the lysosomal β-hexosaminidase subunit α (20.0x ↓) and β (25.6x ↓) upon M. tb infection. The dramatic β-hexosaminidase regulation seemed to counteract the simultaneous, but weaker, down-regulation (6.0 x ↓) of the biosynthetic Mgat2 glycosyltransferase thereby shifting the glycan truncation/elongation equilibrium towards GlcNAc-capping, Fig. 4B. To get deeper insight into the M. tb induced regulation of the lysosomal N-glycoproteome in macrophages we interrogated our proteomics HCD-MS/MS data for the presence of intact Nglycopeptides using established glycoproteomics method (48). The 47 confidently identified Nglycosylated proteins displayed site-specific glycoprofiles that were estimated, by the use of 17 ACS Paragon Plus Environment


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glycopeptide spectral counting, to largely reflect the glycan type distribution established using quantitative glycomics, Fig. 4C. However, in addition to the complex, hybrid, high mannose and paucimannose-type structures, the little reported chitobiose type glycosylation was also observed (44, 49, 50). In agreement with the glycome profiling, this crude method for glycopeptide-based quantitation showed trends towards an infection-induced up-regulation of the complex-type Nglycosylation and a concomitant down-regulation of the truncated paucimannose and chitobiose-type structures albeit not statistically significant. The same trends were observed within the subset of 16 glycoproteins covering 23 unique glycosylation sites and 70 non-redundant glycopeptides that are known to reside specifically in the lysosome, including lysosome-associated membrane glycoprotein 1-2 and prosaposin, Fig. 4C-D (see Supplementary Table S-2 for more glycopeptide data). This suggested that changes in lysosome N-glycosylation contributed to the overall alterations of the Nglycome. The macrophage lysates showed little or no changes in other N-glycosylation features upon M. tb infection including sialylation and core fucosylation, either because the responsible glycosylation enzymes were not regulated or as a result of neutralizing bidirectional regulation of both the biosynthetic glycosyltransferase and the degrading glycosidases as observed for the core fucosylation feature, Fig. 4E-F. These observations also support that the many reported glycoside hydrolases of M. tb are significantly altering the host glycoproteome upon infection (51).

N-glycosylation initiation, but not the N-glycosylation profile, is altered in infected MPs M. tb infection significantly increases the number of MPs released from macrophages (18). Since the total protein amount within the macrophage and the protein amount per MP remain largely unchanged upon infection as estimated by gel electrophoresis (Supplementary Fig. S-1), this indicated an infection-driven elevation of the overall protein synthesis in macrophages facilitated by a more relaxed quality control of the protein folding machinery. This hypothesis was supported by a dramatic 25 fold down-regulation of UDP-glucose:glycoprotein glucosyltransferase 1 that functions to reglucosylate slightly misfolded N-glycoproteins (52). In total, 1,086 human proteins (UI-MP: 95 unique proteins, I-MP: 75 unique proteins and 916 shared proteins) were identified in the MPs, Supplementary Fig. S-1D-E and see also Supplementary Table S-2 and Supplementary Table S-3 18 ACS Paragon Plus Environment

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for more information of the identified peptides/proteins. Quantitative evaluation demonstrated that infection promoted significant regulation of the MP proteome as shown by the detection of 127 (11.7%) down- and 85 (7.7%) up-regulated proteins relative to the uninfected MP proteome, Supplementary Fig. S-1F. Pathway analysis showed that infected MPs were slightly enriched in proteins associated with ER (P = 1.8 x 10-5) and lysosomal processing (P = 2.2 x 10-2). Out of the 19 glycosylation enzymes and receptors identified and quantified in the MPs, the most notable observation was the up-regulation of a cluster of proteins forming the multi-subunit oligosaccharyltransferase (OST) complex including OST48 (10.7x ↑), ribophorin I (4.7x ↑), ribophorin II (5.2x ↑), STT3A (4.3x ↑), DAD1 (4.0x ↑), Fig. 5A. The OST complex is a large ERresident enzyme complex that initiates protein N-glycosylation by the transfer of N-glycan precursors (Glc3Man9GlcNAc2) from dolichol carriers to sequon-located Asn of newly translated proteins in the ER lumen (53, 54). Interestingly, the two ER glucosidases GLU2B (4.2x ↑) and MOGS (3.3x ↑), which are responsible for the subsequent removal of the outermost α1,2-Glc of the glycan precursor early in the biosynthetic machinery and malectin (2.0x ↑), which is binding strongly to the immature Glc2Man9GlcNAc2-glycan and, thus, are associated with the protein synthesis (quality control of protein folding) (52) were also up-regulated as a result of infection. These observations correlate well with the increased rate of MP formation, increased N-glycan density of MP proteins (as measured by higher, but not significant total EIC intensities of the detected glycans), Fig. 5B, and the significantly higher level of the glycan precursor i.e. Glc1Man9GlcNAc2, Fig. 5C. All of these changes mechanistically require a higher level of the OST complex and/or longer reaction times with these glyco-initiating enzymes. The identification of these ER-typic enzymes within infected MPs is particularly intriguing. MPs have been shown to transfer functional proteins between cells. For example, MPs transferred functional CCR5 to CCR5 negative cells via MP delivery to recycling endosomes of the receiving cells (18, 47). It is still unclear how the ER-specific OST enzyme complex and the associated α-glucosidases are packaged into the plasma membrane-rich MPs and whether these are still enzymatically active in the secreted MPs. Further work is also needed to detail whether the OST complex can be integrated into recipient cells and participate in the protein glycosylation initiation process. Conforming to the absence of regulation of other glycosylation 19 ACS Paragon Plus Environment


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enzymes within infected MPs, no significant changes in the glycosylation pattern of the MPs were detected upon M. tb infection Fig. 5D. No consistent infection-driven regulation of the individual OST subunits was identified in the CLs (Table 1) indicating a lack of a link between OST changes in the MP and CL.

