Supplementary Figure 1. Heavy chain sequences of 2G1 and 8M2 aligned with V H 1-69
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1 Supplementary Figure 1. Heavy chain sequences of 2G1 and 8M2 aligned with V H 1-69 germline gene sequence. 2G1 and 8M2 acquired 14 and 18 mutations from the germline gene sequence, respectively. In 2G1, the mutations are mostly located in the HCDR1 and HCDR2 loops. Antibody residues that are in contact with HA are highlighted in red. The signature hydrophobic residues of the V H 1-69 germline, Ile53 and Phe54, are highlighted with red reverse triangles.
2 Supplementary Figure 2. Glycan binding analysis of recombinant escape mutants of H2 HA on the glycan microarray. (a) A/Japan/305+/1957 R137Q. (b) T193K. (c) G135D. (d) K156E. All error bars in the figure are indicative of standard deviation from quadruplicate measurements. Specific bindings for neutral glycans (1 and 2), α2-3- linked glycans (3 to 35), α2-6-linked sialylated glycans (36 to 56) and glycans of α2-6 and α2-3 mixed linkages (57 and 58) are colored in black, blue, red and cyan bars, respectively. The list of glycans on the array is provided in Supplemental Table 1. Note that the panels shown in this figure are on a different scale than Fig. 6B-C due to the relatively low avidity of the mutants. In general, the single mutations abolish glycan binding (or weaken glycan binding to below the detection threshold) for H2 HA, compared with the wild-type HA tested under the same conditions. Mutant R137Q retained some weak binding for one α2-6-linked glycan (#55).
3 Supplementary Figure 3. Electron density maps (2Fo-Fc) of CDR loops in the crystal structures. (a) 2G1 HCDR2, (b) 8M2 HCDR2, (c) 8F8 HCDR3. The maps are contoured at a 2σ level.
4 Supplementary Table 1. Glycans imprinted on the microarray. Glycan # 1 Galβ(1-4)-GlcNAcβ-ethyl-NH2 2 Name Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)- GlcNAcβ(1-4)-GlcNAcβ-Asn- 3 NeuAcα(2-3)-Galβ(1-4)-6-O-sulfo-GlcNAcβ-propyl- 4 NeuAcα(2-3)-Galβ(1-4)-[Fucα(1-3)]-6-O-sulfo-GlcNAcβ-propyl-NH2 5 NeuAcα(2-3)-6-O-sulfo-Galβ(1-4)-GlcNAcβ-ethyl-NH2 6 NeuAcα(2-3)-6-O-sulfo-Galβ(1-4)-[Fucα(1-3)]-GlcNAcβ-propyl-NH2 7 NeuAcα(2-3)-Galβ(1-3)-6-O-sulfo-GlcNAcβ-propyl-NH2 8 NeuAcα(2-3)-Galβ(1-4)-Glcβ-ethyl- 9 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ-ethyl- 10 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ-ethyl- 11 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ-ethyl- NH2 12 NeuAcα(2-3)-GalNAcβ(1-4)-GlcNAcβ-ethyl- 13 NeuAcα(2-3)-Galβ(1-3)-GlcNAcβ-ethyl- 14 NeuAcα(2-3)-Galβ(1-3)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ-ethyl- 15 NeuAcα(2-3)-Galβ(1-3)-GlcNAcβ(1-3)-Galβ(1-3)-GlcNAcβ-ethyl- 16 NeuAcα(2-3)-Galβ(1-3)-GalNAcβ(1-3)-Galα(1-4)-Galβ(1-4)-Glcβ-ethyl- 17 NeuAcα(2-3)-Galβ(1-3)-GalNAcα-Thr- 18 NeuAcα(2-3)-Galβ(1-3)-[GlcNAcβ(1-6)]-GalNAcα-Thr- 19 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-6)-[Galβ(1-3)]-GalNAcα-Thr- 20 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-6)-[Galβ(1-3)]-GalNAcα-Thr- 21 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-GalNAcα-Thr- 22 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-GalNAcα-Thr- 23 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-[NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-6)]-GalNAcα- Thr- 24 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-[NeuAcα(2-3)-Galβ(1-4)- GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-6)]-GalNAcα-Thr- 25 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-2)- Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn- NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[NeuAcα(2-3)- 26 Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)- GlcNAcβ-Asn- NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-2)- 27 Manα(1-3)-[NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)- GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn- 28 NeuAcα(2-3)-[GalNAcβ(1-4)]-Galβ(1-4)-GlcNAcβ-ethyl- 29 NeuAcα(2-3)-[GalNAcβ(1-4)]-Galβ(1-4)-Glcβ-ethyl- 30 Galβ(1-3)-GalNAcβ(1-4)-[NeuAcα(2-3)]-Galβ(1-4)-Glcβ-ethyl- 31 NeuAcα(2-3)-Galβ(1-4)-[Fucα(1-3)]-GlcNAcβ-propyl- 32 NeuAcα(2-3)-Galβ(1-3)-[Fucα(1-4)]-GlcNAcβ(1-3)-Galβ(1-4)-[Fucα(1-3)]-GlcNAcβ-ethyl- 33 NeuAcα(2-3)-Galβ(1-4)-[Fucα(1-3)]-GlcNAcβ(1-3)-Galβ(1-4)-[Fucα(1-3)]-GlcNAcβ-ethyl- 34 NeuAcα(2-3)-Galβ(1-4)-[Fucα(1-3)]-GlcNAcβ(1-3)-Galβ(1-4)-[Fucα(1-3)]-GlcNAcβ(1-3)- Galβ(1-4)-[Fucα(1-3)]-GlcNAcβ-ethyl- 35 NeuGcα(2-3)-Galβ(1-4)-GlcNAcβ-ethyl- 36 NeuAcα(2-6)-Galβ(1-4)-6-O-sulfo-GlcNAcβ-propyl- 37 NeuAcα(2-6)-Galβ(1-4)-Glcβ-ethyl- 38 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ-ethyl-
5 39 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ-ethyl- 40 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ-ethyl- 41 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-[NeuAcα(2-6)]-Galβ(1-4)-GlcNAcβ-ethyl-NH2 42 NeuAcα(2-6)-GalNAcβ(1-4)-GlcNAcβ-ethyl- 43 NeuAcα(2-6)-[Galβ(1-3)]-GalNAcα-Thr- 44 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-6)-[Galβ(1-3)]-GalNAcα-Thr- 45 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-6)-[Galβ(1-3)]-GalNAcα-Thr- 46 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-GalNAcα-Thr- 47 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-GalNAcα-Thr- 48 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-[NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-6)]-GalNAcα- Thr- 49 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-[NeuAcα(2-6)-Galβ(1-4)- GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-6)]-GalNAcα-Thr- 50 Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]- Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn- 51 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]- Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn- 52 GlcNAcβ(1-2)-Manα(1-3)-[NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)- GlcNAcβ(1-4)-GlcNAcβ-Asn- 53 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-2)- Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn- NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[NeuAcα(2-6)- 54 Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)- GlcNAcβ-Asn- NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-2)- 55 Manα(1-3)-[NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)- GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn- 56 NeuGcα(2-6)-Galβ(1-4)-GlcNAcβ-ethyl- 57 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-2)- Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn- 58 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-2)- Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn-
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