Figure 2.
Glycan composition and structure of the lymphoma-derived Fabs. Determination of the crystal structure of L14 and glycan analysis of Fabs L14 and L29 that have N-glycan sites in both the CDR loops and the FR. (A) Schematic representation of the Ig variable heavy (H) and kappa light (κ) chain pairs of L29 and L14 displaying the location of the AGSs and of the natural N57 glycosylation site. Sites are numbered according to the IMGT/V-QUEST numbering system. Distribution of each site is represented relative to CDR or FR and colored by predominant glycan composition. Pie charts are colored according to the proportion of oligomannose-type glycans, processed complex-type glycans, and no glycans at each site. At least 1 CDR AGS is always occupied by glycans terminating at oligomannose-type in both L14 and L29, whereas the site in FR3 of L29 is predominantly but not always occupied by complex glycans. (B) Site-specific glycan compositions detected for L29 and L14 as determined by liquid chromatography-mass spectrometry. Bars represent the relative abundance of each category of glycan. Oligomannose-type (green) glycans are categorized according to the number of mannose residues (M9-M5), hybrids by the presence/absence of fucose (F), and complex-type glycans (magenta) according to the number of N-acetyl hexosamine structures detected (HexNAc) and the presence or absence of fucose. The proportion of AGSs without a glycan attached is shown as a gray bar. (C) Structure of the L14 Fab at 1.65 Å resolution. The Ig heavy chain (HC) is light gray and the Ig κ light chain (KC) is dark gray. CDR loops are shown: KCDR1 (red), KCDR2 (yellow), KCDR3 (light blue), HCDR1 (magenta), HCDR2 (purple), and HCDR3 (blue). The amino acids Q44, N57, and E110 that interact with the glycan at position N38 are shown as sticks. Their carbon atoms are colored according to the CDR on which they are present, whereas their oxygen atoms are red and their nitrogen atoms are blue. The 2 resolved GlcNAc residues at N38 are colored by atom with carbon in green, oxygen in red, and nitrogen in blue. Atoms likely to form electrostatic interactions with the N38 glycan are shown as dashed yellow lines. The representation in the left panel is rotated by 100° on the y-axis and 20° on the x-axis in the right panel to visualize the contacts of the glycan to itself and to the protein. Maximum likelihood-weighted 2Fo-Fc electron density obtained for the glycan at N38 is shown (green mesh).

Glycan composition and structure of the lymphoma-derived Fabs. Determination of the crystal structure of L14 and glycan analysis of Fabs L14 and L29 that have N-glycan sites in both the CDR loops and the FR. (A) Schematic representation of the Ig variable heavy (H) and kappa light (κ) chain pairs of L29 and L14 displaying the location of the AGSs and of the natural N57 glycosylation site. Sites are numbered according to the IMGT/V-QUEST numbering system. Distribution of each site is represented relative to CDR or FR and colored by predominant glycan composition. Pie charts are colored according to the proportion of oligomannose-type glycans, processed complex-type glycans, and no glycans at each site. At least 1 CDR AGS is always occupied by glycans terminating at oligomannose-type in both L14 and L29, whereas the site in FR3 of L29 is predominantly but not always occupied by complex glycans. (B) Site-specific glycan compositions detected for L29 and L14 as determined by liquid chromatography-mass spectrometry. Bars represent the relative abundance of each category of glycan. Oligomannose-type (green) glycans are categorized according to the number of mannose residues (M9-M5), hybrids by the presence/absence of fucose (F), and complex-type glycans (magenta) according to the number of N-acetyl hexosamine structures detected (HexNAc) and the presence or absence of fucose. The proportion of AGSs without a glycan attached is shown as a gray bar. (C) Structure of the L14 Fab at 1.65 Å resolution. The Ig heavy chain (HC) is light gray and the Ig κ light chain (KC) is dark gray. CDR loops are shown: KCDR1 (red), KCDR2 (yellow), KCDR3 (light blue), HCDR1 (magenta), HCDR2 (purple), and HCDR3 (blue). The amino acids Q44, N57, and E110 that interact with the glycan at position N38 are shown as sticks. Their carbon atoms are colored according to the CDR on which they are present, whereas their oxygen atoms are red and their nitrogen atoms are blue. The 2 resolved GlcNAc residues at N38 are colored by atom with carbon in green, oxygen in red, and nitrogen in blue. Atoms likely to form electrostatic interactions with the N38 glycan are shown as dashed yellow lines. The representation in the left panel is rotated by 100° on the y-axis and 20° on the x-axis in the right panel to visualize the contacts of the glycan to itself and to the protein. Maximum likelihood-weighted 2Fo-Fc electron density obtained for the glycan at N38 is shown (green mesh).

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