Human milk glycans (HMGs) are prebiotics, pathogen receptor decoys and regulators of host physiology and immune responses. Mechanistically, human lectins (glycan-binding proteins, hGBP) expressed by dendritic cells (DCs) are of major interest, as these cells directly contact HMGs. To explore such interactions, we screened many C-type lectins and sialic acid-binding immunoglobulin-like lectins (Siglecs) expressed by DCs for glycan binding on microarrays presenting over 200 HMGs. Unexpectedly, DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) showed robust binding to many HMGs, whereas other C-type lectins failed to bind, and Siglec-5 and Siglec-9 showed weak binding to a few glycans. By contrast, most hGBP bound to multiple glycans on other microarrays lacking HMGs. An α-linked fucose residue was characteristic of HMGs bound by DC-SIGN. Binding of DC-SIGN to the simple HMGs 2′-fucosyl-lactose (2′-FL) and 3-fucosyl-lactose (3-FL) was confirmed by flow cytometry to beads conjugated with 2′-FL or 3-FL, as well as the ability of the free glycans to inhibit DC-SIGN binding. 2′-FL had an IC50 of ∼1 mM for DC-SIGN, which is within the physiological concentration of 2′-FL in human milk. These results demonstrate that DC-SIGN among the many hGBP expressed by DCs binds to α-fucosylated HMGs, and suggest that such interactions may be important in influencing immune responses in the developing infant.

INTRODUCTION

Carbohydrates are the most abundant class of biomolecules in human milk. The majority of this total carbohydrate (∼70 g/l) is lactose, a major source of energy for infants, and the remainder (5–20 g/l) consists of non-digestible larger-sized glycans that are derived from lactose [13]. These human milk glycans (HMGs) have been classically defined as prebiotics and receptor decoys that are predicted to prevent infection by blocking pathogen adherence to the infant epithelium [4,5]. However, HMGs may have functions beyond interactions with microbes, as more recent studies suggest that HMGs may regulate multiple physiological functions in infants, including gene expression and immune and allergic responses [6,7]. HMGs also regulate gut motility [8] and enhance learning and memory [9], suggesting their role in neuronal responses and cognition. However, the mechanisms underlying these physiological functions of HMGs are still unclear.

Human lectins (human glycan-binding proteins, hGBP) play numerous roles in physiology and immunity, including regulation of gene expression and immune responses, pathogen sensing, cell–cell interactions and tissue homing [1012]. The glycan specificities of many hGBP have been explored by multiple techniques, but the most powerful new approach has utilized glycan microarrays in which hundreds of structurally defined glycan ligands are displayed on a single slide, as developed by the Consortium for Functional Glycomics (CFG) (http://www.functionalglycomics.org/glycomics/publicdata/primaryscreen.jsp). These studies have shown that each hGBP has a restricted specificity, even within a given hGBP family [13]. The binding of different hGBP to specific glycan determinants allows different hGBP to regulate specific physiological functions.

There have been some previous studies broadly examining hGBP glycan specificity towards HMGs [1416], and such general screening suggests that some HMGs may be recognized by specific hGBP. By extension, we hypothesized that HMGs might serve as general ligands for many hGBP, which could be important in modulating the hGBP downstream effector or signalling functions. The purpose of the present study was to identify hGBP that bind HMGs, investigate glycan determinant specificity and the extent of the human milk metaglycome bound, and determine whether binding occurs at physiologically relevant concentrations.

To address these questions, we focused on those hGBP expressed by dendritic cells (DCs), since such cells may directly contact HMGs in the developing infant intestine via dendrite extension through the intestinal epithelium [17,18]. We screened members of the C-type lectin [19] and I-type lectin [11,20] families for binding to a human milk shotgun glycan microarray as well as defined glycan microarrays. The results of the present study showed that from this large set of hGBP, only DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) was a major binder of HMGs, with multiple α-linked fucose-containing glycans bound on an array consisting of approximately 250 purified HMG structures. This binding of specific HMGs by DC-SIGN suggests that DC-SIGN may serve as an HMG receptor, which may have implications in infant immunity, physiology and development.

MATERIALS AND METHODS

Preparation and screening of microarrays

All of the recombinant hGBP used in the present study were purchased from R&D Systems and are shown in Table 1, which includes information on the amino acid sequences, fusion tags and catalogue numbers used. The proteins were checked for activity by their binding to one or more glycans in various glycan microarrays. The human milk shotgun glycan microarray version 2 (HM-SGM-v2), consisting of 247 purified HMGs structures and 13 controls, has been previously described [21]. The defined HMG microarray, consisting of simple defined HMG structures, was generated as described previously [22]. The recombinant hGBP were screened on CFG glycan microarray version 5.1, HM-SGM-v2 and defined HMG microarray as previously described [23]. Alexa Fluor 488-labelled anti-human IgG (Molecular Probes) or Alexa Fluor 488-labelled anti-pentaHis (Qiagen) antibodies were used at 5 μg/ml for detection of recombinant hGBP carrying an Fc fusion tag or 6–9 × His tag respectively. As a control we screened 10 μg/ml DC-SIGN binding on the HM-SGM-v2, in which Ca2+ was omitted from the binding buffer and replaced with 0.2 mM EDTA to confirm Ca2+-dependent binding. For HMG inhibition experiments, the recombinant hGBP was preincubated with free HMG or N-aminoethyl 2-aminobenzamide (AEAB)-derivatized HMG containing an ‘open-ring’ reducing end or glycosylamide-glycyl-N-aminoethyl 2-aminobenzamide (GGAEAB)-derivatized HMG containing a ‘closed-ring’ reducing end, generated as previously described [24,25], for 1 h prior to screening of the defined HMG microarrays. Detection was performed using 5 μg/ml Alexa Fluor 633-labelled anti-human IgG (Molecular Probes). Rank and average rank calculations of the microarray data were performed as previously described [26]. The microarray data were manually examined for binding motifs and, for the CFG microarray data, were further analysed with Glycopattern (https://glycopattern.emory.edu) [27] to define the CFG glycan microarray binding motif.

Table 1
Recombinant human glycan-binding proteins used in the present study

All recombinant proteins were purchased from R&D Biosystems and were expressed from a mouse myeloma cell line, NS0-derived.

NameLectin familyGenBank accession numberAmino acid sequenceProtein fusion tagR&D catalogue number
DC-SIGN C-type lectin Q9NNX6 Lys62–Ala404 N-terminal MD–human IgG1 Fc–IEGR fusion tag 161-DC-050 
Langerin C-type lectin Q9UJ71 Tyr64–Pro328 N-terminal 9× His tag 2088-LN-050 
Dectin-2 C-type lectin Q6EIG7 Thr46–Leu209 N-terminal 6× His tag 3114-DC-050 
MGL (CLEC10A) C-type lectin Q8IUN9 Gln61–His316 N-terminal 6× His tag 4888-CL-050 
Siglec-1 I-type lectin Q9BZZ2 Ser20–Gln1641 C-terminal 6× His tag 5197-SL-050 
Siglec-5 I-type lectin O15389 Glu17–Thr434 C-terminal IEGRID–human IgG1 Fc fusion tag 1072-SL-050 
Siglec-7 I-type lectin Q9Y286 Gln19–Gly357 C-terminal DIEGRMD–human IgG1 Fc fusion tag 1138-SL-050 
Siglec-9 I-type lectin Q9Y336 Gln18–Gly348 C-terminal DIEGRMD–human IgG1 Fc fusion tag 1139-SL-050 
Siglec-10 I-type lectin Q96LC7 Met17–Thr546 C-terminal IEGRMD–human IgG1 Fc fusion tag 2130-SL-050 
NameLectin familyGenBank accession numberAmino acid sequenceProtein fusion tagR&D catalogue number
DC-SIGN C-type lectin Q9NNX6 Lys62–Ala404 N-terminal MD–human IgG1 Fc–IEGR fusion tag 161-DC-050 
Langerin C-type lectin Q9UJ71 Tyr64–Pro328 N-terminal 9× His tag 2088-LN-050 
Dectin-2 C-type lectin Q6EIG7 Thr46–Leu209 N-terminal 6× His tag 3114-DC-050 
MGL (CLEC10A) C-type lectin Q8IUN9 Gln61–His316 N-terminal 6× His tag 4888-CL-050 
Siglec-1 I-type lectin Q9BZZ2 Ser20–Gln1641 C-terminal 6× His tag 5197-SL-050 
Siglec-5 I-type lectin O15389 Glu17–Thr434 C-terminal IEGRID–human IgG1 Fc fusion tag 1072-SL-050 
Siglec-7 I-type lectin Q9Y286 Gln19–Gly357 C-terminal DIEGRMD–human IgG1 Fc fusion tag 1138-SL-050 
Siglec-9 I-type lectin Q9Y336 Gln18–Gly348 C-terminal DIEGRMD–human IgG1 Fc fusion tag 1139-SL-050 
Siglec-10 I-type lectin Q96LC7 Met17–Thr546 C-terminal IEGRMD–human IgG1 Fc fusion tag 2130-SL-050 

