Glycoproteins and N-glycans from exosomes Júlia Costa Laboratory of Glycobiology WP3: Exosome specific glycosignatures defining specificity in exosomes targeting GlioEx University Medical Center Hamburg Eppendorf 22 May 2014
The erythrocyte glycocalyx Roseman, 2001
Localizations and functions of glycoconjugates Blood and other fluids - Solubility - Stability - Recognition by carbohydrate-binding proteins Golgi apparatus Bacterial infection Cell-cell interactions - Metastasis - Inflammation Lysosomal targeting (Man6P) Nucleus Endoplasmic reticulum Lysosome Viral infection Quality control of glycoprotein folding Luminal/extracellular glycans Cytoplasmic/nuclear glycans Cell-matrix interactions Carrier proteins or lipids Glycan-binding proteins Extracellular matrix
N-glycosylation In N-glycosylation, glycans are covalently bound to asparagine side chains (NXS/T, X P) of proteins. In animal cells, the sugar linked to the asparagine residue is N-acetylglucosamine. trans-golgi and trans-golgi network Endoplasmic reticulum cis-golgi medial-golgi Oligosaccharyltransferase ER glucosidases I, II ER mannosidase Golgi mannosidases IA, IB, IC GlcNActransferase I Golgi mannosidase II GlcNActransferase II Fucosyltransferase VIII Galactosyltransferase Sialyltransferases Peripheral fucosyltransferases Mannose Glucose N-Acetylglucosamine Galactose Fucose N-Acetylneuraminic acid After Taylor and Drickamer, 2006
O-glycosylation In O-glycosylation glycans are covalently bound to the side chains of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side-chains. - Mucins - Proteoglycans - O-linked Man - O-linked GlcNAc
Examples of peripheral glycosylation linked to the LacNAc structure α2,6 α1,2 α1,3/4 6 SO 3 - α2,8 α2,3 β1,4 β1-r β1-4 α1,3 β1,4 N-Acetylglucosamine Galactose Fucose N-Acetylneuraminic acid
Multiple approaches for profiling a cell s glycome Derivatization Liquid chromatography Mass spectrometry (2009) Essentials of Glycobiology
Exosomes Théry et al. (2009) Nat. Rev. Immunol. 9, 581
Exosome separation Ovarian carcinoma SKOV3 cells 150 µm Centrifugation of cell supernatant at 500xg, 10,000xg, and 100,000xg (crude exosomes) 100,000xg pellet Escrevente et al. (2008) BBA 1780, 905
Exosome separation Exosc Exos 100 nm 100 nm Escrevente et al. (2013) PLOS One 8, e78631
Structural analysis of protein glycosylation Lectin blotting Immunoblotting Exosomes Glycoproteins Lectin affinity chromatography Peptide mass fingerprinting Glycan structural analysis Glycan release from tryptic glycopeptides: peptide N-glycosidase F (PNGase F) Glycan labelling: 2-aminobenzamide (2-AB) Glycan separation and structure elucidation: HPAEC-PAD, NP-HPLC, exoglycosidase digestion, mass spectrometry
Sialoglycoproteins from exosomes Ovarian carcinoma cell lines SKOV3 kda Ext Exos c Ext Exos c Ext Exos c Ext Exos c Ext Exos c Ext Exos c Ext Exos c Ext Exos c Embryonic kidney HEK293 Neuroglioma H4 MAL - NeuAcα2,3Gal; SNA - NeuAcα2,6Gal/GalNAc; kda Ext Exos c Ext Exos c kda Ext Exos c Ext Exos c Escrevente et al. (2011) BMC Can. 11, 108
Exosomes have specific glycosylation profiles in comparison with plasma membrane and microsomal fractions Ovarian carcinoma SKOV3 cells kda Ext MF PM IC Cyt Exos Ves c Maackia amurensis lectin (MAL) specificity: NeuAcα2,3Gal Sambucus nigra aglutinin (SNA) specificity: NeuAcα2,6Gal/GalNAc Wisteria floribunda (WFA) specificity: GalNAcβ4GlcNAc (LacdiNAc) Concanavalin A (Con A) specificity: α-mannosyl containing branched glycans
