Functional Glycomic Analysis of Human Milk Glycans Reveals the Presence of Virus Receptors and Embryonic Stem Cell Biomarkers^*

Preparation of Human Milk Glycans

The procedure for isolation of HMGs is illustrated in supplemental Fig. S1A (33). Briefly, human milk was defatted by centrifugation at 6000 × g for 30 min (4 °C); skimmed milk was filtered through glass wool and mixed with 2 volumes of ethanol and allowed to stand at 4 °C overnight to precipitate the bulk of the lactose and proteins. After centrifugation, the supernatant was concentrated and fractionated with Sephadex G-25 column to fraction A, B, and C. Fraction A, enriched with glycans larger than lactose, was applied to a DEAE column equilibrated with 2 mm pyridinium acetate, yielding neutral, monosialyl, and disialyl fractions by eluting sequentially with 2, 20, and 200 mm pyridine acetate, respectively (34). The resulting fractions were lyophilized.

Glycan-AEAB Conjugation and Purification

Standard glycans and the three human milk glycan fractions were conjugated with AEAB as described previously (31). Briefly, 1–10 mg of glycan was mixed with 50–250 μl of freshly prepared 0.35 m AEAB hydrochloride salt and an equal volume of 1 m NaCNBH₄ in DMSO/AcOH (v/v = 7/3). The conjugation reaction was left to proceed for 2 h at 65 °C and was stopped by the addition of 10 volumes of cold acetonitrile and allowed to stand for 30 min at −20 °C. The precipitated glycan-AEAB derivatives were collected after centrifugation at 10,000 × g for 3 min.

High Performance Liquid Chromatography

A Shimadzu HPLC CBM-20A system coupled with a UV detector SPD-20A and a fluorescence detector RF-10AXL was used for HPLC analysis and separation of glycan-AEABs. The AEAB-conjugated milk glycans were first separated by normal phase HPLC on an Agilent NH₂ column (250 × 4.6 mm). The mobile phase linear gradient was from 80% acetonitrile, 10 mm ammonium acetate, pH 4.5, to 10% acetonitrile, 125 mm ammonium acetate, pH 4.5, in 160∼200 min. Individual peaks were collected and dried. Each peak collected on the NH₂ column was further purified in a second dimension by reverse phase HPLC on a porous graphitized carbon column (150 × 4.6 mm) with a gradient of 15–45% acetonitrile (0.1% trifluoroacetic acid) in 30 or 15 min. The effluents were monitored by UV absorption at 330 nm and/or fluorescence at 420 nm with excitation 330 nm. LNFPIII-AEAB or lactose-AEAB was utilized as the standard for the quantification of the AEAB derivatives. The individual glycans after the second dimension chromatography were quantified, dried, and reconstituted in water at a concentration of 200 μm and stored as a HMG-tagged glycan library (TGL).

Printing of the SGM, Binding Assays, and Analysis of Microarray

The printing conditions for the SGM, binding assays, and data analysis of glycan microarrays have been previously described (31, 35, 36). Briefly, each glycan in the TGL was adjusted to 100 μm in sodium phosphate buffer (100 mm, pH 8.5) in a final volume of 10 μl and distributed into the 384-well source plate of the Piezorray printer. The glycans were printed as 0.3-nl aliquots in replicates of 5. The biotinylated lectins or unlabeled antibodies were incubated for 1–3 h on the slides. After washing, the bound lectins were detected by a secondary incubation with cyanine 5-labeled streptavidin (5 μg/ml), and the bound antibodies were detected using Alexa488-labeled goat anti-mouse IgG (5 μg/ml). The fluorescence signals were generated using a ProScanArray Scanner (PerkinElmer Life Sciences) with the excitation/emission wavelength set at 649/670 nm and 495/510 nm for cyanine 5 and Alexa488, respectively.

Virus Preparations and Binding Assays

The human parainfluenza (hPIV) and influenza A were grown in LLC-MK2 cells and MDCK cells, respectively, as previously described (37, 38). Harvested viruses were purified through a sucrose gradient centrifugation (10–60% for hPIV and 10–40% for influenza A) and resuspended in calcium-magnesium saline followed by labeling with Alexa488 succinimidyl ester (Molecular Probes, Invitrogen). For the binding assays, the slides were incubated with labeled viruses for 1 h at 4 °C and directly scanned after washing. The WT and mutant strains of minute virus of mice (MVM) were constructed and prepared as previously described (39–42). Briefly, the WT MVMp empty particles were grown in mouse A9 ouab^r11 fibroblasts, MVMi mutants (MVMi-ggA and MVMi-agD) were grown in NB324K cells, and VLPs (virus-like particles) were grown in sf9 insect cells. The harvested viruses were released by repeated freezing and thawing and then purified by centrifugal sedimentation and density equilibrium gradients. The purity and integrity of prepared viruses were assessed by hemagglutination, SDS-PAGE, and electron microscopy (41).