Preparation and screening of HMG microarray for MAGS

A panel of HMG samples bound by DC-SIGN was printed on separate Nexterion N-hydroxysuccinimide (NHS) H slides (Schott) and screened with lectins, antibodies and DC-SIGN at three different concentrations of each sample. Slide printing and sample screening were performed as previously described [23]. The anti-sialyl-Lewis a antibody was purchased from Abcam. All of the other lectins, antibodies and glycosidases used for metadata-assisted glycan sequencing (MAGS), as well as the concentration(s) and glycosidase treatment procedures, are the same as described in a previous study [21]. Multi-dimensional mass spectrometry on HMG-9, HMG-19 and HMG-36 was performed as previously described [28].

Preparation of HMG-derivatized beads and flow cytometry assessment of binding

HMGs were first derivatized with AEAB [13] by reductive amination as previously described [24]. The HMGs were then coupled to 1.00 μm diameter PolyBead® carboxylate microspheres using the PolyLink Protein Coupling Kit (PolySciences) as follows. Beads (200 μl) were pelleted by gentle centrifugation at 500–1000 g for 5 min and resuspended in 160 μl of PolyLink coupling buffer. Then, 20 μl of 200 mg/ml freshly prepared EDC and 20 μl of freshly prepared sulfo-NHS (Thermo Scientific) were then added and the reaction mixture was incubated at room temperature with gentle rotation for 30 min. The beads were then washed twice with 250 μl of PolyLink Wash/Storage Buffer and then resuspended in 1 mM glycan-AEAB in 100 mM sodium phosphate (pH 8.5). The reaction mixture was incubated at room temperature with gentle mixing for 1–2 h. The beads were washed three times with PolyLink Wash/Storage Buffer and stored at 4°C in the same buffer until use.

For measurement of DC-SIGN binding to the glycan-derivatized beads, lacto-N-tetraose (LNT)-, 2′-fucosyl-lactose (2′-FL)- and 3-fucosyl-lactose (3-FL)-derivatized beads were incubated for 1 h with 5 μg/ml recombinant human DC-SIGN at room temperature, washed three times with PBS, and then incubated for 1 h with 2 μg/ml Alexa Fluor 633-labelled goat anti-human IgG. As a negative control, 2′-FL-derivatized beads were incubated with secondary antibody only (no DC-SIGN). All samples were analysed by flow cytometry with a BD FACSCalibur with the 633 nm laser. A total of 10000 events were counted, and the FL-4 filter was used for detection. The data were analysed using FlowJo. Gating was assigned in FlowJo by running the beads alone compared with buffer alone on a forward-scatter against side-scatter plot, with >99% of the events falling in the gated area for all samples.

RESULTS

Binding of hGBP to the human milk shotgun glycan microarray

A set of eight recombinant hGBP was tested for binding to HMGs, and this set included C-type lectins and sialic acid-binding immunoglobulin-like lectin (Siglec) members of the I-type lectin family (refer to Table 1 for all of the hGBP used in the present study). These hGBP were selected based on their stability, availability and known expression by DCs [10,2931]. The C-type lectins and Siglecs were screened on a human milk shotgun glycan microarray consisting of 247 HMG structures purified from human milk as well as 13 control glycans. This microarray was termed the HM-SGM-v2 [21]. However, only three of these hGBP, i.e. DC-SIGN, Siglec-5 and Siglec-9, showed binding to the HM-SGM-v2 (Figure 1; also refer to Supplementary Table S1 for the data for all concentrations of all hGBP screened). The binding of Langerin was considered inconclusive because high concentrations of protein were needed and the signal/noise ratio was poor (Supplementary Table S1). All other hGBP showed no evidence of binding to the HM-SGM-v2, although most bound to other glycan microarrays.

HM-SGM-v2 data for Siglec-5, Siglec-9 and DC-SIGN

Figure 1
HM-SGM-v2 data for Siglec-5, Siglec-9 and DC-SIGN

Siglec-5, Siglec-9 and DC-SIGN were screened on the HM-SGM-v2 at various concentrations. The results for 90 μg/ml Siglec-5 (a), 10 μg/ml Siglec-9 (b) and 10 μg/ml DC-SIGN with (c) or without (d) Ca2+ are shown; Alexa Fluor 488-labelled anti-human IgG was used for detection. Refer to Supplementary Table S1 for the results at all concentrations screened. For DC-SIGN without Ca2+ (d), Ca2+ was omitted from the binding buffer and 0.2 mM EDTA was added.

Figure 1
HM-SGM-v2 data for Siglec-5, Siglec-9 and DC-SIGN

Siglec-5, Siglec-9 and DC-SIGN were screened on the HM-SGM-v2 at various concentrations. The results for 90 μg/ml Siglec-5 (a), 10 μg/ml Siglec-9 (b) and 10 μg/ml DC-SIGN with (c) or without (d) Ca2+ are shown; Alexa Fluor 488-labelled anti-human IgG was used for detection. Refer to Supplementary Table S1 for the results at all concentrations screened. For DC-SIGN without Ca2+ (d), Ca2+ was omitted from the binding buffer and 0.2 mM EDTA was added.

DC-SIGN bound to many glycans on the HM-SGM-v2 (Figure 1c), specifically all of the glycans containing at least one α-linked fucose residue based on the calculated composition from MALDI–TOF-MS molecular mass measurements [21]. This binding was specific in that all binding required Ca2+ even at the highest DC-SIGN concentration used (Figure 1d). The large number of glycans bound by DC-SIGN on the HM-SGM-v2 necessitated further examination of these bound structures in order to define the HMG determinant recognized by DC-SIGN. To this end, a MAGS approach was used [32], where a number of structures bound by DC-SIGN were printed on a separate microarray and screened with lectins and antibodies that have defined binding to a variety of glycan determinants including α-fucosylated structures, terminal β1-3-linked or β1-4-linked galactose, α2-6-linked sialic acid, Lewis epitopes and Blood Group H Type 1 or Type 2 (Supplementary Table S2). DC-SIGN was also screened on this microarray and confirmed to bind all of the printed structures (Supplementary Table S2).

Based on the MAGS data and mass spectrometry sequencing data for some structures [33], proposed structures for the HMGs bound by DC-SIGN are shown in Figure 2. The key feature of all of these structures is the presence of α-linked fucose, specifically terminal Lewis a (Galβ1-3(Fucα1-4)GlcNAcβ-), terminal Lewis b (Fucα1-2Galβ1-3(Fucα1-4)GlcNAcβ-), terminal Lewis y (Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ-) and/or a terminal Lewis x (Galβ1-4(Fucα1-3)GlcNAcβ-) determinant. Not all fucosylated HMGs were bound though. For example, HMO-8 and HMO-29 contained one fucose, whereas HMO-37 and HMO-80 contained two fucoses but were not bound. The fucosylated glycan determinant present in these four structure was likely Blood Group H Type 1 since the anti-Blood Group H Type 1 antibody, but none of the anti-Lewis antibodies, bound these four structures. Additionally, HMO-23, -31, -41, -47, -48 and -49, containing one or two fucoses, were also not bound and contain only an internal Lewis x determinant (or, in the case of HMO-31, internal Lewis x as the major structures) [21,28]. Therefore, the Lewis x is a binding determinant of DC-SIGN only when present at the non-reducing end of HMGs. The binding of DC-SIGN to HMGs containing terminal Lewis glycan determinants, but not Blood Group H determinants on HMGs, also corroborates previous studies on the glycan specificity of DC-SIGN [34,35]. Additionally, 2′-FL (HMO-3) was also weakly bound on the HM-SGM-v2 (average rank=11), a ligand not seen in previous studies. Overall, these results suggest that DC-SIGN recognizes α-fucosylated HMGs containing Lewis glycan determinants at the non-reducing end as well as 2′-FL, and the high abundance of these structures and determinants in the HMGs metaglycome explains why DC-SIGN binds robustly to the HM-SGM-v2.