Sialoglycoproteins from exosomes Ovarian carcinoma SKOV3 cells Exosomes kda WB: CD9
MAL-binding glycoproteins from exosomes MAL-affinity purified IP: anti-lgals3bp kda Exos c -Ab -Exos c IP MAL Galectin-3 binding protein LGALS3BP Bands 4-8, 10 identified by a proteomics approach (peptide mass fingerprinting, LC-MS/MS) as galectin-3 binding protein. LGALS3BP is a secreted glycoprotein that has been found in the extracellular matrix and in body fluids and exosomes It has been found in several cancers where it has a negative prognostic value and is involved in cell adhesion
NP-HPLC of 2-AB-labeled N-glycans after PNGase F release 7x10 5 M5 M6 M7 M8 M9 Exosomes High mannose glycans 0 5x10 6 0 2x10 6 0 M5 M5 M6 M7 M8 M9 M6 M7 M8 M9 Microsomal fraction Plasma membrane 5 10 15 20 25 30 35 40 45 50 55 60 Mannose N-Acetylglucosamine Minutes Exosomes (23%) Microsomal fraction (41%) Plasma membrane (15%) Exosomes, microsomal fraction and plasma membrane have distinct N-glycosylation signatures.
Digestion of N-glycans from exosomes with exoglycosidases M: α-(1-2,3,6)-mannosidase S: α-(2-3/6)-sialidase G: β(1-3,4)-galactosidase F: α-l-(1-2,3,4,6)-fucosidase Sialic acid containing complex-type N-glycans Exosomes (77%) (approx NeuAc content: 170 pmol/µg total protein) Microsomal fraction (35%) Plasma membrane (26%)
Glycosidases used for structure analysis
MALDI-TOF MS of N-glycans Intens. 600 * 2028.8 * Exosomes 400 200 1257.5 1419.6 1663.7 1581.7 1743.8 1809.8 1850.7 1906.8 2012.9 2174.9 2377.9 2215.8 2539.9 2742.9 0 1500 1743.7 Microsomal fraction 1250 1000 750 1419.6 1581.7 1905.8 2012.9 1809.8 500 250 1257.5 1338.6 1485.7 1663.7 2174.9 2377.9 2028.8 2540.9 2742.9 2906.1 3108.1 3271.0 0 2028.8 Plasma membrane 600 400 200 1338.6 1419.6 1663.7 1809.8 2012.8 1581.7 1743.7 1905.8 2174.9 2377.9 2540.9 2744.0 0 1250 1500 1750 2000 2250 2500 2750 3000 3250 m/z *LC-ESI Ion Trap MS analyses; collision induced fragmentation; fragments identified corresponding to bisecting GlcNAc
Conclusions and perspectives Exosomes have characteristic glycan signatures. The sialoglycoprotein LGALS3BP is an abundant exosome marker. Does LGALS3BP and specific glycans play a role in interaction with target cells? Engineering of exosomes with respect to glycoproteins/glycosylation may improve their interaction with target cells. SKOV3 cell after internalization of CSFE labeled exosomes Escrevente (2012) BMC Can 11, 108
Acknowledgements Laboratory of Glycobiology Present: Margarida Gonçalves Gonçalo Silva Teresa Silva Former post-docs and students: Stefanie Glathe, Francisco Enguita, Carla Jorge, Vitor Sousa, Vanessa Morais, Angelina Palma, Catarina Brito, Catarina Gomes, Eda Machado, Rita Carilho, Ricardo Gouveia, Cristina Escrevente, Adriana Gomes, Joana Batista, Susana Jorge Collaborations in exosomes projects Harald Conradt, GlycoThera, Hannover Peter Altevogt, DKFZ, Heidelberg Paula Alves, Patrícia Alves, ITQB, IBET, Oeiras Funding (past 5 years) FCT, Portugal: PTDC/SAU-NEU/100724/2008; PIC/IC/82765/2007; SOPHIA, JPND/0003/2011 (EU Joint Programme Neurodegenerative Disease Research); GlioEx ENMed/0001/2013 (EuroNanomedII); PhD fellowships