Glycomics-based analysis of chicken red blood cells provides insight into the selectivity of the viral agglutination assay

Abstract

Agglutination of red blood cells (RBCs), including chicken RBCs (cRBCs), has been used extensively to estimate viral titer, to screen glycan-receptor binding preference, and to assess the protective response of vaccines. Although this assay enjoys widespread use, some virus strains do not agglutinate RBCs. To address these underlying issues and to increase the usefulness of cRBCs as tools for studying viruses, such as influenza, we analyzed the cell surface N-glycans of cRBCs. On the basis of the results obtained from complementary analytical strategies, including MS, 1D and 2D-NMR spectroscopy, exoglycosidase digestions, and HPLC profiling, we report the major glycan structures present on cRBCs. By comparing the glycan structures of cBRCs with those of representative human upper respiratory cells, we offer a possible explanation for the fact that certain influenza strains do not agglutinate cRBCs, using specific human-adapted influenza hemagglutinins as examples. Finally, recent understanding of the role of various glycan structures in high affinity binding to influenza hemagglutinins provides context to our findings. These results illustrate that the field of glycomics can provide important information with respect to the experimental systems used to characterize, detect and study viruses.

Fig. 1. MALDI-TOF mass spectra of free, non-reduced N-glycans isolated from cRBCs

Peaks appeared in the mass range of 2000 to 3600 with the most prominent peaks at m/z 2589.1 and 2880.5. Each peak was calibrated as a non-sodiated species using external N-glycan standards as mass calibrants. Proposed glycan structures for each peak using MS annotation software are shown along with their observed m/z value. The number in the bracket for each glycan indicates the percentage of each corresponding glycan within the total glycan pool as estimated by semi-quantitative MALDI MS.

Fig. 2. Qualitative and quantitative linkage analysis of N-glycans from cRBCs

(A) MALDI-MS spectra of sialidase S-treated, unlabeled, N-glycans that were released from cRBCs by PNGase F. (B) ¹H-NMR spectra of sialic acid linkage in the free non reduced N-glycans isolated from cRBCs.

Fig. 2. Qualitative and quantitative linkage analysis of N-glycans from cRBCs

(A) MALDI-MS spectra of sialidase S-treated, unlabeled, N-glycans that were released from cRBCs by PNGase F. (B) ¹H-NMR spectra of sialic acid linkage in the free non reduced N-glycans isolated from cRBCs.

Fig. 3. ¹H-NMR (600 MH_Z, D₂O) spectra of N-glycans from cRBCs

Landmark chemical shifts are identified for each region of interest. The possible structural annotations of each monosaccharide fingerprint proton are labeled in the spectrum.

Fig. 4. HSQC-spectra of N-glycans from cRBCs

The spectrum shows the cross peaks between the anomeric protons (5.25–4.30 ppm) and carbon (89–105 ppm) signals. The cross peaks confirm the presence of primarily bi- and triantennary structures. Notably, there is no cross peak detected at around 4.68–4.71 ppm, indicating the absence of lactosamine repeat units in the cRBC N-glycan pool.

Fig. 5. HPLC profiling of 2AB-linked N-glycan isolated from cRBCs

Glycans are eluted using a normal phase column with a 50 mM ammonium formate/ acetonitrile gradient as eluant. Total run time is 290 minutes. (A) HPLC profiling of glucose homo-polymer for calibration of the column. (B) A mixture of three sialic acid containing N-glycans standards, chosen based on their polarity and molecular weight, are used as benchmarks. Three different species appeared in the retention time between 120–200 min. (C) 2-AB labeled N-glycan pool from cRBCs were analyzed within the calibrated and standardized column system. The acidic N-glycans from cRBCs eluted within the retention time window of 120–200 minutes. Glycan under each peak was determined from the MALDI-MS data of the isolated fraction of their corresponding peaks (Table S3).

Fig. 5. HPLC profiling of 2AB-linked N-glycan isolated from cRBCs

Glycans are eluted using a normal phase column with a 50 mM ammonium formate/ acetonitrile gradient as eluant. Total run time is 290 minutes. (A) HPLC profiling of glucose homo-polymer for calibration of the column. (B) A mixture of three sialic acid containing N-glycans standards, chosen based on their polarity and molecular weight, are used as benchmarks. Three different species appeared in the retention time between 120–200 min. (C) 2-AB labeled N-glycan pool from cRBCs were analyzed within the calibrated and standardized column system. The acidic N-glycans from cRBCs eluted within the retention time window of 120–200 minutes. Glycan under each peak was determined from the MALDI-MS data of the isolated fraction of their corresponding peaks (Table S3).

Fig. 5. HPLC profiling of 2AB-linked N-glycan isolated from cRBCs

Glycans are eluted using a normal phase column with a 50 mM ammonium formate/ acetonitrile gradient as eluant. Total run time is 290 minutes. (A) HPLC profiling of glucose homo-polymer for calibration of the column. (B) A mixture of three sialic acid containing N-glycans standards, chosen based on their polarity and molecular weight, are used as benchmarks. Three different species appeared in the retention time between 120–200 min. (C) 2-AB labeled N-glycan pool from cRBCs were analyzed within the calibrated and standardized column system. The acidic N-glycans from cRBCs eluted within the retention time window of 120–200 minutes. Glycan under each peak was determined from the MALDI-MS data of the isolated fraction of their corresponding peaks (Table S3).

Fig. 6. LC-MS/MS data of selected MS peaks of N-glycans from cRBC

Shown are the MS/MS signals of 2-AB labeled structures: (A) 2620; (B) 2911; (C) 3000; and unlabeled structure: (D) 3083.8. Fragment assignments are shown for each structure.

Fig. 6. LC-MS/MS data of selected MS peaks of N-glycans from cRBC

Shown are the MS/MS signals of 2-AB labeled structures: (A) 2620; (B) 2911; (C) 3000; and unlabeled structure: (D) 3083.8. Fragment assignments are shown for each structure.

Fig. 6. LC-MS/MS data of selected MS peaks of N-glycans from cRBC

Shown are the MS/MS signals of 2-AB labeled structures: (A) 2620; (B) 2911; (C) 3000; and unlabeled structure: (D) 3083.8. Fragment assignments are shown for each structure.

Fig. 6. LC-MS/MS data of selected MS peaks of N-glycans from cRBC

Shown are the MS/MS signals of 2-AB labeled structures: (A) 2620; (B) 2911; (C) 3000; and unlabeled structure: (D) 3083.8. Fragment assignments are shown for each structure.