1. The role of glycosylation in the assembly and transport of human CD1d glycoproteins Crina Paduraru

2. membrane glycoproteins encoded by genes outside the MHC cluster 2 groups: I - CD1a, b, c II - CD1d intermediate - CD1e present lipid-containing ligands (self or non-self) to T cells expressed as heterodimers: CD1 heavy chain (folded in  1,  2 and  3 domains) noncovalently associated with  2-microglobulin different intracellular localization determined by the presence of motifs in the cytoplasmic tail that can interact with adaptor protein (AP) complexes (except CD1a) From “CD1-restricted antigen presentation: an oily matter”, Joyce S and Van Kaer L, Cur Opin Immunol, 2003, 15:95-104 CD1 (cluster of differentiation 1)

3. the binding groove is more hydrophobic, narrower and deeper than in MHC I; differs structurally for each isoform, suggesting a different array of structurally diverse lipid-containing antigens that can be loaded CD1 (cluster of differentiation 1) From “Anatomy of CD1-lipid antigen complexes”, Moody DB, Zajong DM and Wilson IA, Nature, 2005, 5:387-399

4. member of CD1 family membrane glycoprotein with 4 occupied N-glycosylation sites and 6 Cys residues (2 disulfide bridges) correct folding is dependent on the glucose-trimming dependent interaction with CNX/CRT/ERp57 forms non-covalent heterodimers with  2-microglobulin in the ER after the heavy chain is released from the CNX/CRT cycle, but can be expressed poorly at the plasma membrane without it enters the endocytic system by AP2 dependent internalization from the plasma membrane and by trafficking in association with MHC class II presents lipid antigens to NKT cells; antigen loading occurs mainly in the endocytic system From “CD1 assembly and the formation of CD1-antigen complexes”, Hava DL et all, Cur Opin Immunol, 2005, 17:88-94 CD1d

5. Aim of the project investigate nature of the individual N-linked glycans of CD1d use mutagenesis to evaluate their role in assembly and transport of CD1d

6. 1. Single N-glycosylation mutants of CD1d Generation of single N-glycosylation mutants by mutating Asn from Asn-X- Ser/Thr to Ala (N18A (Δ1); N40A (Δ2); N106A (Δ3); N161A (Δ4)) Stable expression in B cell like line C1R – cloning methods of analysis: metabolic labeling, immunoprecipitation, FACS, NKT stimulation, HPLC, MS antibodies: mouse monoclonal D5 – recognizes free CD1d heavy chains mouse monoclonal 51.1.3 – recognizes heavy chain – β2- microglobulin dimers rabbit polyclonal anti human β2-microglobulin Computer generated model of human CD1d with monoglucosylated glycans (Dr. Adina Milac, Dr. Andrei Petrescu) 1 2 3 4 1 2 3 4 Ligand binding site β2m binding site

7. 1.1 All the single N-glycosylation mutants reach the plasma membrane as dimers with β2-microglobulin Extracelullar FACS with 51.1.3 (heavy chain-β2m) antibodies; 2 nd antibody: goat anti- mouse IgG fluor 10 0 1 2 3 4 FL1-H 0 20 40 60 80 100 % of Max 10 0 1 2 3 4 FL1-H 0 20 40 60 80 100 % of Max 10 0 1 2 3 4 FL1-H 0 20 40 60 80 100 % of Max 10 0 1 2 3 4 FL1-H 0 20 40 60 80 100 % of Max 10 0 1 2 3 4 FL1-H 0 20 40 60 80 100 % of Max WT CD1d Δ4 CD1d Δ3 CD1d Δ2 CD1d Δ1 CD1d Negative control 51.1.3 sample

8. 1.2 Kinetics of N-glycan maturation and the interaction with β2-microglobulin D5 51 D5 51 D5 51 D5 51 D5 51 EndoH - + - + - + - + - + - + - + - + - + - + Chase (h) 0 1 2 4 8 WT CD1d Δ1 CD1d Δ4 CD1d Δ3 CD1d Δ2 CD1d

