N-Linked Glycosylation on Non-Consensus Protein Motifs Alain Balland Analytical & Formulation Sciences, Amgen. Seattle, WA CASSS - Mass Spec 2010 Marina Del Rey, CA. September 8 th, 2010
Outline 2 Consensus N-linked Glycosylation: Glycosylation of Asn in the sequence motif NXS/T where X is not Pro. Structural requirements also necessary ( Asparagine turn ). Non-Consensus Glycosylation Discovered on C H 1 domain of recombinant antibody and antibodies pooled from normal human serum Influence of primary sequence around the modification probed by site specific mutants Survey of non-consensus glycosylation sites on recombinant antibodies Protein sequences and structural features of non- consensus glycosylation Conclusion
Typical IgG2 structure with N-linked oligosaccharides in the CH 2 domain of Fc 2 V H C H1 V L Glycosylation occurs in the CH 2 domain on Asparagine (N) on the consensus sequence NST IgG1 and IgG2 can carry extra glycan structures due to presence of consensus site in CDRs C L C H2 C H3
RP mass analysis of the CEX acidic variants identifies HC population with 2 oligosaccharides 7 x10 4 3.75 3.5 3.25 3 2.75 2.5 2.25 2 1.75 1.5 1.25 1 0.75 0.5 0.25 0 + TIC Scan 051608_2.d A B 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5 20 20.5 21 21.5 22 22.5 23 23.5 24 24.5 25 25.5 26 26.5 B A John Valliere-Douglass
2 nd glycosylation site is localized in CH1 domain (Tryptic peptide H15 aa 151-213) HC Fab H15 H15 Tryptic Peptide 151 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSNFGTQTYTCNVDHKPSNTK 213 Reduced RP-MS Reduced papain RP-MS Tryptic digest peptide map V H15b Chymotryptic Peptide H V C H1 NSGAL N162 W/ 162 NSGAL 166 L C L C H2 C H3 Chymo- tryptic digest H 2 O 18 degly. chymotryptic digest
Product ion monitoring of tryptic map profile helps identify glycopeptide gy p 100 % G2F-Sa potential HexNac-Hex product ions (366.15 Da) 366.027_366.246 0.05Da 115 HexNac-Hex product ion spectrum (post PNGase F digestion) 0 116.00 118.00 120.00 122.00 124.00 126.00 128.00 130.00 132.00 134.00 136.00 138.00 140.00 142.00 AMG 393 AV sialidase trypsin Sm (Mn, 1x2) 2: TOF MS ES+ H15+G2F 100 366.039_366.2 0.05Da 777 % HexNac-Hex product ion spectrum (post sialidase digestion) 0 116.00 118.00 120.00 122.00 124.00 126.00 128.00 130.00 132.00 134.00 136.00 138.00 140.00 142.00 AMG 395 AV trypsin Sm (Mn, 1x2) 2: TOF MS ES+ H15+G2F Sa 100 366.023_366.212 0.05Da 123 H15+G2F H15+G2F-Sa % HexNac-Hex product ion spectrum 0 116.00 118.00 120.00 122.00 124.00 126.00 128.00 130.00 132.00 134.00 136.00 138.00 140.00 142.00 Time
Determination of N-linked site by deglycosylation in the presence of H 2 18 O Conversion of asparagine to aspartic by PNGase F in the presence of H 2 O 18 results in the incorporation of a 3 Da mass tag on the previously occupied asparagine 1 HO HO H 2 N O H O 18 H H 2 N O O PNGase F O NH O 18 H Glycan Gonzalez, J.et al., Anal Biochem. 1992 Aug 15;205(1):151-158
Deglycosylation of Antibody A in H 18 2 O results in 18 O incorporation at HC Asn 162 8 Valliere-Douglass et al (2009) J. Biol. Chem. 284, 32493-32506
Non-consensus glycosylation is observed on the IgG1 C H 1 domain peptide in human serum IgG2 IgG1 IgG2/IgG1 CH1 tryptic peptide amino acids 151-213 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK 9 Lectin enrichment of natural IgGs: Wesley Wang
Site specific mutations to probe influence primary sequence around modification 9/29/2010 C H 1 Consensus mutants C H 1 Non-Consensus Wild Type C H 1 Non-Consensus mutants (expected change) WNSS WNSG ANSG ( ) WNGT WNTG ( ) WNGS WNAG ( 0 ) Statistical analysis of glycoprotein sequence in PDB * Trp in -1 correlated with greater likelihood of occupancy in consensus motifs Ser/Thr in +2functions as proximal hydrogen acceptor, Thr correlated ltdwith greater likelihood of occupancy versus Ser Small hydrophobic amino acids (Ala) are preferred in +1, aromatic amino acids (Trp) correlated with non-occupancy * Petrescu et al., (2004) Glycobiology 14, 103-114 Site directed mutagenesis and expression in HEK293 cells: Mirna Mujacic and Pranhitha Reddy 10
