Fig 2: AMR binding of IAP glycoforms. (A–C) Representative fields of view from sections of fresh frozen liver overlayed with Alexa Fluor 647 conjugated preparations of sialylated and desialylated IAP α2-3 Bi (A), IAP α2-6 Bi (B), and IAP α2-3 Multi (C) followed by a DAPI nuclear stain. (Scale bar, 40 μm.) Livers originated from littermate male mice 12 wk of age. The signal captured in the Alexa Fluor 647 channel is displayed in yellow and DAPI in blue. (D) Sections were imaged using a TissueGnostics workstation and analyzed for cell-associated (DAPI+) Alexa Fluor 647 signal. For each section, at least 130,000 events were recorded. Data are representative of three to six separate littermate cohorts of the indicated genotypes and are presented as means ± SD. The unpaired t test was used to analyze for difference (***P < 0.001; **P < 0.01; *P < 0.05). (E) Microplates were coated with an antibody targeting the cytoplasmic tail of the AMR to orientationally capture the receptor. Following AMR capture and incubation with 2.5-fold serial dilutions of IAP, AP enzyme activity was determined using the p-nitrophenyl phosphate substrate. (F) Dot blot analysis of twofold serially diluted IAP overlaid with purified AMR followed by AMR primary and secondary detection antibodies. The membrane was scanned at 700 nm using a LiCorBio Odyssey CLx imager to quantify AMR binding and then overlaid with a biotinylated IAP antibody followed by IRDye 800CW streptavidin before being scanned at 800 nm. The blot and data are representative of three independent experiments.

Fig 3: AMR function in the circulating half-lives of desialylated and sialylated IAP glycoforms. Sialic acids were removed from IAP using agarose-coupled neuraminidase. (A) WT, (B) Asgr1−/−, and (C) Asgr2−/− littermate male and female mice 12 to 14 wk of age were intravenously administered 1 µg desialylated IAP. Blood samples were collected at indicated timepoints beginning 30 s after injection for WT mice, and 10 min after injection for Asgr1−/− and Asgr2−/− mice. (D) WT, (E) Asgr1−/−, and (F) Asgr2−/− littermate male and female mice 12 to 14 wk of age were intravenously administered 1 µg sialylated IAP. Blood samples were collected at indicated timepoints beginning 10 min after injection. Relative plasma IAP concentration was quantified by fluorescent western blot. The graphed points are presented as mean ± SD and the lines represent a two-phase exponential decay curve fitted to the points. The half-life for each animal was calculated from its specific measurements and summarized in the tables adjoining the figure legends. Where indicated, the unpaired t test was used to analyze the difference in half-lives (****P < 0.0001).

Fig 4: Modeling IAP glycoforms to detect AMR ligands. (A) Schematic representation of the AMR depicting the separation distance of the galactose-binding lectin domains as first proposed by Lee et al. (12) and modified from (37). (B) An ensemble of 200 desialylated biantennary N-glycan conformers was modeled at each N-glycan site (N141, N268, and N429) on two complementary IAP monomers. All three N-glycan conformers of IAP monomers exhibit biantennary galactose linkages separated by more than 25 angstroms (colored yellow) and thus are unlikely to be AMR ligands. (C) Dimerization of IAP monomers shown in panel B forms the active IAP homodimer and results in juxtapositioning of biantennary N-glycans linked to N141 and N429. This juxtapositioning facilitates the formation of trivalent and higher valency galactose linkages separated by (C) less than 25 Å or (D) less than 15 Å consistent with the presence of AMR ligands (red color). The percentage of N-glycan conformers that overlap to produce AMR ligands is indicated at each N-glycosylation site as the mean ± SD of six analyses. (E) The IAP homodimer modeled with desialylated tetra-antennary N-glycans carries multivalent galactose ligands of the AMR at all six N-glycosylation sites.