9. 1.3 Association with β2-microglobulin is partly impaired for delta 1 and 2 WT CD1d 0 20 40 60 80 100 0246810 chase (h) % from total 0 20 40 60 80 100 0246810 chase (h) % from total Δ4 CD1d 0 20 40 60 80 100 0246810 chase (h) % from total Δ3 CD1d 0 20 40 60 80 100 120 0246810 chase (h) % from total Δ2 CD1d 0 20 40 60 80 100 120 0246810 chase (h) % from total Δ1 CD1d D5 – free heavy chains 51.1.3 – heavy chains associated with β2m Triton X 100 extraction

10. Pulse – 15 min; Chase – 4h 1.4 CD1d heavy chain – β2-microglobulin dimers are more stable in digitonin than in Triton X 100 Delta 2 heavy chain- β2-microglobulin dimers are unstable even in digitonin. D5 β2m D5 β2m D5 β2m D5 β2m D5 β2m Digitonin TritonX Digitonin TritonX Digitonin TritonX Digitonin TritonX Digitonin TritonX WT CD1d Δ1 CD1d Δ2 CD1d Δ3 CD1d Δ4 CD1d

11. Purification of steady state wt CD1d – β2-microglobulin complexes from C1R.CD1d cells on 51.1.3 column Purification on ConA RP-HPLC C18 column Collect non overlapping peaks Sequence the peptides (Edman sequencing) +/- EndoH 2. Identification of the EndoH sensitive glycan EndoH - + Trypsin digest

12. Glycan 2 is not processed to complex form TDGLAWLGELQTHSWSNDSDTVR glycosylation site 2 (Asn40) TDGLAXLGELQTHS TDGLAXLGELQTH

13. 3. N-glycan sequencing for wt CD1d and single N- glycosylation mutants Glycoprotein Enzymatic sequencing MS HPLC 2AB labeling PNGaseF release of the N-linked glycans WAX fractionation

14. GU 6 7 8 9 10 11 12 13 14 WT CD1d neutral glycans monosialylated glycans bisialylated glycans 3.1 WAX fractionation of 2AB-labeled N-glycans from WT CD1d

15. GU 5 6 7 8 9 10 11 12 13 14 WT CD1d WT CD1d ABS BTG WT CD1d ABS BTG GUH WT CD1d ABS BTG GUH BKF WT CD1d ABS Complex polylactoseamine, possibly bisected and core fucosylated, with 1 or 2 sialic acid residues 3.2 Identification of main 2AB-labeled N-linked glycans from WT CD1d by exoglycosidase digestions

16. WT CD1d Δ3 CD1d Δ4 CD1d Δ1 CD1d Δ2 CD1d 3.3 Comparison of WT CD1d and single N-glycosylation mutants N-linked glycans GU 6 7 8 9 10 11 12 13 14

17. Glycosylated CD1d – 3D structure 3D glycosylated model for human CD1d based on the crystal structure of mouse CD1d1 (Dr. Mark Wormald, Dr. Adina Milac, Dr. Andrei Petrescu) 1 4 3 2 Β2-microglobulin Ligand binding site Heavy chain GlcNAc Man Gal NANA Fuc

18. Conclusions: 1.Deletion of any of the four glycans does not prevent surface expression as active dimers with β2- microglobulin 2.When glycan 2 is deleted the CD1d-β2-microglobulin dimer is less stable than wild type and the other single mutants 3.Except glycan 2 all the other glycans are very accessible to glycosidases 4.Glycan 2 remains EndoH sensitive in wild type CD1d, predominantly as a high mannose structure (Man6) 5.Glycan 2 is proximal to the β2-microglobulin binding site 6.The data suggests that glycan 2 shields the CD1d- β2-microglobulin contact region

19. Future plans: Study double, triple and quadruple N-glycosylation mutants (and the mutation controls) activity kinetics of N-glycan maturation stability of the interaction with β2-microglobulin calnexin and calreticulin interaction arrival at the plasma membrane

20. Acknowledgements Immunobiology Department, Yale University Peter Cresswell Weiming Yuan All the cresswellians Glycobiology Institute, Oxford University Raymond Dwek Pauline Rudd Catherine Radcliffe David Harvey Louise Royle Mark Wormald Everyone from Rudd lab Institute of Biochemistry, Bucharest Stefana Petrescu Adina Milac Andrei Petrescu