MPs are derived from the macrophage plasma membrane and carry cell surface-like Nglycosylation signatures The proteomics data demonstrated that although the MPs share strong protein signatures with their macrophage origins they display a unique proteome (I-/UI-MP: 327 unique proteins, I-/UI-CL: 736 unique proteins and 759 shared proteins), Supplementary Fig. S-1G. Localization analysis using GO annotation of the identified proteins showed that the MP proteins are preferentially being packaged from the plasma membrane in agreement with our current understanding of these EVs (17). Interestingly, M. tb infection reduced the contribution of plasma membrane proteins and, in turn, increased the contribution of ER proteins in the MPs (data not shown) in line with the infectioninduced increase of the ER-typic OST complex in MPs. The increased ER signature in the infected MPs correlates with the observation that cells, upon apoptosis, secrete MPs arising from the ER membrane rather than deriving exclusively from the plasma membrane (55). Direct proteomics comparisons of the CLs and MPs also showed that de novo protein synthesis and/or degradation (or depletion via secretion) contribute more to the M. tb infection-induced proteome alterations than protein re-routing as evidenced by the increase in “same direction” protein regulations within CLs and their paired MPs, Supplementary Fig. S-1H rather than promoting “opposite direction” regulation of CL and MP proteins, Supplementary Fig. S-1I. Our glycome data allowed a detailed comparative paired analysis of the glycosylation of the macrophages and their corresponding MPs. Irrespective of infection status, we observed a dramatic enrichment of complex-type N-glycosylation and a concomitant depletion of paucimannosylation in the secreted MPs, Fig. 6A. Even when adjusting for this additional degree of complex-type glycosylation this analysis still demonstrated a significant enrichment of α2,3- and α2,6-sialylation, Fig. 6B, at the expense of terminal galactosylation, Fig. 6C, and terminal GlcNAc epitopes, Fig. 6D. 20 ACS Paragon Plus Environment

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We and other research groups have previously shown that the cell surface glycoproteins have a higher degree of complex-type glycosylation and specifically a higher degree of sialylation relative to glycoproteins residing in the intercellular compartments as evaluated by N-glycome profiling of cell surface enriched and microsomal (total membrane) protein preparations and established that this subcellular-specific differential processing, in part, is driven by differences in site accessibility (5658). Although these glycosylation signatures and the associated mechanisms may be cell-type dependent, the MPs of macrophages display cell-surface-like glycosylation signatures in agreement with their plasma membrane origin. Only relatively few glycosylation-centric studies have reported on the glycosylation signatures of EVs (59). These have predominantly been performed on isolated exosomes of various cellular origins where enrichment of multiple glycosylation features including high mannose and complex-type glycosylation were reported (60-63). Interestingly, these smaller EVs were also shown to contain ER signatures including the presence of exoglycosidases (64). Only just recently was the glycocalyx of vascular cell MPs reported using lectin-based characterization, which indicated that the glycosylation of MPs differ significantly from the glycosylation patterns of the parents cells (65). These observations are in agreement with our findings demonstrating for the first time the detailed glycan structures and the associated proteome profile of MPs relative to their cellular source of origin.

CONCLUSIONS In this system-wide study of macrophage glycobiology conducted in the context of TB pathology, we propose that, upon infection, M. tb seemingly induces profound alterations of the glycosylation machinery of macrophages resulting in significant changes in the N-glycoproteome signatures, Fig. 7. While these preliminary findings are exciting by suggesting some interesting host-pathogen response mechanisms, protein glycosylation is particular sensitive to factors such as cellular origin and local and systemic physiology and, importantly, the structure of individual protein carriers (66). Further in vivo and ex vivo research investigating the M. tb infection-induced regulation of the glycosylation machinery of tissue derived alveolar macrophages and their released MPs are required to confirm if the in vitro-based findings reported here are reflected in vivo in the usual host macrophages of 21 ACS Paragon Plus Environment


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individuals with M. tb infection. Alveolar macrophages have indeed shown immune-regulatory properties that may delay optimal protective host immune responses (67). From our observations, we cannot determine if the manipulation of the N-glycosylation machinery of human macrophages is driven by the pathogen to promote infection or by the host to facilitate protection from mycobacteria. In conclusion, we demonstrate that the protein N-glycosylation of macrophages undergoes rapid and significant changes following M. tb infection. The hypothesis-generating nature of this work has left many intriguing research questions unanswered including, not least, if the infection-specific glycosylation signatures are reflected in TB patients and, if so, is this aberrant protein glycosylation causing, or arising from, the pathogenicity of the bacteria? Further, which of these glycosylation changes can be attributed to a host-response and which to microbial manipulation? Addressing these questions will undoubtedly bring us yet another step closer to understanding the intriguing complexity of M. tb pathogenesis.

SUPPORTING INFORMATION: The following files are available free of charge at ACS website http://pubs.acs.org: Supplemental Table S-1: Overview of primer design for qPCR. Supplemental Table S-2: Overview of the CL and MP proteome, N-glycome and intact glycopeptides of lysosomal glycoproteins (provided as a separate excel file). Supplemental Table S-3: Overview of the identified peptides and their spectral counts for all proteins identified from the proteomics experiments. Supplemental Fig. S-1. Overview of the proteome changes of the macrophage CLs and in their secreted MPs upon M. tb infection.