Proposed structures of HMGs bound by DC-SIGN on the HM-SGM-v2

Figure 2
Proposed structures of HMGs bound by DC-SIGN on the HM-SGM-v2

A portion of the HM-SGM-v2 structures bound by DC-SIGN was printed on a separate microarray and interrogated by MAGS, where multiple lectins and antibodies specific for particular glycan determinants were screened. Proposed structures for these HMGs samples are shown. HMG samples in bold-face font were further analysed by multi-dimensional mass spectrometry (MSn) to more accurately determine the structures(s) within these samples; HMG-20, HMG-21 and HMG-28 were previously sequenced by MSn [21,28], and HMG-9, HMG-19 and HMG-36 were also by sequenced by MSn in a more recent study [33]. Refer to Supplementary Table S2 for the lectin and antibody screening data.

Figure 2
Proposed structures of HMGs bound by DC-SIGN on the HM-SGM-v2

A portion of the HM-SGM-v2 structures bound by DC-SIGN was printed on a separate microarray and interrogated by MAGS, where multiple lectins and antibodies specific for particular glycan determinants were screened. Proposed structures for these HMGs samples are shown. HMG samples in bold-face font were further analysed by multi-dimensional mass spectrometry (MSn) to more accurately determine the structures(s) within these samples; HMG-20, HMG-21 and HMG-28 were previously sequenced by MSn [21,28], and HMG-9, HMG-19 and HMG-36 were also by sequenced by MSn in a more recent study [33]. Refer to Supplementary Table S2 for the lectin and antibody screening data.

Siglec-5 bound weakly to four sialylated HMGs: HMO-157, HMO-213, HMO-118 and HMO-237 (Figure 1a). However, this binding required a high Siglec-5 concentration of 90 μg/ml and the signal was detectable but weak. Although the structures of these four HMGs have not been completely defined, HMO-157, HMO-213 and HMO-237 were bound by GM35-specific monoclonal antibody [21], which we have shown to bind to the sialyl-Lewis a determinant (Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ-) and so-called ‘sialyl-Lewis c’ (Neu5Acα2-3Galβ1-3GlcNAcβ) determinant [36]. These data suggest that Siglec-5 binds to a restricted set of HMGs structures containing the sialyl-Lewis c determinant, but the reason that Siglec-5 bound to only a restricted subset of all structures containing this determinant is unclear, since several other glycans on the array also were bound by GM35 but not Siglec-5. HMO-118 is likely to be a mixture that contains 3′-sialyl-lactose (3′-SL) based on its predicted composition; however, 3′-SL itself was not bound on the defined HMG microarray by Siglec-5 (described in more detail below), suggesting that trace glycans within HMO-118 may have contributed to binding. Siglec-9 bound an extensive number of HMGs, all of which are sialylated, although three of the four HMGs bound by Siglec-5 (HMO-157, HMO-213 and HMO-118) were consistently the strongest Siglec-9 binders as well (Figure 1b). However, the binding of Siglec-9 was only weakly dose-dependent (Supplementary Table S1) and oddly depended on reducing end derivatization of the glycans, as discussed below. Thus, the results indicate that DC-SIGN robustly recognizes a number of α-fucosylated HMGs, whereas Siglec binding is weak and may not be significant. In regard to Siglec binding, the significance was further tested below.

Binding of hGBP to the CFG glycan microarray

To confirm that all hGBP were active, they were concurrently screened on the microarray from CFG. Most hGBP tested on the CFG microarray showed binding to at least two glycans on that microarray (Supplementary Table S3). However, the CFG microarray data for Siglec-1 was deemed relatively inconclusive as no specific candidate glycans were identified. Many of the hGBP that did not bind to the HM-SGM-v2 bound glycan determinants on the CFG microarray that were not found on the HM-SGM-v2, verifying hGBP activity. For example, Dectin-2 is specific for mannan structures containing the motif Manα1-2Manα1-6(Manα1-3)Manα- or Manα1-2Manα1-6Manα1-6(Manα1-3)Manα-, although Dectin-2 also weakly bound Man8–9GlcNAc2 N-glycan structures. A common feature of all structures bound by macrophage galactose/N-acetylgalactosamine-type lectin (MGL, CLEC10A) was the presence of D-N-acetylgalactosamine (GalNAc), particularly at the reducing and/or non-reducing end, although not all these GalNAc-containing structures were bound. Early studies suggested that Blood Group A and B antigens and enzymes with Blood Group A and B activity may be present in human milk (reviewed in [37]), but this has not been confirmed in more recent studies [38]. Indeed, we have screened the HM-SGM-v2 microarray with an antibody recognizing Blood Group A Type 1–3 determinants and saw no binding of this antibody (results not shown), confirming more recent studies that Blood Group A (and most likely Blood Group B) determinants are not present at detectable amounts on HMGs. Thus, since mannose and GalNAc are not found on free HMGs, it is logical that Dectin-2 and MGL-1 did not bind the HMGs microarrays.

For the Siglecs, most showed a broad binding pattern on the CFG glycan microarray but no binding to the few HMGs present on the CFG microarray. Siglec-5 bound some, but not all, complex N-glycans containing terminal β1-3-linked galactose; beyond that, Siglec-5 did not bind to a common motif. In contrast with the HM-SGM-v2, we did not observe Siglec-5 binding to sialylated HMGs on the CFG microarray, including a lack of binding to 3′-SL and all of the non-HMG glycans containing the SLec determinant. Siglec-5 also did not bind a microarray consisting of defined HMG structures (as described below), suggesting that Siglec-5 may poorly bind HMGs and thus was possibly binding trace contaminants on the HM-SGM-v2 or only binds to only specific glycan presentations such as glycans with specific linkers. Siglec-7 bound to a variety of sialylated structures, the strongest of which was sialyl-Lewis x containing 6-O-sulfated GlcNAc; some N-glycan structures and α2-8-sialylated structures were also bound. However, no motifs found on HMGs were bound by Siglec-7. Siglec-10 showed a very broad binding pattern, including binding to both sialylated and non-siaylated glycans. The biological and biochemical significance of the Siglec-10 binding to non-sialylated glycans is currently unclear, but we believe that the binding may have been artificially induced by the presentation and/or aglycone component (refer to the Discussion for more information). Langerin not only strongly bound mannan and high-mannose N-glycan structures (Man6–9GlcNAc2) but also lactose that was 6-O-sulfated on the galactose, but neither determinant is found on HMGs. Additionally, Langerin bound weakly to glycans containing terminal β-linked GlcNAc, Blood Group H Types 1 and 2, Blood Group A and B Type 2, Lewis y and other sulfated glycan determinants. These results are in good agreement with previous glycan microarray results for Langerin [39]. Although Type 1 Blood Group H and Lewis y are found on some HMGs, Langerin binding to the HMGs microarrays was inconclusive (Supplementary Table S1).