Preliminary Characterization of the HMGs on the SGM by Lectins and Antibodies

Defined glycan microarrays are used to explore the specificity of GBPs, including lectins and anti-glycan antibodies (23). In turn, GBPs with well defined binding specificity can assist in the elucidation of glycan structures. To interrogate the SGM and evaluate the structural diversity of isolated HMGs, 10 biotinylated lectins, each at several concentrations in the range of 0.001–10 μg/ml, were applied to the array. No significant binding was observed with concanavalin A, Vicia villosa lectin, Griffonia simplicifolia lectin II, and Maackia amurensis lectin I. These lectins recognize glycans containing mannose (43–45), terminal GalNAc (46), terminal GlcNAc (47), and terminal Neu5Acα2–3Galβ1–4GlcNAc (48), respectively; the absence of their binding is consistent with the lack of such structures in human milk free glycans (6). Independently the binding of these lectins to a defined glycan microarray from the CFG (v5.0) was evaluated, and the data are available online as glycan array data on the CFG website. The other six lectins, Aleuria aurantia lectin (AAL), Sambucus nigra agglutinin (SNA), Lotus tetragonolobus lectin (LTL), Ulex europaeus agglutinin I (UEA-I), Ricinus communis agglutinin I (RCA-I), and Erythrina cristagalli Lectin (ECL), exhibited binding to many of the HMGs on the SGM as discussed below, and the binding histograms showing average relative fluorescence units (RFU) for selected concentrations of each lectin are shown in Fig. 2, and the binding data are listed in supplemental Table S2.

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FIGURE 2.

Plant lectins binding to human milk SGM. The human milk SGM microarray was characterized with biotinylated lectins AAL (0.1 μg/ml; A), UEA-I (10 μg/ml; B), LTL (10 μg/ml; C), SNA (5 μg/ml; D), RCA-I (10 μg/ml; E), and ECL (10 μg/ml; F). A total of 140 glycans was printed on the microarray. Glycans 1–73 are sialylated glycans (tan), 74–127 are neutral glycans (violet), and 128–140 are controls of structurally defined glycans (light green). The structures in symbols indicate the binding specificity of each lectin identified by defined glycan microarray (CFG v5.0). Considering the variation of binding affinity, the histogram shows the data at the concentration that yielded the best signal/background ratio instead of the data at the same concentration.

Three fucose binding lectins, AAL, LTL, and UEA-I, were used to reveal the fucosylated glycans on the SGM. Approximately 61 glycans (48%) showed strong binding with AAL (RFU > 10,000), which binds to terminal α-linked l-fucose in 1–2, 1–3, and 1–4 linkages (49), and an additional 24 glycans (19%) had weaker binding (RFU 1,000–8,000). Together, the fucosylated glycans recognized by AAL made up 67% of the total isolated HMGs, which is close to the percentage found by using HPLC-ChIP/MS method (21). In addition, most of the neutral glycans and about 40% of the sialylated glycans are fucosylated, in agreement with the results from glycan composition analysis based on mass (supplemental Table S1). UEA-I, which is specific for α1–2-linked fucose on a type 2 chain (50, 51), bound very weakly (<4,000 RFU) to several multifucosylated, neutral glycans (Fig. 2B), suggesting the presence of α1–2 fucose on our HMG array. However, this weak binding was presumably due to the cross-reactivity of UEA-I with other fucose-containing glycans as shown in Table 1, and supplemental Table S2 and actually not from Fucα1–2. This conclusion was supported by the observation that glycans were resistant to α1–2 fucosidase digestion in solution (data not shown). This result indicated the absence of H type 2 (Fucα1–2Galβ1–4GlcNAc) and Le^y (Fucα1–2Galβ1-4(Fucα1–3)GlcNAc) structures in the SGM. LTL recognizes α1–3-linked fucose within type 2 glycans (52, 53), like Le^x and Le^y determinants. It showed strong binding to three neutral glycans (H-75, H-83, and H-87) and weak binding to another five glycans (Fig. 2C). The three high affinity binders likely contain terminal Le^x determinants because the absence of UEA-I binding (Fig. 2B) excludes the possibility of α1–2-linked fucose.