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3. Hmama, Z.; Pena-Diaz, S.; Joseph, S.; Av-Gay, Y., Immunoevasion and immunosuppression of the macrophage by Mycobacterium tuberculosis. Immunol Rev 2015, 264, (1), 220-32. 4. Weiss, G.; Schaible, U. E., Macrophage defense mechanisms against intracellular bacteria. Immunol Rev 2015, 264, (1), 182-203. 5. Loke, I.; Kolarich, D.; Packer, N. H.; Thaysen-Andersen, M., Emerging roles of protein mannosylation in inflammation and infection. Mol Aspects Med 2016, 51, 31-55. 6. Kallenius, G.; Correia-Neves, M.; Buteme, H.; Hamasur, B.; Svenson, S. B., Lipoarabinomannan, and its related glycolipids, induce divergent and opposing immune responses to Mycobacterium tuberculosis depending on structural diversity and experimental variations. Tuberculosis (Edinb) 2016, 96, 120-30. 7. Torrelles, J. B.; Schlesinger, L. S., Diversity in Mycobacterium tuberculosis mannosylated cell wall determinants impacts adaptation to the host. Tuberculosis (Edinb) 2010, 90, (2), 84-93. 8. Smith, G. T.; Sweredoski, M. J.; Hess, S., O-linked glycosylation sites profiling in Mycobacterium tuberculosis culture filtrate proteins. J Proteomics 2014, 97, 296-306. 9. Arya, S.; Sethi, D.; Singh, S.; Hade, M. D.; Singh, V.; Raju, P.; Chodisetti, S. B.; Verma, D.; Varshney, G. C.; Agrewala, J. N.; Dikshit, K. L., Truncated hemoglobin, HbN, is post-translationally modified in Mycobacterium tuberculosis and modulates host-pathogen interactions during intracellular infection. J Biol Chem 2013, 288, (41), 29987-99. 10. Schlesinger, L. S.; Kaufman, T. M.; Iyer, S.; Hull, S. R.; Marchiando, L. K., Differences in mannose receptor-mediated uptake of lipoarabinomannan from virulent and attenuated strains of Mycobacterium tuberculosis by human macrophages. J Immunol 1996, 157, (10), 4568-75. 11. Maeda, N.; Nigou, J.; Herrmann, J. L.; Jackson, M.; Amara, A.; Lagrange, P. H.; Puzo, G.; Gicquel, B.; Neyrolles, O., The cell surface receptor DC-SIGN discriminates between Mycobacterium species through selective recognition of the mannose caps on lipoarabinomannan. J Biol Chem 2003, 278, (8), 5513-6. 12. Krishnan, S.; Liu, F.; Abrol, R.; Hodges, J.; Goddard, W. A., 3rd; Prasadarao, N. V., The interaction of N-glycans in Fcgamma receptor I alpha-chain with Escherichia coli K1 outer membrane protein A for entry into macrophages: experimental and computational analysis. J Biol Chem 2014, 289, (45), 30937-49. 13. Kang, P. B.; Azad, A. K.; Torrelles, J. B.; Kaufman, T. M.; Beharka, A.; Tibesar, E.; DesJardin, L. E.; Schlesinger, L. S., The human macrophage mannose receptor directs Mycobacterium tuberculosis lipoarabinomannan-mediated phagosome biogenesis. J Exp Med 2005, 202, (7), 987-99. 14. Rajaram, M. V.; Ni, B.; Morris, J. D.; Brooks, M. N.; Carlson, T. K.; Bakthavachalu, B.; Schoenberg, D. R.; Torrelles, J. B.; Schlesinger, L. S., Mycobacterium tuberculosis lipomannan blocks TNF biosynthesis by regulating macrophage MAPK-activated protein kinase 2 (MK2) and microRNA miR-125b. Proc Natl Acad Sci U S A 2011, 108, (42), 17408-13. 15. Lee, T. H.; Mitchell, A.; Liu Lau, S.; An, H.; Rajeaskariah, P.; Wasinger, V.; Raftery, M.; Bryant, K.; Tedla, N., Glycosylation in a mammalian expression system is critical for the production of functionally active leukocyte immunoglobulin-like receptor A3 protein. J Biol Chem 2013, 288, (46), 32873-85. 16. Amaral, E. P.; Lasunskaia, E. B.; D'Imperio-Lima, M. R., Innate immunity in tuberculosis: how the sensing of mycobacteria and tissue damage modulates macrophage death. Microbes Infect 2016, 18, (1), 11-20. 17. Morel, O.; Jesel, L.; Freyssinet, J. M.; Toti, F., Cellular mechanisms underlying the formation of circulating microparticles. Arterioscler Thromb Vasc Biol 2011, 31, (1), 15-26. 18. Walters, S. B.; Kieckbusch, J.; Nagalingam, G.; Swain, A.; Latham, S. L.; Grau, G. E.; Britton, W. J.; Combes, V.; Saunders, B. M., Microparticles from mycobacteria-infected macrophages promote inflammation and cellular migration. J Immunol 2013, 190, (2), 669-77. 19. Hare, N. J.; Chan, B.; Chan, E.; Kaufman, K. L.; Britton, W. J.; Saunders, B. M., Microparticles released from Mycobacterium tuberculosis-infected human macrophages contain increased levels of the type I interferon inducible proteins including ISG15. Proteomics 2015, 15, (17), 3020-9. 20. Chang, T. C.; Zeitels, L. R.; Hwang, H. W.; Chivukula, R. R.; Wentzel, E. A.; Dews, M.; Jung, J.; Gao, P.; Dang, C. V.; Beer, M. A.; Thomas-Tikhonenko, A.; Mendell, J. T., Lin-28B 23 ACS Paragon Plus Environment


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ACKNOWLEDGEMENTS This research was facilitated through access to the Australian Proteomics Analysis Facility (APAF). We thank Dr. Ardeshir Amirkhani and Mr. Dylan Xavier (APAF) and Dr. Ben Crossett (University of Sydney) for assistance with LC-MS/MS acquisition and data handling. M.T.-A. was supported by a fellowship from the Cancer Institute, NSW, Australia and a Macquarie University Research Development Grant (MQRDG). Ian Loke was supported by an international Macquarie University Research Scholarship (iMQRES) and an Australian Cystic Fibrosis postgraduate studentship. This work was also funded in part by the NHMRC Centre for Research Excellence in Tuberculosis Control (APP1043225) (www.tbcre.org.au) and an NHMRC project grant (APP570771).

Abbreviations 4-MU, 4-methylumbelliferone; ACN, acetonitrile; AGC, automatic gain control; CID, collision induced dissociation; CL, cell lysate; CM, culture media; DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin; EIC, extracted ion chromatogram; ESI, electrospray ionization; EV, extracellular vesicles, FDR: false discovery rate; Fuc, fucose; Gal, galactose; Glc, Glucose; GlcNAc, N-acetylglucosamine; HCD, higher-energy collision dissociation; Hex, hexose; Hex A, hexosaminidase A, HexNAc, N-acetylhexosamine; IFIT, interferon-induced protein with tetratricopeptide repeats; LC-MS/MS, liquid chromatography tandem mass spectrometry; Man, mannose; miRNA, microRNA; MR, mannose receptor; M. tb, Mycobacterium tuberculosis; MP, microparticles, NeuAc, N-acetylneuraminic acid; OST, oligosaccharyltransferase; PGC, porous graphitized carbon; PMA, phorbol 12-myristate 13-acetate; qPCR, quantitative real-time polymerase chain reaction; SD, standard deviation; SEM, standard error of the mean; TB, tuberculosis.

