Figure 3.

Site-Specific Glycoform Composition of MT145K Trimer

(A) Site-specific glycoform quantification of the MT145K SOSIP soluble trimer. MT145K trimers from transiently transfected HEK293F cell expressed supernatants were affinity purified by the quaternary trimer-specific antibody, PGT145. The purified MT145K trimers were treated separately with three proteases—trypsin, chymotrypsin, and elastase—and the digests were enriched for glycopeptides and analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS). The individual glycan compositions of the N-linked glycan sites (n = 26) are represented by bar graphs that indicate the relative abundance of each glycoform species and are derived from the mean of two analytical replicates. The pie charts summarize the proportion of glycoforms for each site and this information is color coded: oligomannose type in green and complex and/or hybrid glycans in pink. The glycoforms at N262 and N268 positions (indicated by “^∗”) could not be separately determined by enzymatic digestion and the bars represent the average glycan compositions across both sites.

(B) Hydrophilic interaction ultra-performance liquid chromatography (HILIC-UPLC) profiles of the total N-linked glycans released from the MT145K trimers. The proportions of oligomannose plus hybrid glycan contents and complex-type glycans are represented in green and pink colors, respectively.

(C) Modeled glycan shields for the MT145K and BG505 SOSIP trimers. Man9GlcNAc2 oligomannose-type glycans were docked and rigid-body fitted at each of the corresponding Env glycan positions using the MT145K structure (determined in this study [PDB: 6OHY]) and the unliganded BG505 SOSIP.664 trimer structure (Kwon et al., 2015; PDB: 4ZMJ). Top and side views of the trimers are shown and the individual glycan sites are labeled and color coded based on the content of oligomannose: green, 100%–80%; orange, 79%–20%; and pink, 19%–0%.