TABLE 1

Heat map of lectin-binding data for structural analysis

A heat map summary of the binding data from analyses of the HMG subarray with different plant lectins before and after specific exoglycosidase digestion(s) is shown. A total of 40 glycans were printed on the HMG subarray, including 18 control glycans and 22 selected HMGs. The color-coded numbers highlight the binding intensity (the darker the color, the higher the binding intensity). The color scale for the Excel spreadsheet was set using 0 as minimum and 60,000 RFU as maximum. The untreated HMG subarrays were tested with eight lectins. The HMG subarrays treated with non-specific neuraminidase and α2-3-specific neuraminidase were tested with SNA and ECL. The β1–3 galactosidase (β1–3 galase)- or β1–4 galactosidase (β1–4 galase, from either New England Biolabs or Prozyme)-treated slides were tested with GSL-II. The lectin concentrations used were 10 μg/ml with the exception of AAL, which was 1 μg/ml.

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SNA, which binds to the Neu5Acα2–6Galβ1–4GlcNAc determinant (23, 54), bound well to 14 (of 73) sialylated glycans on the SGM along with three control glycans (2–6-DS-NA2, LSTc, and fetuin, structures shown in supplemental Table S1), indicating the existence of α2–6-sialylated type 2 structure in 19% of the glycans (Fig. 2D). Both RCA-I and ECL recognize terminal Galβ1–4GlcNAc (55–59), but the former has much higher affinity and also binds to Neu5Acα2–6Galβ1–4GlcNAc with slightly lower affinity, whereas the latter can also bind Fucα1–2Galβ1–4Glc (60). Consistent with these features, the binding pattern of RCA-I on this SGM was similar to the combined pattern of SNA and ECL (Fig. 2, D–F) together, suggesting that ∼46% of glycans have terminal type 2 or sialylated type 2 structures. In summary, the results of lectin binding both validated the preparation of the SGM and provided significant structural information on individual HMGs.

The structures of HMGs reflect the Lewis blood type and secretor status of the mothers (61, 62). Certain fucosylated glycans that were suggested to possess important biological functions (3) occur only in the milk of Lewis positive and/or Secretor-positive mothers due to the expression of FUT-3 and FUT-2, respectively (19). Because there are no lectins specific for Lewis blood group antigens, we analyzed the SGM with several blood group-related monoclonal antibodies. As shown in Fig. 3A, the mAb to Le^a bound to 34 glycans, including 9 sialylated ones, with fluorescence signals in the range of 500–15,000 RFU. To reveal additional Le^a-containing glycans, we interrogated the array with an anti-sialyl Le^a (SLe^a) antibody. According to the data from the structurally defined CFG glycan microarray, this antibody also binds to LSTa (Neu5Acα2–3Galβ1–3GlcNAcβ1–3Galβ1–4Glc) moiety with lower signal. At the concentration of 10 μg/ml, 31 sialylated glycans showed binding signals higher than 30,000 RFU with the antibody, and another 19 glycans had signals >5,000 RFU (Fig. 3B). Although we could not simply assign SLe^a and LSTa structures by signal intensity, it is certain that many of these sialyl glycans are fucosylated based on their AAL binding (Fig. 2A) and may contain the SLe^a moiety. Nevertheless, the abundance of Le^a-containing glycans showed that the SGM was from a Lewis positive donor. To determine the secretor status of the donor, we interrogated the SGM with anti-Le^b and anti-blood group H type 1 antibodies. There was little to no binding observed with the anti-Le^b antibody at any concentration (data not shown), which indicates that the milk sample is from a secretor negative donor. This finding was confirmed by assaying with anti-H1 antibody, which bound only the control LNFP I (Fucα1–2Galβ1–3GlcNAcβ1–6Galβ-4Glc) (Fig. 3C), the precursor of Le^b antigen.

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FIGURE 3.

Antibodies binding to human milk SGM. The human milk SGM was interrogated with antibodies: anti-Le^a antibody (10 μg/ml; A), anti-SLe^a/LSTa antibody (10 μg/ml; B), anti-blood group H1 antibody (1:10 dilution; C), and anti-CD15 antibody (10 μg/ml; D). The microarray was also used to test the binding specificity of anti-TRA-1–60 antibody (50 μg/ml; E) and anti-TRA-1–81 antibody (50 μg/ml; F). Glycans 1–73 are sialylated glycans (tan), 74–127 are neutral glycans (violet), 128–140 are controls of structurally defined glycans (light green).

We also interrogated the array with anti-CD15 antibody, known to recognize Le^x antigen. Strong binding was observed to glycans H-98 and H-103 with weaker binding to several other neutral glycans (Fig. 3D). When comparing CD15 antibody with lectin LTL, the two Le^x-recognizing proteins showed distinct specificity toward different HMGs, although with some overlap. This could indicate that the recognition does not solely depend on the Le^x determinant for complex glycans, and the nearby residues or branches could affect the binding.