Conflict of Interest Disclosure The authors have no conflict of interest to declare with respect to the performed study.



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FIGURE LEGENDS Figure 1. Experimental design of the study. Infected (I) and uninfected (UI) macrophage cell lysates (CL) and microparticles (MPs) were collected for glycomics, proteomics and intact glycopeptide analysis 72 h post-infection in biological paired triplicates. In addition, qPCR measurements of the macrophage CLs and hexosaminidase activity determinations of the culture media (CM) were performed at the indicated time points. *The extracted proteins were separated by one-dimensional gel electrophoresis prior to proteomics and glycopeptide analyses. Figure 2. M. tb infection of macrophages manipulates the lysosomal exoglycosidases. A. Overview of the sequential truncation of terminal monosaccharide residues of N-glycoproteins by individual exoglycosidases in the lysosome. B. Infection-dependent down-regulation of the lysosomal exoglycosidases at the proteome (top, 72 h post-infection) and transcript level (bottom, 4 h, 24 h, 48 h and 72 h post-infection). Data points are presented on a log10 scale as mean ± SD, n = 3. C-D. Total hexosaminidase (Hex A and B) and Hex A activities were measured in the CM at the indicated time points, respectively. Data points are presented as mean ± SEM, n = 3. For all panels, *** P < 0.0001, ** P < 0.01, * P < 0.05, ns (not significant) P ≥ 0.05. See Fig. 3 for key to monosaccharide symbols.

Figure 3. Structural overview of the N-glycomes of human macrophages and their MPs as characterised in details by PGC-LC-MS/MS. The biosynthetic constraints of the N-glycosylation machinery assisted the characterisation, but some structural ambiguity remains in particular for terminal glycosidic linkages and branch points e.g. the antennae-fucosylation (i.e. Glycan #22 and #24) can be either LewisX or LewisA. The biosynthetic relatedness of the individual N-glycans are displayed by arrows indicating single or multiple enzymatic reactions in the glycosylation machinery. The glycan numbers correspond to the structures presented in Table 1. The low mass glycan structures (a-d) were only observed at the intact glycopeptide level. See insert for key to monosaccharide symbols and glycosidic linkages. The glycans are represented in their respective glycan types (dark grey shading except for complex-type glycans and the glycan precursor).


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Figure 4. Regulation of macrophage protein N-glycosylation upon M. tb infection. A. Distribution of N-glycan types in UI-CL (white bars) and I-CL (black bars, valid for all panels) as evaluated by glycan quantitation. B. Regulation of GlcNAc truncation (to paucimannose) and elongation (to complex type) as driven by the down-regulation of the responsible enzymes i.e. β-hexosaminidase subunit α and β and Mgat2 (top). The relative quantities of the individual glycans (bottom) confirm the overall direction of regulation towards GlcNAc-capped structures upon infection. C. Distribution of N-glycan types as evaluated by glycopeptide quantitation of all identified glycoproteins and specifically of the lysosomal glycoproteins (overlaid, open grey bars). D. Overview of the lysosomal N-glycoproteins, the number of non-redundant glycoforms and the type of the N-glycosylation identified in macrophage lysates. Key to glycan types: “P” denotes paucimannose-type glycans, “C” denotes complex-type glycans, “Ch” denotes chitobiose-type glycans and “HM” denotes highmannose-type glycans. E. Degree of core fucosylation (normalized to the level of complex, hybrid and paucimannosidic-type glycosylation that can receive core fucosylation). F. Equal infection-driven regulation of the fucosyltransferase and α-fucosidase responsible for the addition and removal of α1,6-fucosylation. For all panels: Data points are presented as mean ± SD, n = 3, * P < 0.05, ns (not significant) P ≥ 0.05.. See Fig. 3 for key to monosaccharide symbols.

Figure 5. M. tb infection of macrophages regulates the degree of protein N-glycosylation, but not the N-glycosylation profile in secreted MPs. A. Illustration of the enzymatic initiation of protein Nglycosylation and the infection-driven regulation of the responsible enzymes in particular the multisubunit OST complex within secreted MPs. In contrast, no consistent infection-driven regulation of the individual OST subunits was identified in the CLs (see Table 1). B. Protein-normalized glycan density as measured by the total EIC areas of all N-glycans detected in the MP glycome. C. Relative abundance of the immature N-glycan precursor (Glc1Man9GlcNAc2) of the total MP glycome. D. Distribution of the N-glycan types of the MP glycome. For all panels: Data points are presented as mean ± SD, n = 3, * P < 0.05. See Fig. 3 for key to monosaccharide symbols.



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Figure 6. MPs display cell surface-like N-glycosylation signatures that differ from their parent macrophage glycosylation. A. Distribution of N-glycan types of CLs (white bars) and MPs (black bars, valid for all panels) without (top) and with (bottom) M. tb infection. B-D. Degree of total and linkage-specific sialylation, total terminal galactosylation and total terminal GlcNAcylation (normalized to the amount of hybrid and complex type glycans) without (top) and with (bottom) M. tb infection. Since the valency of terminal monosaccharide epitopes per glycan structure was taken into account in these calculations the degree of modification can exceed 100%. For all panels: Data points are presented as mean ± SD, n = 3, *** P < 0.001, ** P < 0.01, * P < 0.05.

Figure 7. Simplified overview of the pathogen- and/or host-driven manipulation of the Nglycosylation machinery and N-glycosylation signatures of macrophages upon M. tb infection as proposed by our multi-‘omics datasets. This simplistic glyco-centric model illustrates the profound manipulation of the macrophages and their secreted MPs upon infection with M. tb as indicated with red and green arrows to represent infection-induced down- and up-regulation, respectively. Many of these observations and their exact involvement in the host-pathogen response require targeted followup experiments to confirm the proposed regulation and their molecular relationships. Please note that aspects relating to phago-lysosomal fusion mechanisms were not investigated in this work, but the M. tb-driven inhibition of this event is well supported by existing literature (see text for more). It remains to be investigated if the proposed regulation of lysosomal exoglycosidases is related to the attenuation of the phago-lysosomal fusion event.

