WCBP Pauline M Rudd Dublin-Oxford Glycobiology group. Conway Institute

Transcription

1 WCBP linked glycans Pauline M Rudd Dublin-xford Glycobiology group Conway Institute From Back Eoin Cosgrave, Weston Struwe, scar Potter, Michael Schomburg, Patrick Jennings, Karina Marino, Jayne Telford, Niaobh McLoughlin, John Rourke, Barbara Keegan, Ciara McManus, Mark Hilliard, Jayesh Kattla, Rebecca Duke, Barbara Adamczyk, Radka Fahey, Joanne Withers Missing from group photograph - Eugene Dempsey, Margaret Doherty, Natalia Artemenko, Tharmala Tharmalingam, Marie Galligan, Giorgio Carta

2 Carbohydrate Peptide linkages in Glycoproteins Nt N-Glycan Asn-X-Ser - GalNAc CXSXPC -glucose Human erythrocyte CD59 CXXGG(S/T)C -fucose Ser - GalNAc Thr Gelatinase B -linked glycan Ser/Pro/Thr/Ser domain N-linked glycans Asn-X-Thr Asn C Et P N-Glycan N Glycan -Inositol EP P N-linked glycan GPI anchor -linked glycan tpa CC

3 Biosynthesis of -glycans in Golgi Specific transferases add initial monosaccharide (GalNAc) to Ser/Thr -H groups accessible on fully folded protein GalNAc Gal GlcNAc Ser/Thr Core I Core II Core III Core IV. XII Sequential additions of monosaccharides by other transferases give branching, elongation and termination

4 Major -glycan core structures Core 2 Core 1 - Gal(β1,3)GalNAc Core 2 - Galβ1,3[GlcNAcβ1,6]GalNAc Core 3 - GlcNAc(β1,3)GalNAc Core 4 - GlcNAcβ1,3[GlcNAcβ1,6]GalNAc

5 Symbolic notation- embedded monosaccharide sequence, linkage and anomericity pen symbols: hexose Filled in: HexNAc GlcNAc Gal Linkage position Antigenicity Blood Group Antigens A GalNAc Fuc (deoxy galactose) NeuNAc 3 2 Linkage type β-linkage k α-linkage unknown B

6 Functions for -glycans -linked glycans protect hinge IgD: a secreted and membrane bound glycoprotein IgD forms part of the B-Cell Receptor antigen capture mechanism on lymphocytes 6 13/11/ :44

7 T cell -linked glycans extend peptide linkers: CD8 80A 140 A CD8αβ MHC I Lec CH K1 APC Merry, Gilbert, Wormald, Dwek, Classen, Rudd, Davis J. Biol. Chem. P values (shape) s values (size)

8 Recognition: siga binds pathogenic gut bacteria Secretory component 7N-links -glycosylated hinge region -glycosylated hinge region sig 4N-links A J-chain I N-link siga 4 N-links

9 Secretory IgA -glycans Galβ1-3GlcNAc GU 10

10 siga -glycans bind bacteria E.coli Actinomyces Naeslundii Helicobacter pylori

11 Effect of glycosyltransferase changes on -glycan processing control cells tumour cells Extended d glycan Core 2 β6gnt Core 2 β6gnt Truncated glycan α6st1 SLe x Elongation of 1,6 GlcNAc arm - expression of Lewis structures Stop signal for β3galt Premature termination on both arms -SialylTn antigen

12 Loss of Golgi organisation: may relate to ph changes and contribute to glycan changes in malignant cells or culture Healthy controll cancer Golgi apparatus disorganised i d in cancer cells T, Tn, STn cancer associated Absent / low carbohydrate Ag Golgi ph6: normal pathway expression High Golgi ph in tumour cells may: be a result of reduced proton pump activity be sub-optimal for glycosyltransferase activity interfere with ER / Golgi recycling High levels Ser/Thr Ser/Thr Ser/Thr T Tn Sialyl Tn KelloKumpu et al, FEBS (2002) 516;

13 Site occupancy: verexpression of MUC-1 with increased - glycosylation aids detachment in breast cancer MUC-1 ubiquitous cell surface expression increased sialylation MUC-1 -glycan site occupancy doubled

14 Challenges No enzymes available for generic release of linked glycans glycosidase removes: Gal(β1 3)GalNAc Butrequires exoglycosidases to remove other sugars from core NeuAc GalNAc β1,3 Ser/Thr +Sialidase Gal NeuAc glycosidase Chemical methods available Hydrazine Ammonia based β elimination Ser/Thr

15 LC-MS: -glycan Release Chemistry -glycan Chemical Release: R 2 H R 1 AcNH X H -H H 2 Rc NHR n -H Aldose H R 1 R 2 AcNH H Reductive β-elimination NaBH 4 R 2 H Alditol R 1 H AcNH H Fluorescent labelling not possible R 2 H R 1 AcNH X Rc NHRn H 2 H R 1 H R 2 H AcNH -H Peeling Product H H AcNH R 1 H + R H 2 Further degradation Non-reductive β-elimination

