Human DC-SIGN binds specific human milk glycans

Abstract

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.

Keywords: dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN); glycan microarrays; glycan recognition; glycan-binding proteins; human milk glycans; lectins.

Fig. 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 multiple concentrations. The results for 90μg/ml Siglec-5 (a), 10μg/ml Siglec-9 (b), and 10μg/ml DC-SIGN with (c) or without (d) Ca²⁺ are shown; Alexa Fluor 488-labeled anti-human IgG was used for detection. Refer to Supplementary File 1 for the results at all concentrations screened. For DC-SIGN without Ca²⁺ (d), Ca²⁺ was omitted from the binding buffer and 0.2mM EDTA was added.

Fig. 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 were printed on a separate microarray and interrogated by metadata-assisted glycan sequencing (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 analyzed by multi-dimensional mass spectrometry (MSⁿ) to more accurately determine the structures(s) within these samples; HMG-20, -21, and -28 were previously sequenced by MSⁿ [21, 28] and HMGs-9, -19, and -36 were also by sequenced by MSⁿ in a more recent manuscript [33]. Refer to Supplementary File 2 for the lectin and antibody screening data.

Fig. 3. Defined HMGs Microarray Screening Data for DC-SIGN and Siglec-9

10μg/ml and 50μg/ml DC-SIGN (a) and 10μg/ml and 90μg/ml Siglec-9 (b) were screened on the defined HMGs microarray, and Alexa Fluor 488-labeled anti-human IgG was used for detection. Graphs on the left show the lower concentrations and the right graphs show the higher concentrations. Refer to Supplementary File 4 for the raw data for these screenings.

Fig. 4. DC-SIGN Binding to HMGs-Derivatized Microspheres

DC-SIGN was incubated with microspheres (beads) derivatized with 2′-FL, 3-FL, or LNT. Alexa Fluor 633-labeled anti-human IgG was used for detection of recombinant DC-SIGN. 2′-FL microspheres incubated with the Alexa Fluor 633-labeled anti-human IgG alone was used as the negative control. All samples were analyzed by Flow Cytometry with a 633nm 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.

Fig. 5. Inhibition of DC-SIGN Binding to HMGs Microarrays with Free, Underivatized HMGs

1μg/ml DC-SIGN was preincubated with or without 0.1, 1, or 10mM of free, underivatized 2′-FL and then screened on the defined HMGs microarray (a) or the HMGs MAGS microarray (b) described in Supplementary File 2. The results for 0mM 2′FL (no inhibitor), 1mM 2′-FL, and 10mM 2′-FL on both microarrays are shown; refer to Supplementary File 5 for the results of all other screenings, including 0.1mM 2′-FL.

Fig. 6. Inhibition of Siglec-9 Binding to Defined HMGs Microarray with Free, Underivatized HMGs and Free, Derivatized 6′-Sialyllactose

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