Table 1. Protein regulation of glycosylation enzymes (glycosidases and glycosyltransferases) and lectins involved in the N-glycosylation machinery and in glycan recognition. Infection-induced fold changes (≥2) are colour coded (red, down-regulation and green, up-regulation) and significance levels indicated; *** P < 0.0001, ** P < 0.01, * P < 0.05, not significant (ns) P ≥ 0.05. ND, not detected. ^Please see Supplementary Table S-2 and Supplementary Table S-3 for further details of the identified peptides/proteins and their spectral counts, as well as quantitative information of the fold change and the statistical significance of the reported proteins. 30 ACS Paragon Plus Environment

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Table 2. N-glycome profiles of M. tb infected (I) and uninfected (UI) MPs and CLs. *Key to glycan types: “P” denotes paucimannose-type glycosylation, “C” is complex-type, “H” is hybrid-type and “HM” is high-mannose-type glycosylation. The glycan IDs correspond to the structures presented in Fig. 3. **This glycan precursor is here classified as a high mannose type structure. ‡Glycans that were significantly regulated are indicated with * (P < 0.05, student t-test, type 2), please see Supplementary Table S-2 for details regarding the relative abundance and statistical significance of the reported N-glycome regulation.



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Table 1

Protein name (subcellular location, if known)

UniProtKB Function/Activity (Gene)

I-CL : UI-

I-MP : UI-

CL^ Fold P

MP^ Fold P

change Lysosomal exoglycosidases (hydrolytic/degrading) Lysosomal α-mannosidase O00754 Cleaves terminal α1,2/3/6-Man

change

4.1x ↓ *** 1.7x ↑

ns

(lysosome) β-mannosidase

(MAN2B1) of N-glycoproteins O00462 Cleaves terminal β-mannose of

10.7x ↓ **

1.6x ↓

ns

(lysosome) β-hexosaminidase subunit α

(MANBA) N-glycoprotein P06865 Cleaves terminal β-HexNAc

20.0x ↓ *** 2.3x ↓

ns ns

(lysosome) β-hexosaminidase subunit β

(HEXA) P07686

of glycoproteins/glycolipids Cleaves terminal β-HexNAc

25.6x ↓ *** 1.5x ↑

(lysosome) β-galactosidase

(HEXB) P16278

of glycoproteins/glycolipids Cleaves terminal β-Gal of

18.7x ↓ ***

ND

(lysosome) Tissue α-L-fucosidase

(GLB1) P04066

N-glycoprotein and glycolipids Cleaves terminal α1,6-Fuc of

4.0x ↓

ND

(lysosome) Lysosomal α-glucosidase

(FUCA1) N-glycoproteins P10253 Cleaves terminal α1,4/6-Glc

*

12.5x ↓ ***

ND

(lysosome) Glucosylceramidase

(GAA) P04062

of glycogen and glycans Cleaves terminal β-linked Glc

18.7x↓ ***

ND

(lysosome) α-galactosidase A

(GBA) P06280

of glycolipids Cleaves terminal α-Gal

20.0x ↓ ***

ND

(lysosome) β-glucuronidase

(GLA) P08236

of glycolipids Cleaves β-glucuronosides

6.7x ↓

*

ND

(lysosome) α-N-acetylgalactosaminidase

(GUSB) P17050

of dermatan/keratin sulfates Cleaves terminal α1,3-GalNAc

6.0x ↓

*

ND

(lysosome) α-N-acetylglucosaminidase

(NAGA) P54802

of N-glycoproteins/glycolipids Cleaves terminal α-GlcNAc

5.0x ↓

ns

ND

4.0x ↓

ns

ND

(lysosome) (NAGLU) of heparan sulfate ER/Golgi glycosidases (biosynthetic) α-mannosidase 2 Q16706 Cleaves terminal α1,3/6-Man (Golgi) Mannosyl-oligosaccharide

(MAN2A1) of N-glycoproteins P33908 Cleaves terminal α1,2-Man of

3.0x ↓

ns

ND

1,2-α-mannosidase IA (Golgi) Neutral α-glucosidase AB

(MAN1A1) high mannose N-glycoproteins Q14697 Cleaves terminal α1,3-Glc

1.1x ↑

ns

1.3x ↑

ns

(Golgi) Glucosidase 2 subunit β (GLU2B)

(GANAB) of maturing N-glycoproteins P14314 Cleaves terminal α-glucose

1.3x ↑

ns

4.2x ↑

*

(ER) Mannosyl-oligosaccharide

(PRKCSH) of maturing N-glycoproteins Q13724 Cleaves terminal α1,2-Glc

1.8x ↑

ns

3.3x ↑

ns

glucosidase (ER) ER/Golgi glycosyltransferases

(MOGS)

of maturing N-glycoproteins


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Dolichyl-diphosphooligosaccharideprotein glycosyltransferase 48 kDa

P39656

Subunit of the OST complex

1.0x → ns 10.7x ↑ ***

(DDOST) which initiated the

subunit (ER) Dolichyl-diphosphooligosaccharide-

P46844

N-glycosylation machinery Subunit of the OST complex

protein glycosyltransferase subunit

(RPN2)

which initiated the

2 (Ribophorin-2 (ER) Dolichyl-diphosphooligosaccharide-

P46977



(STT3A)

which initiated the

STT3A (ER) Dolichyl-diphosphooligosaccharide-

P61803



(DAD1)

which initiated the

(ER) Dolichyl-diphosphooligosaccharide

P46843



(RPN1)

which initiated the

1 (Ribophorin-1) (ER) β-1,2-N-acetylglucosaminyl-

Q10469

N-glycosylation machinery Catalyzes formation of complex

1.8x ↑

ns

5.2x ↑ ***

1.2x ↑

ns

4.3x ↑

*

3.1x ↑

*

4.0x ↑

ns

2.0x ↓ *** 4.7x ↑ ***

6.0x ↓

*

ND

ns

ND

transferase II (GnT-II) (Golgi) α-(1,6)-fucosyltransferase

(MGAT2) N-glycoproteins Q9BYC5 Catalyzes formation of core

4.7x ↓

(Golgi) UDP-glucose:glycoprotein

(FUT8) fucosylated N-glycoproteins Q9NYU2 Re-glucosylates slightly

25.0x ↓ ***

glucosyltransferase 1 (ER) (UGGT1) misfolded N-glycoproteins Lectins Cation-independent mannose-6- P11717 Responsible for glycoprotein phosphate receptor (Golgi/lysosome) Cation-dependent mannose-6-