The screening of DC-SIGN on the CFG microarray revealed three major motifs (Table 2 and Supplementary Table S3). The first motif was terminal α1-2-linked mannose on mannan backbones, including high-mannose N-glycans, although the mannans containing α-linked mannose at the reducing end were bound slightly stronger than the high mannose N-glycans. The second motif was the Lewis a determinant, including Lewis b structures. The third motif was the Lewis x determinant at the non-reducing end of glycan structures, which also included Lewis y structures. Notably, the Lewis a and non-reducing end Lewis x determinants were also the major HMG-binding determinants revealed by the HM-SGM-v2 screening (Table 2). Sialyl-Lewis a and especially sialyl-Lewis x structures were typically poorly bound by DC-SIGN, although some sialylated fucosylated HMGs were bound on the HM-SGM-v2 whose structures remain to be determined (Figure 1 and Supplementary Table S1). Glycans containing Blood Group H Type 1 and  2 determinants, as well as 2′-FL, were poorly bound by DC-SIGN on the CFG microarray, a finding also seen in previous studies [35]. This further suggests that α1-2 fucosylated glycan structures are lower-affinity than Lewis a- and Lewis x-containing structures. Binding motifs for Siglec-9 on the CFG microarray were sialyl-Lewis x on N-glycans as well as 3′- or 6′-sialyl-lactosamine (Neu5Acα2-3/6Galβ1-4GlcNAcβ-) that was 6-O-sulfated on the GlcNAc, but binding was slightly stronger to 6-O-sulfo-sialyl-Lewis x as previously seen on this array (refer to the CFG website, http://www.functionalglycomics.org/glycomics/publicdata/primaryscreen.jsp); importantly, these motifs are not found on HMGs. Siglec-9 also weakly bound 3′-sialyl-lactosamine and 6′-sialyl-lactosamine as well as the sialyl-Lewis x tetrasaccharide (Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc), although the HMGs 3′-SL and 6′-sialyl-lactose (6′-SL) were poorly, if at all, bound. Siglec-9 binding to the CFG microarray was also poorly dose-dependent, as seen when Siglec-9 was screened on the defined HMG microarray, suggesting that the binding may not be specific. Therefore, only DC-SIGN was concluded to be a strong HMG receptor, whereas Siglec-5, Siglec-9 and Langerin are likely to be poor HMG receptors.

Table 2
DC-SIGN CFG glycan microarray-binding motifs
graphic
graphic

Binding of hGBP to a defined HMG microarray

To further investigate the binding of hGBP to HMGs, the hGBP were also screened on a microarray consisting of a selection of chemically defined HMG-related glycans and galacto-oligosaccharides (GOSs) that are commonly used or under experimental testing as supplements in infant formula. This microarray was termed the ‘defined HMG microarray’. As expected from the HM-SGM-v2 screenings, DC-SIGN and Siglec-9 bound structures on the defined HMG microarray (Figure 3), whereas all other hGBP showed no binding (refer to Supplementary Table S4 for the defined HMG glycan microarray data at all concentrations of all hGBP screened). In contrast with the HM-SGM-v2 data, Siglec-5 at 90 μg/ml did not bind to the defined HMG microarray even though HMO-118 (likely 3′-SL) was bound by Siglec-5 on the HM-SGM-v2. This suggests that Siglec-5 binds 3′-SL with low affinity and HMO-118 may contain trace contaminants that improved Siglec-5 binding. Siglec-9 bound to both 3′-SL and 6′-SL.

Defined HMG microarray screening data for DC-SIGN and Siglec-9

Figure 3
Defined HMG microarray screening data for DC-SIGN and Siglec-9

Samples of 10 μg/ml and 50 μg/ml DC-SIGN (a) and 10 μg/ml and 90 μg/ml Siglec-9 (b) were screened on the defined HMG microarray, and Alexa Fluor 488-labelled anti-human IgG was used for detection. Histograms on the left show the lower concentrations and those on the right show the higher concentrations. Refer to Supplementary Table S4 for the raw data for these screenings.

Figure 3
Defined HMG microarray screening data for DC-SIGN and Siglec-9

Samples of 10 μg/ml and 50 μg/ml DC-SIGN (a) and 10 μg/ml and 90 μg/ml Siglec-9 (b) were screened on the defined HMG microarray, and Alexa Fluor 488-labelled anti-human IgG was used for detection. Histograms on the left show the lower concentrations and those on the right show the higher concentrations. Refer to Supplementary Table S4 for the raw data for these screenings.

DC-SIGN bound the fucosylated HMGs 2′-FL and 3-FL, whereas very weak binding was seen towards the Blood Group H Type 1-containing glycan lacto-N-fucopentaose I (LNFPI). It should be noted that 2′-FL (HMO-3) but not 3-FL (HMO-2) was bound on the HM-SGM-v2, although 2′-FL was weakly bound relative to Lewis a and Lewis x structures (average rank=11; Supplementary Figure S1). However, DC-SIGN binding to 3-FL was highly dependent on maintaining the ring structure of the reducing end glucose because DC-SIGN poorly bound to reductively aminated 3-FL (Figure 3 and Supplementary Table S4), which was the only ring form of glycans on the HM-SGM-v2. Thus, the actual strength of binding to 3-FL was probably underestimated on the HM-SGM-v2. In contrast with the HMG microarray results, 2′-FL was not bound by DC-SIGN on the CFG glycan microarray (Chart ID 77, rank < 10; Supplementary Figure S3). The reason for this non-binding on the CFG glycan microarray is unknown, but may have to do with differences in the linker or other presentation issues compared with the HMG microarrays. 3-FL was absent from the CFG glycan microarray. This suggests that 2′-FL and 3-FL are weaker ligands than structures containing terminal Lewis a or Lewis x determinants, although more studies are needed to confirm this observation. Overall, these results suggest that 2′-FL and 3-FL are also ligands for DC-SIGN. Despite their potentially lower binding strength than Lewis a and Lewis x-containing HMGs, 2′-FL and 3-FL are much more abundant than these Lewis a- and Lewis x-containing HMG structures in human milk, with concentrations ranging from approximately 0.5 to 5 mM for these two HMGs [2]. The overall results of this experiment show that Siglec-9 and DC-SIGN, but not Siglec-5, may also bind to simple defined HMG structures.

Binding of hGBP to the beads derivatized with HMGs

To confirm the binding of DC-SIGN to the defined HMGs 2′-FL and 3-FL in a different format, polystyrene beads were derivatized with 2′-FL, 3-FL or LNT and binding of DC-SIGN to these derivatized beads was measured by flow cytometry (Figure 4). As expected, DC-SIGN bound to the 2′-FL- and 3-FL-derivatized beads but not the LNT-derivatized beads (non-fucosylated HMGs control), which further confirmed the binding of DC-SIGN to fucosylated HMGs.

DC-SIGN binding to HMG-derivatized microspheres

Figure 4
DC-SIGN binding to HMG-derivatized microspheres

DC-SIGN was incubated with microspheres (beads) derivatized with 2′-FL, 3-FL or LNT. Alexa Fluor 633-labelled anti-human IgG was used for detection of recombinant DC-SIGN. 2′-FL microspheres incubated with the Alexa Fluor 633-labelled anti-human IgG alone was used as the negative control. All samples were analysed by flow cytometry with a 633 nm laser and FL-4 filter for detection. Histograms of DC-SIGN binding to LNT beads (thick line), 2′-FL beads (thin black line) and 3-FL beads (thin grey line), as well as secondary antibody alone binding to 2′-FL beads (filled line), are shown.

Figure 4
DC-SIGN binding to HMG-derivatized microspheres

DC-SIGN was incubated with microspheres (beads) derivatized with 2′-FL, 3-FL or LNT. Alexa Fluor 633-labelled anti-human IgG was used for detection of recombinant DC-SIGN. 2′-FL microspheres incubated with the Alexa Fluor 633-labelled anti-human IgG alone was used as the negative control. All samples were analysed by flow cytometry with a 633 nm laser and FL-4 filter for detection. Histograms of DC-SIGN binding to LNT beads (thick line), 2′-FL beads (thin black line) and 3-FL beads (thin grey line), as well as secondary antibody alone binding to 2′-FL beads (filled line), are shown.

HMG inhibition of hGBP binding

Experiments using glycan microarrays and beads are useful for defining glycan specificity and potential binding of hGBP to HMGs. However, the glycan microarray screenings themselves have a few important limitations. Specifically, the glycans on the microarray are synthetically derivatized with a bifunctional linker at the reducing end and presented in a solid-phase format, which is in contrast with HMGs that occur as free reducing glycans in human milk. To confirm that DC-SIGN and Siglec-9 can also bind to free underivatized HMGs in solution, DC-SIGN and Siglec-9 were screened on the defined HMG microarray in the presence or absence of various concentrations of 2′-FL and 6′-SL respectively; lactose was used as a negative control for non-specific HMGs inhibition. DC-SIGN binding to both the defined HMG microarray (Figure 5a) and the MAGS array (Figure 5b) was inhibited by 2′-FL (refer to Supplementary Table S5 for the total data for DC-SIGN inhibition) in a dose-dependent manner and with an approximate IC50 of 1 mM for 2′-FL, confirming that DC-SIGN specifically binds to natural 2′-FL and in solution. Lactose (1–10 mM) caused little or no inhibition of DC-SIGN binding to the defined HMG microarray (Supplementary Table S5), confirming that the presence of α-linked fucose is required for DC-SIGN binding. The data also confirm binding to all of the HMGs on the HM-SGM-v2 and defined HMG microarrays was specific.