Detection of HMGs That Are Epitopes of Anti-TRA-1 Antibodies, Specific for Human Pluripotent Stem Cells

Recently, Natunen et al. (63) predicted epitopes for mAbs anti-TRA-1–60 and anti-TRA-1–81 based on binding data from Version 4.2 of the CFG glycan microarray. These mAbs, which are specific for human pluripotent stem cells, bound only to two glycans, both containing the type 1 lactosamine epitope, Galβ1–3GlcNAcβ1–3Galβ1–4GlcNAc, on that version of the CFG glycan microarray. We further examined the two mAbs on Version 5.0 of the CFG array, which contains many multiantennary glycans with poly-N-acetyllactosamine (64) and observed strong binding by three additional glycans (supplemental Fig. S2). Importantly, these three glycans (#522, #572, and #573) are multiantennary glycans with two to three type 1 lactosamine repeats at their non-reducing ends. In addition, we observed weak but significant binding to 387, a glycan with a fucosylated type 1 lactosamine chain. Considering that HMG is a rich source of type 1 and type 2 lactosamine structures, we interrogated the SGM with the two anti-TRA-1 antibodies at several concentrations (1–100 μg/ml), and the results at 50 μg/ml are shown in Fig. 3, E and F. Consistent with the CFG array data, TRA-1–60 and TRA-1–81 share similar receptor specificity as both bind glycans H-71, H-99, H-108, H-111, H-125, and H-127. However, unlike the CFG results, which showed no significant difference in the signal intensity and binding patterns for the two mAbs at 50 μg/ml, we observed that with the SGM the signal intensity for TRA-1–60 binding was always severalfold higher than that of TRA-1–81 at the same concentration and that there are three more low affinity binders for TRA-1–60, H-109, H-112, and H-126 (Fig. 3E). To define the glycan epitope in HMG for TRA-1 antibodies, we retrieved glycans H-71, H-99, H-108, H-111, H-125, H-126, and H-127 from the TGL for further characterization. With the exception of sialylated glycan H-71, all of the TRA-1-bound glycans are neutral fucosylated structures consisting of 2–4 lactosamine repeats. We predict from previous studies (63) that these glycans possess type 1 lactosamine. Our data also indicate that the two anti-TRA-1 mAbs recognize complex glycans, as described below.

Investigation of Virus Binding to the SGM

The HMG-derived SGM provides a library of 127 naturally occurring glycans that permits us to investigate the binding properties of biologically relevant proteins and pathogens and to provide interesting insights into the potential function of HMGs. To explore the general application of this HMG-derived SGM for exploring pathogen interactions, we examined MVM and influenza virus, both of which attach to the sialic acid on the surface of their target cells at the initial stage of infection (40, 65).

Two prototype strains, empty capsid (MVMp-WT) and virus-like particle (MVMp-VLP), and two immunosuppressive strain mutants, MVMi-agD and MVMi-ggA (39), were tested at 200 μg/ml concentration and detected by a rabbit anti-MVM capsid antibody. All of the MVM viruses recognized glycans H-30, H-31, H-34, H-43, H-60, and H-73 with each non-WT strain binding several additional glycans (Fig. 4). Interestingly, the initial MALDI data (supplemental Table S1) showed that the 6 common binders are disialylated glycans, and the lectin and antibody binding data revealed that all of the binders were also recognized by the anti-SLe^a/LSTa antibody, suggesting that the terminal sialyl α2–3-linked type 1 motif might be part of the binding determinant. This is a new finding compared with the previous report with CFG Glycan Array Version 3.0 (∼180 glycans), which concluded that MVMs specifically recognized α2–3-linked type 2 motif and MVMi also bound to α2–8-linked multisialylated glycans (41). These data are complementary to the data from the CFG array as there is very little overlap between the structures found on the CFG array and HMG-derived SGM. In fact, these observations suggest that the repertoire of glycan receptors of MVM is broader than originally reported, and these viruses prefer highly charged or possibly multisialylated glycans. We retrieved glycans that were bound by all MVM strains from the TGL for more detailed structural analysis discussed below.

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FIGURE 4.