ND

1.9x ↓ *** 1.1x ↓

ns

(IGF2R) P20645

trafficking to lysosome Responsible for glycoprotein

1.5x ↑

ns

2.8x ↑

**

phosphate receptor (Golgi/lysosome) (M6PR) Galectin-1 P09382

trafficking to lysosome Binds β-Gal and other glyco-

1.4x ↑

ns

1.8x ↓

**

(secreted) Galectin-3

(LGALS1) P17931

epitopes in many processes Galacto-specific lectin which

1.6x ↑

ns

3.7x ↓

ns


(LGALS3) P47929

binds IgE in cellular processes Involved in cell-cell and cell- 4.0x ↓

*

4.3x ↓

ns


(LGALS7) O00182

matrix interactions Binds galactosides in many

1.0x → ns

(secreted) Sialoadhesin

(LGALS9) Q9BZZ2

cellular processes Mediates sialic acid-dependent

6.0x ↑

*

ND

(cell membrane/secreted) Malectin

(SIGLEC1) antigen presentation and binding Q14165 Binding to immature 2.2x↓

ns

2.0x ↑

(ER) C-type mannose receptor 2

(MLEC) N-glycoprotein (quality control) Q9UBG0 Binds and internalizes

*

ND

(cell surface)

(MRC2)

5.3x ↓

5.2x ↑ ***

ns

mannosylated ligands



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 42

Table 2

Glycan RT # (min) m/z 1 38.1 749.3 2 50.7 895.4 3 45.4 911.4 4 52.9 1057.5 5 48.6 1114.5 6 48.3 1235.5 7a 44.9 1260.5 7b 55.5 1260.5 8 41.8 698.3 9 47.1 731.3 10a 40.7 779.3 10b 41.5 779.3 11 44.4 799.8 12 46 820.3 13a 40.8 860.3 13b 41.4 860.3 14 43.6 864.3 15 51.4 893.4 16 41.5 941.4 17a 48 945.4 17b 55.4 945.4 18a 49.6 965.9 18b 56.7 965.9 19 42.6 994.9 20 43.4 1022.4 21a 54.1 1038.9 21b 61 1038.9 22 47 1039.4 23a 50.5 1111.4 23b 58.7 1111.4 23c 62.5 1111.4 24 51.1 1112 25 49.5 1148.4 26a 55.2 1184.5 26b 62.1 1184.5 26c 63.6 1184.5 27a 58.2 1221.4 27b 61.7 1221.4 28 61.8 1294 29 62 1367 30a 61.4 1439.5 30b 63.5 1439.5

Z 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

Obs mass (Da) 750.3 896.4 912.4 1058.5 1115.5 1236.5 1261.5 1261.5 1398.6 1464.6 1560.6 1560.6 1601.6 1642.6 1722.6 1722.6 1730.6 1788.8 1884.8 1892.8 1892.8 1933.8 1933.8 1991.8 2046.8 2079.8 2079.8 2080.8 2224.8 2224.8 2224.8 2226.0 2298.8 2371.0 2371.0 2371.0 2444.8 2444.8 2590.0 2736.0 2881.0 2881.0

Theo mass (Da) 750.3 896.3 912.3 1058.4 1115.4 1236.4 1261.5 1261.5 1398.5 1464.5 1560.5 1560.5 1601.6 1642.6 1722.6 1722.6 1730.6 1788.6 1884.6 1892.7 1892.7 1933.7 1933.7 1991.7 2046.7 2079.7 2079.7 2080.8 2224.8 2224.8 2224.8 2225.8 2298.8 2370.8 2370.8 2370.8 2444.9 2444.9 2589.9 2736.0 2881.0 2881.0

Δm Monosacch. composition (Da) Hex HexNAc Fuc NeuAc 0.0 2 2 0.1 2 2 1 0.1 3 2 0.1 3 2 1 0.1 3 3 0.1 5 2 0.0 3 3 1 0.0 3 3 1 0.1 6 2 0.1 3 4 1 0.1 7 2 0.1 7 2 0.0 6 3 0.0 5 4 0.0 8 2 0.0 8 2 0.0 5 3 1 0.2 5 4 1 0.2 9 2 0.1 6 3 1 0.1 6 3 1 0.1 5 4 1 0.1 5 4 1 0.1 5 5 1 0.1 10 2 0.1 5 4 1 1 0.1 5 4 1 1 0.0 5 4 3 0.0 5 4 2 0.0 5 4 2 0.0 5 4 2 0.2 5 4 2 1 0.0 6 5 1 0.2 5 4 1 2 0.2 5 4 1 2 0.2 5 4 1 2 -0.1 6 5 1 1 -0.1 6 5 1 1 0.1 6 5 2 0.0 6 5 1 2 0.0 6 5 3 0.0 6 5 3

UI-CL I-CL Rel abundance Type* Av SD Av SD P 4.5 0.9 3.2 1.2 P 23.0 1.3 23.2 3.8 P 9.6 1.2 4.2 0.3 P 13.0 0.7 9.6 1.6 C 0.5 0.2 HM 3.8 0.8 3.5 1.6 C 0.3 0.2 0.6 0.3 C 1.0 0.3 1.6 0.5 HM 3.9 0.6 3.4 1.4 C 0.4 0.3 2.0 0.9 HM 5.8 1.3 5.4 0.6 HM 1.5 0.4 1.6 0.2 H 1.4 0.7 1.5 0.2 C 0.9 0.2 0.2 0.2 HM 6.2 1.3 8.4 0.6 HM 1.5 0.5 0.7 0.1 H 0.8 0.1 C 1.3 0.5 1.4 0.5 HM 13.3 1.3 11.3 0.9 H 1.7 0.6 0.8 0.7 H 0.4 0.4 C 1.5 0.5 1.3 0.3 C 0.6 0.7 0.6 0.6 C 0.4 0.0 HM** 1.7 0.6 1.9 0.4 C 3.1 1.2 4.5 1.2 C C 1.4 0.5 C 0.1 0.1 C 0.3 0.3 C C 1.4 0.5 C C 0.8 0.4 C 1.1 0.3 C C 0.8 0.3 C 0.4 0.4 C C 0.5 0.1 C 0.3 0.5 C 100.0 100.0