16 A ptimization of β-elimination release for Bovine Submaxillary Mucin -glycans β-elimination ptimization with Bovine Submaxillary Mucin Total Area % Peeled Temperature ( o C) Time (Hours) Area (μv*sec) 6e+8 5e+8 4e+8 3e+8 2e+8 1e Temperature ( o C) Time (Hours) 0 1e+8 2e+8 3e+8 4e+8 5e+8 6e+8 Total % of Peaks 2-5 Total area unpeeled glycans Peeling: Blue: minimum Recovery: Red maximum Tharmala Tharmalingam, Li Liu, Jodie Abrahams, Margaret Doherty, Jonathan Tharmala Tharmalingam, Li Liu, Jodie Abrahams, Margaret Doherty, Jonathan Bones, Niaobh McLoughlin

17 Evaluation of Immobilisation

18 ptimised -glycan release Cervical Mucin Highly reproducible, robust basis for glycan analysis Used for Mucins Bovine Cervical Porcine Ileum and Colon Chicken mucins

19 Robotic compatible platform for -glycan release and labelling Ammonium hydroxide saturated 3. Evaporate with Ammonium carbonate ammonia Glycoprotein sample 1. Dehydrated mucins Wash 2. Release glycans Incubate 65 o C for 16 hours 4. Salt cleanup Carbon filter to clean up salts Waste 1 Dry 5. Formic Acid Treatment 10. Structure Assignment Compare GU values with Glycobase Fluorescence Fluorescence fluorescent label Time (mins) 8.NPHPLC profile of glycan pool 7.Elute glycans Transfer to SPE plate autogu cence Fluoresc Fluorescence 9. Exoglycosidase digestions Dry, redissolve in set volume Time (mins)

20 Exoglycosidase Digestions: -glycans Bovine cervical mucins

21 Preliminary assignments for bovine cervical mucin -glycans - HPLC

22 Unicarb DB: Glycan LC MS Database Glycan Structure LC-MS Details Biological Information References Composition Sequence Information Hayes C., Campbell, M., Struwe W., Karlsson, N., Rudd, PM et al. Bioinformatics, 2011 Possible Structural Isomers Giorgio Carta John Rourke

23 LC-MS: -glycan Sample Prep Workflow -glycoprotein gy Purification Glycan Release - Reductive β-elimination: 50 mm NaH and 0.50 M NaBH 4 at 50 C for 16 h Peptide Removal and Desalting: DWEX AG-50 cation exchange resin on the top of a C18 Zip-tip (Millipore). (resin was washed with 1 ml MeH, primed with 1M HCl and equilibrated with 1 ml H 2 ) Borate Removal: 5 washes of 60 μl 1% acetic acid in MeH glycan samples were reconstituted in 10 μlh 2 prior to LC-MS analysis LC-MS Analysis Analysis of glycan alditols performed in negative mode using an ion trap mass spectrometer with an Agilent 1100 HPLC binary pump and HTC-PAL autosampler. HPLC analytical columns consisted of 5 μm porous graphatized carbon in a fused silica capillary (20 cm x 180 μm i.d.; id;od o.d. 375μm) μm). The solvent flow rate was between 8-12 μl per minute. Structural Annotation glycan analysis was performed with the aid of the Unicarb-DB glycan database ( com) Weston Struwe

24 Mucin -glycans from Chicken Intestines Analysis of chicken intestinal mucin glycans: C. jj jejuniis a commensal bacteria in chickens Chicken mucin attenuated bacterial binding & is attributed to mucin glycosylation (Alemka et al.) Large intestine inhibited C. jejuni binding andinternalization better than small Intestine and caecum Goal: To Identify glycosylation differences in the chicken intestinal tract as it pertains to C. jejuni colonization

25 Chicken Mucins are Highly Sulphated and Sialylated H 301 H H NHAc H H 315 H H H NHAc H 505 H H H S4- H S4- H NHAc H H H H NHAc

26 Mucin -glycans are extended core III and core IV Large Intestine contains more glycan diversity More charged structures in the LI Large Intestine contains N-glycolylneuranimic acid Sulphation is present throughout 18 structures present in Large ageintestine e 13 structures present in Small Intestine 13 structures present in Caecum

27 FDA Approved Glycoprotein Biomarkers PRTEIN/MARKER α-fetoprotein (AFP) CA 125 (MUC16) CA 15.3 (MUC1) CANCER TYPE gastric, liver, testicular gastric, lung, ovarian breast CA colorectal, l gastric, liver, pancreatic Carcinoembryonic antigen (CEA) Complement factor H protein Human chorionic gonadotropin-β Kallikrein 3 (prostate specific antigen, PSA) Prolactin Thyroglobulin ErbB2, Her2/neu breast, colorectal, gastric, lung, pancreatic, thyroid bladder -glycosylated therapeutucs testicular prostate Follicle Stimulating Hormone Luteinising Hormone Von Willebrand factor Thyroid stimulating hormone endometrial carcinoma thyroid breast

28 Glycan analysis is complex The question determines the technological approach Marino, Rudd et al Nature Chemical Biology 2010 Instrument Acquisitions Agilent TLA scar Potter Beckmann CE Giorgio Carta Glycobase Key Personnel Jonathan Bones Mark Hilliard Stefan Weston Mittermayer Struwe Andras Guttman group Margaret Docherty Michael Schomberg