Inhibition of DC-SIGN binding to HMG microarrays with free underivatized HMGs

Figure 5
Inhibition of DC-SIGN binding to HMG microarrays with free underivatized HMGs

DC-SIGN at 1 μg/ml was preincubated with or without 0.1, 1 or 10 mM of free underivatized 2′-FL and then screened on the defined HMG microarray (a) or the HMG MAGS microarray (b) described in Supplementary Table S2. The results for 0 mM 2′-FL (no inhibitor), 1 mM 2′-FL and 10 mM 2′-FL on both microarrays are shown; refer to Supplementary Table S5 for the results of all other screenings, including 0.1 mM 2′-FL.

Figure 5
Inhibition of DC-SIGN binding to HMG microarrays with free underivatized HMGs

DC-SIGN at 1 μg/ml was preincubated with or without 0.1, 1 or 10 mM of free underivatized 2′-FL and then screened on the defined HMG microarray (a) or the HMG MAGS microarray (b) described in Supplementary Table S2. The results for 0 mM 2′-FL (no inhibitor), 1 mM 2′-FL and 10 mM 2′-FL on both microarrays are shown; refer to Supplementary Table S5 for the results of all other screenings, including 0.1 mM 2′-FL.

On the other hand, Siglec-9 binding to the defined HMG microarray was not inhibited by even 10 mM 6′-SL (Figure 6a), although binding could be inhibited by 1 mM 6′-SL derivatized with the AEAB linker at the reducing end (Figure 6b; also see Supplementary Table S6 for the total data for Siglec-9 inhibition). Therefore, Siglec-9 did not appear to bind the natural form of 6′-SL (and probably 3′-SL), only the chemically derivatized version; this suggests that Siglec-9 binding to the defined HMG microarray only occurs because of this HMG derivatization. The solution Kd of Siglec-9 for free 6′-SL and 3′-SL was determined to be >10 mM, which is likely to not be physiologically relevant.

Inhibition of Siglec-9 binding to defined HMG microarray with free underivatized HMGs and free derivatized 6′-sialyl-lactose

Figure 6
Inhibition of Siglec-9 binding to defined HMG microarray with free underivatized HMGs and free derivatized 6′-sialyl-lactose

Siglec-9 at 2 μg/ml was preincubated with 1 mM or 10 mM free underivatized 6′-SL or no inhibitor and screened on the defined HMG microarray (a). Siglec-9 at 2 μg/ml was preincubated with free 1 mM 6′-SL-AEAB (Neu5Acα2-6Galβ1-4Glcitol-AEAB) or 6′-SL-GGAEAB (Neu5Acα2-6Galβ1-4Glc-GGAEAB) [25] or no inhibitor and screened on the defined HMG microarray (b). Experiments in (a) and (b) were performed on separate slides but on the same day and at the same time. Refer to Supplementary Table S6 for all other free inhibition of Siglec-9 binding to the defined HMG microarray.

Figure 6
Inhibition of Siglec-9 binding to defined HMG microarray with free underivatized HMGs and free derivatized 6′-sialyl-lactose

Siglec-9 at 2 μg/ml was preincubated with 1 mM or 10 mM free underivatized 6′-SL or no inhibitor and screened on the defined HMG microarray (a). Siglec-9 at 2 μg/ml was preincubated with free 1 mM 6′-SL-AEAB (Neu5Acα2-6Galβ1-4Glcitol-AEAB) or 6′-SL-GGAEAB (Neu5Acα2-6Galβ1-4Glc-GGAEAB) [25] or no inhibitor and screened on the defined HMG microarray (b). Experiments in (a) and (b) were performed on separate slides but on the same day and at the same time. Refer to Supplementary Table S6 for all other free inhibition of Siglec-9 binding to the defined HMG microarray.

DISCUSSION

A major finding of the present study is that DC-SIGN is the only hGBP tested that showed specific binding to HMGs and binding was most robust towards α-fucosylated glycans. A striking observation was the proportion of HMGs bound by DC-SIGN. About half of the HMG structures on the HM-SGM-v2 were bound by 10 μg/ml DC-SIGN (Figure 1, Supplementary Table S1), suggesting that DC-SIGN binds to nearly half of the structures in the HMG metaglycome. The strongest binding was towards HMGs containing a Lewis glycan determinant at the non-reducing end (Figures 13, Supplementary Tables S1–S3). Potentially weaker but probably physiologically significant binding of DC-SIGN to 2′-FL and 3-FL was also observed (Figures 1, 3 and 5, Supplementary Tables S1, S4 and S5). HMGs containing only internal Lewis x or Blood Group H Type 1 were poorly, if at all, bound by DC-SIGN. Therefore, DC-SIGN appears to be a receptor for specific fucosylated HMGs.

The approximate IC50 of DC-SIGN for 2′-FL inhibition of binding to the glycan microarray was 1 mM (Figure 5). Given the typical concentration of 1–5 mM (0.5–2.5 g/l) 2′-FL in secretor-positive human milk [2], this suggests that the binding is within the physiological range. Taking into account that DC-SIGN also binds half of the total HMGs metaglycome in secretor- and Lewis-positive individuals (Figure 1) as well as glycoproteins in human milk such as bile salt-stimulated lipase [40] and MUC1 [17], the actual concentration of DC-SIGN glycan determinants in human milk is probably much higher (∼5–10 mM), suggesting that DC-SIGN may be close to ligand saturation when exposed to human milk (assuming an average Kd of 1 mM). Total HMGs have been previously shown to block DC-SIGN binding to HIV virions [41], further suggesting that some HMGs are DC-SIGN ligands and can block DC-SIGN functions.

Human intestinal DCs express DC-SIGN [42], and DC-SIGN expression is known to occur on cells (probably DCs) in infant gastrointestinal (GI) tract tissue [17]. DCs can extend their dendrites from the lamina propria into the intestinal lumen to ‘sample’ microbes [18]. Since HMGs are not significantly digested by the human repertoire of digestive mechanisms and enzymes in the GI tract [43,44], DC-SIGN on DCs may be exposed to and bind HMGs to near saturable levels in the small intestine of breast-fed infants. DC-SIGN is also known to modulate immune responses, although this binding is not yet known to be a direct stimulator of gene expression [10]. However, it is possible that the interaction of DC-SIGN with HMGs may cause changes in the DC-SIGN-mediated modulation of immune responses and may also help to mechanistically explain how HMGs promote changes in gene expression and immune responses [6]. Notwithstanding, how such interactions occur and whether the HMGs act as agonists or antagonists of DC-SIGN activity is still not fully understood. Interestingly, approximately 20% of individuals lack the secretor enzyme responsible for producing α1-2-fucosylated HMGs and 2′-FL, approximately 10% of individuals lack the Lewis enzyme responsible for producing α1-4-fucosylated HMGs (Lewis a structures), and ∼1% of individuals lack both enzymes [45]. Thus, milk from secretor-negative and/or Lewis-negative individuals may not be capable or interacting as well with DC-SIGN as milk from secretor- and Lewis-positive mothers, although this might be at least partially compensated for by the increased 3-FL concentration in non-secretor compared with secretor human milk as 3-FL is also a DC-SIGN ligand (Figures 3 and 5, Supplementary Tables S3 and S5). The physiological consequence of lacking the secretor and/or Lewis enzyme on DC-SIGN binding in vivo is thus unclear.