P‡

* * *

*

* * * * *

*

*

* * * *

*

UI-MP I-MP Rel abundance Av SD Av SD P‡ 0.6 0.1 0.5 0.2 4.8 0.9 4.7 1.6 2.4 0.2 2.1 0.5 4.0 0.8 4.5 0.3 0.2 0.2 0.1 0.2 5.5 1.4 4.9 1.2 0.0 0.1 0.3 0.0 * 0.6 0.2 1.0 0.2 * 4.8 0.7 3.9 0.4 0.3 0.5 0.5 0.2 4.2 0.4 4.7 0.7 2.2 0.9 1.4 0.2 2.0 0.6 2.1 0.4 1.0 0.2 1.1 0.2 8.1 1.4 6.2 0.6 * 0.8 0.1 0.6 0.1 1.6 0.3 1.4 0.2 2.5 0.5 2.5 0.4 12.2 2.2 13.7 1.2 1.8 0.7 2.9 0.4 * 1.1 0.3 0.7 0.1 2.6 0.4 3.0 0.8 1.3 0.4 1.2 0.5 0.6 0.2 0.4 0.0 1.3 0.4 2.2 0.5 * 7.0 1.2 5.6 0.6 2.5 1.1 2.0 0.6 1.1 0.6 2.1 0.5 * 2.0 0.8 1.7 0.6 4.3 1.9 4.1 1.9 1.2 0.5 0.8 0.2 2.7 0.7 3.7 0.7 0.2 0.4 1.4 0.5 1.4 0.2 3.2 1.6 3.2 0.5 1.7 0.9 0.6 0.3 0.3 0.4 0.8 0.3 0.7 0.2 * 1.4 1.5 1.7 0.4 0.7 0.4 0.4 0.1 1.2 0.9 2.2 1.2 2.7 2.2 2.6 1.1 100.0 100.0


Page 35 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1

Human macrophages (PMAdifferentiated THP-1 cells)

M. tb infection

Incubation (72 h)

(4 h)

No infection (4 h)

Incubation (72 h) 24 h 48 h 72 h

Infected cell lysate (I-CL) Infected microparticles (I-MP) Infected culture media (I-CM) Uninfected cell lysate (UI-CL) Uninfected microparticles (UI-MP) Uninfected culture media (UI-CM)

- N-Glycomics (CL, MP) - Proteomics (CL, MP)* - N-Glycopeptide analysis (CL)* - qPCR of glycosylation enzymes (CL) - Hexosaminidase activity assay (CM)



Figure 2

(A) α6

α6

β4

β4

β4

β2

β2

β2

β4 β2

α3

α6

α3 β4

α-neuraminidase β4

(Not observed)

α6

- Asn -

β2

β2

α6

α3

β4

α6

β4

β-hexosaminidase α/β β4

α6

α6

β4

β-galactosidase β4

α3

β4

- Asn -

α-mannosidase β4

α6

- Asn -

β-mannosidase β4

α6

- Asn -

β4

α6

- Asn -

α6

- Asn -

Time post infection (h)

4000

3000

10.7x ↓**

MAN2B1

MANBA

HEXA

*

HEXB


Total Hex (A: αβ and B: ββ) activity

ns


(D) 400

UI-CM UI-SN I-CM I-SN

2000

1000

ns

300

MANBA_HUMAN

*


Hex A (αβ) activity

Copies/100,000 18SrRNA

4.1x ↓***

Hex A activity

Total Hex activity

(C)

MA2B1_HUMAN

25.6x ↓***



*

HEXB_HUMAN

20.0x ↓***

(4-MU release, arb. values)

GLB1

HEXA_HUMAN


(72 h)

18.7x ↓***


Proteome

BGAL_HUMAN

Transcriptiome

Glyco-enzyme regulation

(B)

(4-MU release, arb. values)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 42

UI-CL I-CL *


***

UI-SN UI-CM I-SN I-CM

200

* 100 0

0 24 h

48 h

Time post infection

72 h

24 h

48 h

72 h

Time post infection


Page 37 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3 Precursor

α6 α6

α6 α3

α3/6 α3/6

23a

20

α6

α3/6 α3/6 α3/6 α3/6

23c

23b

α3

α6

α3

α3

18b

18a

α3/6 α3/6

α3/6

12

α6 α6 α6

α6 α6

α3/6

α3/6

30a

28

25

α6 α6 α3

α3/6

30b

High mannose Complex

16

13a 13b α6

α6

8

10a 10b

6

Paucimannose

α3/6

α3/6

Chitobiose

5 1

3

Hybrid

α3

d

c

a

b 4 17a

14

17b

2

7a 7b

11

Key

α6

Glc

α2 α2 α2 α2 α3/6

19 α6 α6

α6 α3

α3/6

26a

α3/6

22

α3/6 α3/6

21a

α3

α3/6

21b

15

Man Gal Neu5Ac Fuc

α6 α6

α3/6 α3/6

27a

GlcNAc

β4/6

β4 α6

α6

α3/6

β2

α3 α6 β4

β4

9

α3

α6

α3/6

26c

α6 β2

α3 α6 β4

24 α3 α3

26b

α3/6

α3

β4 β4 β4

α3/6

27b

α3/6

α3/6

29



Figure 4

*

THP-1 UI-CL Uninfected Lysate THP-1 I-CL Infected Lysate

β2

β4

30%

*

0% High mannose

(C) 80%

Hybrid

Complex

40% 20% 0%

(E)

Paucimannose

Chitobiose

(F)