Non-consensus glycosylation is conveniently quantified by reduced capillary electrophoresis C H 1 Wild Type and Mutants 2.00 2.00 3.5 1.4 1.4 40 1.75 1.2 3.0 1.2 35 1.50 LIF - Channel 1 1 HC 3.5 1.4 1.4 40 1.75 1.2 3.0 1.2 35 1.50 Heavy Chain C H 1Sequence WNSG / WNAG / ANSG / WNSS 1.0 1.0 2.5 30 1.25 1.0 1.0 2.5 30 1.25 RFU U RFU 0.8 0.8 25 2.0 1.00 0.6 0.6 20 0.75 1.5 0.4 0.4 15 0.50 1.0 0.2 0.2 10 025 0.25 0.5 0.0 0.0 5 0.00-0.2-0.2 0.0 0-0.25 LC NGHC -0.4-0.4-0.5 21.50 2 21.75 4 22.00 6 22.25 8 22.50 10 22.75 12 23.00 14 23.25 23.50 23.75 24.00 24.25 24.50 24 24.75 26 25.00 28 25.25 30-0.5 25.50 16 18 20 22 21.50 21.75 22.00 22.25 22.50 22.75 23.00 23.25 Minutes 23.50 Minutes 23.75 24.00 24.25 24.50 24.50 24.75 24.75 25.00 25.00 25.25 25.25 25.50 Minutes 0.8 0.8 25 2.0 1.00 0.6 0.6 20 C 1mutant 0.75 1.5 H post-hc 0.4 WNSS 0.4 98.30% 15 0.50 WNGT 1.0 97.81% 0.2 0.2 10 WNGS 97.84% 025 0.25 RFU RFU U 0.5 ANSG 3.15% 0.0 0.0 5 0.00 WNAG 1.18% -0.2-0.2 0.0 WNSG (wt) 0 0.62% -0.25 WNTG 0.55% Quantitation N-linked glycosylation by CE: Paul Kodama
Development of non-consensus glycosylation enrichment and detection strategies Remove consensus C H 2 glycans with endoglycosidase treatment Enrich non-consensus glycosylated species using lectin affinity chromatography Identify the modification sites in enriched populations by ion mapping and electron transfer dissociation (ETD) mass spectrometry Catalogue the 2 o structural motifs associated with nonconsensus glycosylation using IgG2 homology models Goal In-silico prediction of non-consensus glycosylation based on sequence homology and 2 o structure 12
Enrichment scheme results in the discovery of additional non-consensus Glycosylation y sites Lectin Enriched Antibody-A Did Not Have Detectable Glycosylated CH2 Glycopeptide Retention on Ricin Column is Thus Due to the Presence of Non-Consensus N-Links 13 Valliere-Douglass et al. (2010) J. Biol. Chem. 285, 1612-1622
Non-consensus N-linked site ID strategy Lectin enriched fractions are digested with endo-f2 Peptide maps run on nanomate LTQ-ETD with fraction collection Peptides modified with GlcNAc-Fuc are fragmented by CID-MS 2 and then by ETD-MS 3 CID-MS 2 N 14 ETD-MS 3 Endo-F2 GlcNAc = 203 Da digestion on modified aa
Non-Consensus Glycosylation Site Identification in Antibody A Lectin Eluate V L CDR L 1 15
9/29/2010 Sites of Glycosylation y on Antibody A Motif Modified Domain Sequence Residue Asn (Non Cons) N162 C H 1 VSWNSGAL Asn (NC) N360 C H 3 MTKNQVSLTC Asn (NC) N35 CDR L 1 SSSNENFL Asn (NC) N164 C L QSGNSQE Gln (C) Q106 V L TFGQGTR Asn (C) N296* C H 2 EQFNSTF 16
Non-consensus glycosylation occurs on loops and turns near transitions in secondary structure Antibody A Fab Antibody A Fc Asn 164 (9/9 loop) Gln 106 (11/12 loop) LC Asn 35 (11/14 loop) HC HC HC Asn 360 (3/3 turn) 17 IgG2 homology models generated from known crystal structure Catherine Eakin
The Non-Consensus sequence motif is a reverse Consensus motif with S/T in position (-2) 9/29/2010 Asn Ser Modified Residue Sequence Distance S/T to N (Angstroms) Distance N to S/T (Angstroms) N162 (C H 1) VSWNSGA 83 8.3 69 6.9 N360 (C H 3) MTKNQVS 8.2 13.1 N164 (C L ) QSGNSQ 7.2 6.3 Ser N35 (CDR L 1) SSSNENF 77 7.7 -- Distances Reported N (Consensus) PDB database* N X (S/T) -- 7.3 (avg) * Petrescu et al., (2006) Curr. Opin. Struct. Biol. 10, 980-
Conclusions Non-consensus glycosylation Found on antibodies from various recombinant expression systems as well as from natural human sources occurs on Asn residues preceded by S/T in the -2 position (reverse consensus motif (S/T)X N) is observed on loops and turns within 3 residues of a change in 2 o structure Can occur in CDR s; the consequences to potency are under evaluation The presence of non-consensus glycosylation on antibodies from human serum indicates it is likely not immunogenic The in-vivo half life and stability of recombinant mabs with non-consensus glycosylation in C H 1 domain are under evaluation Glycosylation can occur on Glutamine residues (Q-glycosylation) Enrichment strategy and sensitive mass spectrometry techniques instrumental in showing that protein glycosylation governed by more diversified requirements than previously appreciated 19
9/29/2010 Acknowledgements John Valliere-Douglass Boxu Yan, Lowell Brady Jennifer Kerr Wesley Wang Mirna Mujacic, Pranhitha Reddy Paul Kodama Catherine Eakin, Randy Ketchem Alison Wallace, Mike Treuheit Confidential l Internal Amgen Use Only 20