Unexpectedly, given the fact that sialic acid is a common residue in HMGs, the only Siglecs tested that showed some binding to HMGs were Siglec-5 and Siglec-9 (Figure 1), consistent with the possibility that glycan recognition by Siglecs is complex and the presence of sialic acid is necessary but not sufficient in most cases. Siglec-5 binding was weak and only occurred at high Siglec-5 concentrations, whereas Siglec-9 binding was stronger but binding to the free underivatized HMGs 6′-SL was still weak (Figure 6). Instead, Siglec-9 bound strongly to 6′-SL derivatized at the reducing end with an aglycone linker, AEAB and especially GGAEAB. This finding suggests that the aglycone component and/or multivalent presentation may be an important factor in Siglec-9 and other Siglecs for binding glycoconjugate ligands, or that specific sialylated glycans yet to be identified are strong ligands for Siglecs. This finding of the potential importance of the aglycone in Siglec binding may also explain why the binding of Siglecs to the CFG microarray in the present study has a generally weak broad binding pattern. This result may be due to differences in glycan presentation, which may have positively or negatively affected by the presence of specific aglycone linker units. Thus, the weak broad binding pattern of Siglecs to the CFG microarrays was likely to be because of non-preferential glycan presentation and/or aglycone components as opposed to poor Siglec activity or the recombinant Siglec construct used. Future studies in our laboratory are aimed at understanding the functional importance of aglycone components, especially natural aglycone components such as lipids and peptides, in Siglec binding. This future study may also unravel why some Siglecs, especially Siglec-10, bind to a few non-sialylated glycans.

The glycan presentation in multivalent forms may be most important for Siglec binding, as prior studies showed that Siglec-1, -3, -5, -7, and -9 all bound 3′-SL and 6′-SL-derivatized beads with micromolar affinity constants by surface plasmon resonance [15]. The multivalent presentation and/or aglycone bead component may contribute to this strong binding, since an IC50 of ∼1 mM was calculated for free 6′-SL inhibition [15], which was ∼100–1000-fold higher than for the Kd for 6′-SL beads. Siglec-5 binds 3′-SL and 6′-SL with a Kd of 2–4 μM but to free 3′-SL and 6′-SL with a Kd of ∼8 mM [46], which is high relative to the concentrations of these two sialylated glycans in human milk. Based on these current and previous findings, we conclude that the binding of DC-expressed Siglecs to free underivatized HMGs is weak and likely to be non-physiological. We speculate that this low-affinity binding is due to the lack of an aglycone component on and/or multivalent presentation of HMGs, which normally exist as free reducing glycan structures in solution. These findings also stress the importance of using other methodologies besides glycan microarrays to confirm binding of samples to HMGs, which naturally exist as free underivatized structures in solution.

In addition to C-type lectins and Siglecs used in the present study, other hGBP have been screened on the HMG microarrays. These include galectins, most of which showed binding and, in some cases, robust binding to neutral HMGs [22]. Preliminary HMG microarray screenings of the three human selectins (P-, E- and L-selectin), which are known to bind sialyl-Lewis x and sialyl-Lewis a determinants in solution with relatively low affinity [47,48], were negative. This suggests that human selectins are poor HMGs receptors, consistent with previous studies showing that, although selectins may bind HMGs, the interaction and effects are weak [4951]. Preliminary screenings with Siglec-11 also revealed no binding of these hGBP to the HMG microarrays. Future studies are aimed at examining other receptors that bind glycoconjugates, including Toll-like receptors and cytokine receptors.

The present study adds DC-SIGN to the list of hGBP that may act as HMG receptors. Given the physiological concentration of HMGs binding to DC-SIGN and galectins, as well as the anatomical localization and expression patterns of these hGBP, these interactions may be important mechanisms underlying the known HMGs functions of regulating gene expression and immune responses [6]. Therefore, future studies to understand the interactions of these hGBP with HMGs and subsequent physiological effects are currently underway.

AUTHOR CONTRIBUTION

Alexander Noll, Ying Yu, Geralyn Duska-McEwen, Rachael Buck, David Smith and Richard Cummings proposed and designed experiments. Ying Yu performed experiments. Alexander Noll, Ying Yu, David Smith and Richard Cummings analysed data. Yi Lasanajak, Geralyn Duska-McEwen and Rachael Buck provided critical reagents and support. Alexander Noll and Richard Cummings organized data and wrote the paper with review, comments and contributions from all authors.

We thank Jamie Heimburg-Molinaro for a critical review of the paper and Sandra Cummings and Hong Ju for technical assistance.

CONFLICT OF INTEREST

R.D.C. and D.F.S. are consultants for Abbott Nutrition. G.D.M. and R.H.B. are employees of Abbott Nutrition. The other authors declare that they have no conflict of interest in the work reported.

FUNDING

This work was supported by the National Institutes of Health [grant number P41GM103694 (to R.D.C.)]; and Abbott Nutrition, Columbus, OH.

Abbreviations

     
  • AEAB

    N-aminoethyl 2-aminobenzamide

  •  
  • CFG

    Consortium for Functional Glycomics

  •  
  • DC

    dendritic cell

  •  
  • DC-SIGN

    dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin

  •  
  • 2′-FL

    2′-fucosyl-lactose

  •  
  • 3-FL

    3-fucosyl-lactose

  •  
  • Fuc

    L-fucose

  •  
  • Gal

    D-galactose

  •  
  • GalNAc

    D-N-acetylgalactosamine

  •  
  • GGAEAB

    glycosylamide-glycyl-N-aminoethyl 2-aminobenzamide

  •  
  • GI

    gastrointestinal

  •  
  • Glc

    D-glucose

  •  
  • GlcNAc

    D-N-acetylglucosamine

  •  
  • hGBP

    human glycan-binding protein

  •  
  • HMG

    human milk glycan

  •  
  • HM-SGM-v2

    human milk shotgun glycan microarray version 2

  •  
  • LNT

    lacto-N-tetraose

  •  
  • MAGS

    metadata-assisted glycan sequencing

  •  
  • MGL

    macrophage galactose/N-acetylgalactosamine-type lectin

  •  
  • Neu5Ac

    5-N-acetylneuraminic acid

  •  
  • NHS

    N-hydroxysuccinimide

  •  
  • Siglec

    sialic acid-binding immunoglobulin-like lectin

  •  
  • 3′-SL

    3′-sialyl-lactose

  •  
  • 6′-SL

    6′-sialyl-lactose

References

References
1
Kunz
 
C.
Rudloff
 
S.
Baier
 
W.
Klein
 
N.
Strobel
 
S.
 
Oligosaccharides in human milk: structural, functional, and metabolic aspects
Annu. Rev. Nutr.
2000
, vol. 
20
 (pg. 
699
-
722
)
[PubMed]
2
Urashima
 
T.
Asakuma
 
S.
Leo
 
F.
Fukuda
 
K.
Messer
 
M.
Oftedal
 
O.T.
 
The predominance of type I oligosaccharides is a feature specific to human breast milk
Adv. Nutr.
2012
, vol. 
3
 (pg. 
473S
-
482S
)
[PubMed]
3
Newburg
 
D.S.
 
Glycobiology of human milk
Biochemistry (Mosc.)
2013
, vol. 
78
 (pg. 
771
-
785
)
[PubMed]
4
Newburg
 
D.S.
Ruiz-Palacios
 
G.M.
Morrow
 
A.L.
 
Human milk glycans protect infants against enteric pathogens
Annu. Rev. Nutr.
2005
, vol. 
25
 (pg. 
37
-
58
)
[PubMed]
5
Zivkovic
 
A.M.
German
 
J.B.
Lebrilla
 
C.B.
Mills
 
D.A.
 
Human milk glycobiome and its impact on the infant gastrointestinal microbiota
Proc. Natl. Acad. Sci. U.S.A.
2011
, vol. 
108
 
Suppl. 1
(pg. 
4653
-
4658
)
[PubMed]
6
Bode
 
L.
 
Human milk oligosaccharides: every baby needs a sugar mama
Glycobiology
2012
, vol. 
22
 (pg. 
1147
-
1162
)
[PubMed]
7
Castillo-Courtade
 
L.
Han
 
S.
Lee
 
S.
Mian
 
F.M.
Buck
 
R.
Forsythe
 
P.
 
Attenuation of food allergy symptoms following treatment with human milk oligosaccharides in a mouse model
Allergy
2015
, vol. 
70
 (pg. 
1091
-
1102
)
[PubMed]
8
Bienenstock
 
J.
Buck
 
R.H.
Linke
 
H.
Forsythe
 
P.
Stanisz
 
A.M.
Kunze
 
W.A.
 