MVM viruses binding to human milk SGM. The binding preferences of several strains of MVM, MVMp-WT (prototype strain, empty capsid; A), MVMp-VLP (prototype strain, virus-like particles (B)), MVMi-ggA (capsid protein mutant of immunosuppressive strain, empty capsid; C), and MVMi-agD (Non-structural protein mutant of immunosuppressive strain, empty capsid; D) were evaluated on the human milk SGM. Each virus was tested at 200 μg/ml and detected by anti-MVM capsid antibody. Panel E shows that there was no background binding from the anti-MVM capsid antibody to HMG microarray. Glycans 1–73 are sialylated glycans (tan), 74–127 are neutral glycans (violet), and 128–140 are controls of structurally defined glycans (light green).

Recently, we utilized the CFG glycan microarray to determine the receptor binding properties of 2009 swine isolates along with some other strains of influenza viruses (37). Our results showed that the highest binding of H1N1 isolates was toward sialylated poly-N-acetyllactosamine structures, which are abundant among HMGs. To determine if HMGs contained natural glycans capable of binding influenza A virus, we interrogated the SGM with three seasonal human H1N1 strains (A/Brisbane/59/2007, A/Pennsylvania/08/2008, and A/Oklahoma/447/08), one human pandemic H1N1 strain (A/California/04/2009), and one H3N2 virus (A/Oklahoma/483/08) for comparison. Consistent with our previous findings, A/Brisbane isolate showed the broadest binding specificity and preferred glycans with terminal α2–6-linked sialic acid (Fig. 5A). Interestingly, the glycans recognized by A/Brisbane/59/2007 (H1N1) were the same glycans that bound SNA, which is specific for the determinant, Neu5Acα2–6Galβ1–4GlcNAc. The binding profile of A/Oklahoma/447/08 (H1N1) virus is similar to the A/Brisbane virus but displayed a much higher signal to noise ratio (Fig. 5B). When compared with binding data from lectins and antibodies, A/Oklahoma/447/08 (H1N1) displayed a clear preference for glycans with α2–6 sialic acid, binding strongly to 2–6-DS-NA2, LSTc, and all of the glycans bound by SNA. Additionally, like A/Brisbane/59/2007, several glycans were recognized by the anti-SLe^a/LSTa antibody, suggesting certain specificity toward α2–3 sialic acid-containing glycans. A/Pennsylvania/08/2008, which was shown to preferentially bind glycans having terminal α2–6 sialic acid when assayed on the CFG defined glycan array, differed from the other H1N1 strains, A/Oklahoma/447/08 and A/Brisbane/59/2007, in that it did not bind some of the HMGs (such as glycan H-12, H-15, H-16, H-53, and H-55) that possess the Neu5Acα2–6Galβ1–4 motif (Fig. 5C). This result suggests that the virus binding does not solely rely on the sialic acid linkage. In the case of A/California/04/2009, a human pandemic H1N1 isolate, the binding pattern overlaps with SNA (Fig. 5D), which confirms the results from CFG microarray that A/California/04/2009 has a restricted binding preference for α2–6 sialic acid-linked type 2 glycans. Furthermore, as a comparison to the H1N1 A/Oklahoma/447/08 virus, we tested the A/Oklahoma/483/08 H3N2 virus isolate (Fig. 5E) and obtained a more restricted binding pattern where all the bound glycans contain Neu5Acα2–6Galβ1–4GlcNAc structure and were recognized by the H1N1 A/Oklahoma/447/08.

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FIGURE 5.

Influenza viruses binding to human milk SGM. The binding preferences of various influenza virus isolates, A/Brisbane/59/2007 H1N1 (A), A/Oklahoma/447/2008 H1N1 (B), A/Pennsylvania/08/2008 (C), A/California/04/2009 H1N1 (D), A/Oklahoma/483/2008 H3N2 (E), and hPIV2 were evaluated with HMG microarray (F). Glycans 1–73 are sialylated glycans (tan), 74–127 are neutral glycans (violet), and 128–140 are controls of structurally defined glycans (light green).

Finally, we evaluated the binding properties of three human parainfluenza viruses on the human milk SGM. It is known that these viruses require the presence of Neu5Acα2–3Galβ1-4GlcNAc motif (66). However, our M. amurensis lectin I binding data showed that this structure is not a component of the HMG. Thus, as we would predict, no binding was observed with the type 1 and 3 (hPIV1 and hPIV3) viruses. By contrast, unlike type 1 and 3, the type 2 parainfluenza virus (hPIV2) displayed a very strict preference for α2–6-sialic acid-containing glycans (Fig. 5F), with receptor specificity similar to the H1N1 strains. Overall, the virus binding experiments demonstrated that many of the HMGs might function as decoys for cell-bound receptors and that the elements in the HMGs might be found on cell-bound receptors. Glycans that bound viruses were selected for more detailed characterization as described below.