100% 80%

β2

β2

α3 β4

α6

20%

β4

Asn

0%

Degree of core fucosylation

β2

β2

Fut8 (4.8x↓) Golgi

40%

UniProt Q13510 P08236 P06865 P07711 P20645 P11717 P08962 P11117 P10253

P11279 P13473

ns

60%

α-fucosidase (4.0x↓) Lysosome

No overall regulation

*

8% 0%

Golgi

Glycan #4

β4 α6

Hex α/β (20.0/25.6x↓)

Asn 4% 2% 0%

α6

Glycan #7a-b

Lysosome

β4

α6

Asn 4%

*

2% 0%

Glycan #9

(D)

60%

Complex

Lysosome

β4

α3

Overall direction of regulation

16 lysosomal N-glycoproteins

Hybrid

16%

Paucimannose

Glycopeptide quantitation (all 47 N-glycoproteins)

High mannose

α6

Asn

20%

Mgat2 (6.0x↓)

α6

β4

Hex α/β (20.0/25.6x↓)

β2

β2

α3

β4

40%

10%

Rel. Abundance

α6

α3

Rel. abundance

50%

Complex Paucimannose

Rel. abundance

Rel. Abundance

60%

(B)

Glycan quantitation

Rel. abundance

(A)

Rel. Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 42

α3

P15586

α6

β4

P50897

β4

P07602

Asnα6

O00584 H7C469

Protein name Sites Glycoforms‡ Type* Acid ceramidase N259 1 HM β-glucuronidase N272 1 HM β-hexosaminidase subunit alpha (Hex α) N157 2 HM Cathepsin L1 N221 3 P, HM Cation-dependent mannose-6-phosphate receptor N57 4 HM, C Cation-independent mannose-6-phosphate receptor N400 2 HM CD63 antigen N130 1 HM Lysosomal acid phosphatase N167 1 HM Lysosomal α-glucosidase N390 1 P N470 1 HM N882 1 P Lysosome-associated membrane glycoprotein 1 N261 6 HM, C Lysosome-associated membrane glycoprotein 2 N275 2 C N356 7 HM, C N-acetylglucosamine-6-sulfatase N279 1 Ch N362 8 HM, C N405 3 P, HM Palmitoyl-protein thioesterase 1 N197 3 P, HM, C N212 5 P, HM Prosaposin N216 4 Ch, P N332 6 Ch, P, HM, C Ribonuclease T2 N106 3 Ch, P, HM Uncharacterized protein N63 4 Ch, P, HM


Page 39 of 42

Figure 5

(A) α2

α2

α3

α3

α3

α3

α3

α3 α2

α2

α2

α2

α3

α3

α6 α6

β4

OST complex DAD1 (4.0x*↑) STT3A (4.3x↑) RibI (4.7x↑) RibII (5.2x↑) OST48 (10.7x↑)

α2

β4

Quality control lectin

α2

α2

α6 α6

MLEC (2.0x*↑)

α2

α2

α3

α3

GLU2B (4.2x↑) MOGS (3.3x*↑)

β4

Dolichol

(B)

α2

α2

β4

α6

α3 β4

α2

α6

α3

GANAB (1.1x ↑)

Asn

*

(D)

α2

α2

β4

Asn

THP-1… UI-MP (C) 3% THP-1 Infected MP I-MP

α2

α2

α3

α3 β4

α6 α6

No significant changes in glycoenzymes in MPs

β4

Asn

50%

1.0E+08

5.0E+07

2%

1%

Rel. abundance

Rel. abundance

1.5E+08 Total EIC intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


40% 30% 20% 10%

0%

0.0E+00

N-glycan density Total intensity

0%

N-glycan Strictly immature highprecursor mannose precursor High mannose (GlcNAc2Man9Glc1)

Hybrid

Complex

Paucimannose



Figure 6

**

40%

20%

50%

Hybrid

THP-1 I-CL Infected Lysate THP-1 I-MP Infected MP

Complex

Paucimannose

***

***

30% 20%

*

10% High mannose

Hybrid

25%

0%

60%

15%

40%

10%

150% 100% 50%

5%

0%

100%

* *

**

40%

80% 60%

40% 20%

Complex

Paucimannose

0%

0%

Total terminal galactosylation

Total α2,6α2,3sialylation sialylation sialylation

Total α2,6α2,3sialylation sialylation sialylation

0%

*

20%

80%

Rel. abundance

High mannose

*

20%

40%

0%

*

Rel. abundance

50%

**

*

10%

(D)

100%

*

100%

30%

0%

(C) Rel. abundance

**

50%

150%

Rel. abundance

(B)

THP-1 UI-CL Uninfected Lysate THP-1 UI-MP Uninfected MP

Rel. abundance

Rel. abundance

60%

Rel. abundance

(A)

Rel. abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 42

Total terminal galactosylation

Total terminal GlcNAcylation

**

30% 20% 10% 0%

Total terminal GlcNAcylation


Page 41 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7

MP proteins (sequestered from plasma membrane with increased ER proteins upon M. tb infection)

Immature N-glycans N-glycan density N-glycan profile (differs from host cell but unchanged upon M. tb infection)

MP (100-1000 nm)

OST Complex (N-glyco-initiation) OST48 Ribophorin I Ribophorin II STT3A DAD1 MP formation

Sialoadhesin

Classical secretory pathway

Lysosomal N-glycoproteins

Lysosomal Lysosomal exoglycosidases N-glycoprotein β-mannosidase truncation β-hexosaminidase α/β α-mannosidase Lysosome β-galactosidase Release Etc.. Fusion of lysosomal inhibited exoglycosidases by M. tb upon infection

Quality control (UGT1)

Golgi ER

N-glycome (cell lysate) Complex-type Paucimannose-type

Phagocytosis

Hex A activity

M. tb

Lysosomal trafficking (CI-M6PR)

Mannose receptor

Phagosome

Macrophage

Exoglycosidase gene expression MANBA MAN2B1 HEXA HEXB GLB1

Nucleus



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 42

TOC Graphics • Glycomics • Proteomics

Microparticles

• Glycopeptide analysis • qPCR and activity assay of exoglycosidases

Oligosaccharyl---transferase complex

Exoglycosidases

Golgi

ER

Lysosomes Secretion

M. tb

Glyco-genes Phagosome

Human Macrophage

Nucleus


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