Fucosylated but not sialylated milk oligosaccharides diminish colon motor contractions
PLoS One
2013
, vol. 
8
 pg. 
e76236
 
[PubMed]
9
Vazquez
 
E.
Barranco
 
A.
Ramirez
 
M.
Gruart
 
A.
Delgado-Garcia
 
J.M.
Martinez-Lara
 
E.
Blanco
 
S.
Martin
 
M.J.
Castanys
 
E.
Buck
 
R.
, et al 
Effects of a human milk oligosaccharide, 2′-fucosyllactose, on hippocampal long-term potentiation and learning capabilities in rodents
J. Nutr. Biochem.
2015
, vol. 
26
 (pg. 
455
-
465
)
[PubMed]
10
Geijtenbeek
 
T.B.
Gringhuis
 
S.I.
 
Signalling through C-type lectin receptors: shaping immune responses
Nat. Rev. Immunol.
2009
, vol. 
9
 (pg. 
465
-
479
)
[PubMed]
11
Crocker
 
P.R.
Paulson
 
J.C.
Varki
 
A.
 
Siglecs and their roles in the immune system
Nat. Rev. Immunol.
2007
, vol. 
7
 (pg. 
255
-
266
)
[PubMed]
12
McEver
 
R.P.
 
Adhesive interactions of leukocytes, platelets, and the vessel wall during hemostasis and inflammation
Thromb. Haemost.
2001
, vol. 
86
 (pg. 
746
-
756
)
[PubMed]
13
Song
 
X.
Xia
 
B.
Stowell
 
S.R.
Lasanajak
 
Y.
Smith
 
D.F.
Cummings
 
R.D.
 
Novel fluorescent glycan microarray strategy reveals ligands for galectins
Chem. Biol.
2009
, vol. 
16
 (pg. 
36
-
47
)
[PubMed]
14
Guo
 
Y.
Feinberg
 
H.
Conroy
 
E.
Mitchell
 
D.A.
Alvarez
 
R.
Blixt
 
O.
Taylor
 
M.E.
Weis
 
W.I.
Drickamer
 
K.
 
Structural basis for distinct ligand-binding and targeting properties of the receptors DC-SIGN and DC-SIGNR
Nat. Struct. Mol. Biol.
2004
, vol. 
11
 (pg. 
591
-
598
)
[PubMed]
15
Zou
 
Z.
Chastain
 
A.
Moir
 
S.
Ford
 
J.
Trandem
 
K.
Martinelli
 
E.
Cicala
 
C.
Crocker
 
P.
Arthos
 
J.
Sun
 
P.D.
 
Siglecs facilitate HIV-1 infection of macrophages through adhesion with viral sialic acids
PLoS One
2011
, vol. 
6
 pg. 
e24559
 
[PubMed]
16
Brinkman-Van der Linden
 
E.C.
Varki
 
A.
 
New aspects of siglec binding specificities, including the significance of fucosylation and of the sialyl-Tn epitope. Sialic acid-binding immunoglobulin superfamily lectins
J. Biol. Chem.
2000
, vol. 
275
 (pg. 
8625
-
8632
)
[PubMed]
17
Koning
 
N.
Kessen
 
S.F.
Van Der Voorn
 
J.P.
Appelmelk
 
B.J.
Jeurink
 
P.V.
Knippels
 
L.M.
Garssen
 
J.
Van Kooyk
 
Y.
 
Human milk blocks DC-SIGN-pathogen interaction via MUC1
Front. Immunol.
2015
, vol. 
6
 pg. 
112
 
[PubMed]
18
Rescigno
 
M.
Urbano
 
M.
Valzasina
 
B.
Francolini
 
M.
Rotta
 
G.
Bonasio
 
R.
Granucci
 
F.
Kraehenbuhl
 
J.P.
Ricciardi-Castagnoli
 
P.
 
Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria
Nat. Immunol.
2001
, vol. 
2
 (pg. 
361
-
367
)
[PubMed]
19
Cummings
 
R.D.
McEver
 
R.P.
 
Varki
 
A.
Cummings
 
R.D.
Esko
 
J.D.
Freeze
 
H.H.
Stanley
 
P.
Bertozzi
 
C.R.
Hart
 
G.W.
Etzler
 
M.E.
 
C-type lectins
Essentials of Glycobiology
2009
Cold Spring Harbor
Cold Spring Harbor Laboratory Press
(pg. 
439
-
458
)
20
Varki
 
A.
Crocker
 
P.R.
 
Varki
 
A.
Cummings
 
R.D.
Esko
 
J.D.
Freeze
 
H.H.
Stanley
 
P.
Bertozzi
 
C.R.
Hart
 
G.W.
Etzler
 
M.E.
 
I-type lectins
Essentials of Glycobiology
2009
Cold Spring Harbor
Cold Spring Harbor Laboratory Press
(pg. 
459
-
474
)
21
Yu
 
Y.
Lasanajak
 
Y.
Song
 
X.
Hu
 
L.
Ramani
 
S.
Mickum
 
M.L.
Ashline
 
D.J.
Prasad
 
B.V.
Estes
 
M.K.
Reinhold
 
V.N.
, et al 
Human milk contains novel glycans that are potential decoy receptors for neonatal rotaviruses
Mol. Cell. Proteomics
2014
, vol. 
13
 (pg. 
2944
-
2960
)
[PubMed]
22
Noll
 
A.J.
Gourdine
 
J.P.
Yu
 
Y.
Lasanajak
 
Y.
Smith
 
D.F.
Cummings
 
R.D.
 
Galectins are human milk glycan receptors
Glycobiology
2016
[PubMed]
23
Heimburg-Molinaro
 
J.
Song
 
X.
Smith
 
D.F.
Cummings
 
R.D.
 
Preparation and analysis of glycan microarrays
Curr. Protoc. Protein Sci.
2011
 
Chapter 12, 12.10.11–12.10.29
[PubMed]
24
Song
 
X.
Heimburg-Molinaro
 
J.
Smith
 
D.F.
Cummings
 
R.D.
 
Derivatization of free natural glycans for incorporation onto glycan arrays: derivatizing glycans on the microscale for microarray and other applications (ms# CP-10-0194)
Curr. Protoc. Chem. Biol.
2011
, vol. 
3
 (pg. 
53
-
63
)
[PubMed]
25
Song
 
X.
Lasanajak
 
Y.
Xia
 
B.
Smith
 
D.F.
Cummings
 
R.D.
 
Fluorescent glycosylamides produced by microscale derivatization of free glycans for natural glycan microarrays
ACS Chem. Biol.
2009
, vol. 
4
 (pg. 
741
-
750
)
[PubMed]
26
Smith
 
D.F.
Song
 
X.
Cummings
 
R.D.
 
Use of glycan microarrays to explore specificity of glycan-binding proteins
Methods Enzymol
2010
, vol. 
480
 (pg. 
417
-
444
)
[PubMed]
27
Agravat
 
S.B.
Saltz
 
J.H.
Cummings
 
R.D.
Smith
 
D.F.
 
GlycoPattern: a web platform for glycan array mining
Bioinformatics
2014
, vol. 
30
 (pg. 
3417
-
3418
)
[PubMed]
28
Ashline
 
D.J.
Yu
 
Y.
Lasanajak
 
Y.
Song
 
X.
Hu
 
L.
Ramani
 
S.
Prasad
 
B.V.
Estes
 
M.K.
Cummings
 
R.D.
Smith
 
D.F.
Reinhold
 
V.N.
 
Structural characterization by MSn of human milk glycans recognized by human rotaviruses
Mol. Cell. Proteomics
2014
, vol. 
13
 (pg. 
2961
-
2974
)
[PubMed]
29
Lock
 
K.
Zhang
 
J.
Lu
 
J.
Lee
 
S.H.
Crocker
 
P.R.
 
Expression of CD33-related siglecs on human mononuclear phagocytes, monocyte-derived dendritic cells and plasmacytoid dendritic cells
Immunobiology
2004
, vol. 
209
 (pg. 
199
-
207
)
[PubMed]
30
Izquierdo-Useros
 
N.
Lorizate
 
M.
Puertas
 
M.C.
Rodriguez-Plata
 
M.T.
Zangger
 
N.
Erikson
 
E.
Pino
 
M.
Erkizia
 
I.
Glass
 
B.
Clotet
 
B.
, et al 
Siglec-1 is a novel dendritic cell receptor that mediates HIV-1 trans-infection through recognition of viral membrane gangliosides
PLoS Biol.
2012
, vol. 
10
 pg. 
e1001448
 
[PubMed]
31
Li
 
N.
Zhang
 
W.
Wan
 
T.
Zhang
 
J.
Chen
 
T.
Yu
 
Y.
Wang
 
J.
Cao
 
X.
 
Cloning and characterization of Siglec-10, a novel sialic acid binding member of the Ig superfamily, from human dendritic cells
J. Biol. Chem.
2001
, vol. 
276
 (pg. 
28106
-
28112
)
[PubMed]
32
Smith
 
D.F.
Cummings
 
R.D.
 
Application of microarrays for deciphering the structure and function of the human glycome
Mol. Cell. Proteomics
2013
, vol. 
12
 (pg. 
902
-
912
)
[PubMed]
33
Agravat
 
S.B.
Song
 
X.
Rojsajjakul
 
T.
Cummings
 
R.D.
Smith
 
D.F.
 
Computational approaches to define a human milk metaglycome
Bioinformatics
2016
[PubMed]
34
Appelmelk
 
B.J.
van Die
 
I.
van Vliet
 
S.J.
Vandenbroucke-Grauls
 
C.M.
Geijtenbeek
 
T.B.
van Kooyk
 
Y.
 
Cutting edge: carbohydrate profiling identifies new pathogens that interact with dendritic cell-specific ICAM-3-grabbing nonintegrin on dendritic cells
J. Immunol.
2003
, vol. 
170
 (pg. 
1635
-
1639
)
[PubMed]
35
van Die
 
I.
van Vliet
 
S.J.
Nyame
 
A.K.
Cummings
 
R.D.
Bank
 
C.M.
Appelmelk
 
B.
Geijtenbeek
 
T.B.
van Kooyk
 
Y.
 
The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x
Glycobiology
2003
, vol. 
13
 (pg. 
471
-
478
)
[PubMed]
36
Brazil
 
J.C.
Liu
 
R.
Sumagin
 
R.
Kolegraff
 
K.N.
Nusrat
 
A.
Cummings
 
R.D.
Parkos
 
C.A.
Louis
 
N.A.
 
alpha3/4 fucosyltransferase 3-dependent synthesis of Sialyl Lewis A on CD44 variant containing exon 6 mediates polymorphonuclear leukocyte detachment from intestinal epithelium during transepithelial migration
J. Immunol.
2013
, vol. 
191
 (pg. 
4804
-
4817
)
[PubMed]
37
Kobata
 
A.
 
Structures and application of oligosaccharides in human milk
Proc. Jpn. Acad. Ser. B Phys. Biol. Sci.
2010
, vol. 
86
 (pg. 
731
-
747
)
[PubMed]
38
Jiang
 
X.
Huang
 
P.
Zhong
 
W.
Tan
 
M.
Farkas
 
T.
Morrow
 
A.L.
Newburg
 
D.S.
Ruiz-Palacios
 
G.M.
Pickering
 
L.K.
 
Human milk contains elements that block binding of noroviruses to human histo-blood group antigens in saliva
J. Infect. Dis.
2004
, vol. 
190
 (pg. 
1850
-
1859
)
[PubMed]
39
Holla
 
A.
Skerra
 
A.
 
Comparative analysis reveals selective recognition of glycans by the dendritic cell receptors DC-SIGN and Langerin
Protein Eng. Des. Sel.
2011
, vol. 
24
 (pg. 
659
-
669
)
[PubMed]
40
Naarding
 
M.A.
Dirac
 
A.M.
Ludwig
 
I.S.
Speijer
 
D.
Lindquist
 
S.
Vestman
 
E.L.
Stax
 
M.J.
Geijtenbeek
 
T.B.
Pollakis
 
G.
Hernell
 
O.
Paxton
 
W.A.
 
Bile salt-stimulated lipase from human milk binds DC-SIGN and inhibits human immunodeficiency virus type 1 transfer to CD4+ T cells
Antimicrob. Agents Chemother.
2006
, vol. 
50
 (pg. 
3367
-
3374
)
[PubMed]
41
Hong
 
P.
Ninonuevo
 
M.R.
Lee
 
B.
Lebrilla
 
C.
Bode
 
L.
 
Human milk oligosaccharides reduce HIV-1-gp120 binding to dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN)
Br. J. Nutr.
2009
, vol. 
101
 (pg. 
482
-
486
)
[PubMed]
42
Jameson
 
B.
Baribaud
 
F.
Pohlmann
 
S.
Ghavimi
 
D.
Mortari
 
F.
Doms
 
R.W.
Iwasaki
 
A.
 
Expression of DC-SIGN by dendritic cells of intestinal and genital mucosae in humans and rhesus macaques
J. Virol.
2002
, vol. 
76
 (pg. 
1866
-
1875
)
[PubMed]
43
Engfer
 
M.B.
Stahl
 
B.
Finke
 
B.
Sawatzki
 
G.
Daniel
 
H.
 
Human milk oligosaccharides are resistant to enzymatic hydrolysis in the upper gastrointestinal tract
Am. J. Clin. Nutr.
2000
, vol. 
71
 (pg. 
1589
-
1596
)
[PubMed]
44
Gnoth
 
M.J.
Kunz
 
C.
Kinne-Saffran
 
E.
Rudloff
 
S.
 
Human milk oligosaccharides are minimally digested in vitro
J. Nutr.
2000
, vol. 
130
 (pg. 
3014
-
3020
)
[PubMed]
45
Thurl
 
S.
Henker
 
J.
Siegel
 
M.
Tovar
 
K.
Sawatzki
 
G.
 
Detection of four human milk groups with respect to Lewis blood group dependent oligosaccharides
Glycoconj. J.
1997
, vol. 
14
 (pg. 
795
-
799
)
[PubMed]
46
Zhuravleva
 
M.A.
Trandem
 
K.
Sun
 
P.D.
 
Structural implications of Siglec-5-mediated sialoglycan recognition
J. Mol. Biol.
2008
, vol. 
375
 (pg. 
437
-
447
)
[PubMed]
47
Poppe
 
L.
Brown
 
G.S.
Philo
 
J.S.
Nikrad
 
P.V.
Shah
 
B.H.
 
Conformation of sLex tetrasaccharide, free in solution and bound to E-, P-, and L-selectin
J. Am. Chem. Soc.
1997
, vol. 
119
 (pg. 
1727
-
1736
)
48
Nelson
 
R.M.
Dolich
 
S.
Aruffo
 
A.
Cecconi
 
O.
Bevilacqua
 
M.P.
 
Higher-affinity oligosaccharide ligands for E-selectin
J. Clin. Invest.
1993
, vol. 
91
 (pg. 
1157
-
1166
)
[PubMed]
49
Bode
 
L.
Kunz
 
C.
Muhly-Reinholz
 
M.
Mayer
 
K.
Seeger
 
W.
Rudloff
 
S.
 
Inhibition of monocyte, lymphocyte, and neutrophil adhesion to endothelial cells by human milk oligosaccharides
Thromb. Haemost.
2004
, vol. 
92
 (pg. 
1402
-
1410
)
[PubMed]
50
Bode
 
L.
Rudloff
 
S.
Kunz
 
C.
Strobel
 
S.
Klein
 
N.
 
Human milk oligosaccharides reduce platelet-neutrophil complex formation leading to a decrease in neutrophil beta 2 integrin expression
J. Leukoc. Biol.
2004
, vol. 
76
 (pg. 
820
-
826
)
[PubMed]
51
Schumacher
 
G.
Bendas
 
G.
Stahl
 
B.
Beermann
 
C.
 
Human milk oligosaccharides affect P-selectin binding capacities: in vitro investigation
Nutrition
2006
, vol. 
22
 (pg. 
620
-
627
)
[PubMed]

Author notes

1

These authors contributed equally to this work.

2

Present address: Perinatal Institute, Cincinati Children's Hospital Medical Center, Cincinnati, OH 45242, U.S.A.

3

Present address: Department of Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02115, U.S.A.