Galectins are human milk glycan receptors

Abstract

The biological recognition of human milk glycans (HMGs) is poorly understood. Because HMGs are rich in galactose we explored whether they might interact with human galectins, which bind galactose-containing glycans and are highly expressed in epithelial cells and other cell types. We screened a number of human galectins for their binding to HMGs on a shotgun glycan microarray consisting of 247 HMGs derived from human milk, as well as to a defined HMG microarray. Recombinant human galectins (hGal)-1, -3, -4, -7, -8 and -9 bound selectively to glycans, with each galectin recognizing a relatively unique binding motif; by contrast hGal-2 did not recognize HMGs, but did bind to the human blood group A Type 2 determinants on other microarrays. Unlike other galectins, hGal-7 preferentially bound to glycans expressing a terminal Type 1 (Galβ1-3GlcNAc) sequence, a motif that had eluded detection on non-HMG glycan microarrays. Interactions with HMGs were confirmed in a solution setting by isothermal titration microcalorimetry and hapten inhibition experiments. These results demonstrate that galectins selectively bind to HMGs and suggest the possibility that galectin-HMG interactions may play a role in infant immunity.

Keywords: galectins; glycan microarrays; glycan recognition; human milk glycans; shotgun glycomics.

Fig. 1.

Summary of HM-SGM-v2 microarray binding by galectins. Data are examples of one concentration of each biotinylated galectin screened on the HM-SGM-v2 shotgun microarray, with Streptavidin-Cy5 used for detection. The concentrations fell within the approximate linear range of binding to highlight the strongest bound samples. Error bars represent the standard deviation of binding to four technical replicates printed on the array. Refer to Supplementary data, File 1 for the total data from these screenings at all concentrations of all galectins.

Fig. 2.

CFG glycan microarray Version 5.1 results for galectin-2 binding. (A) Galectin-2 binding to the CFG microarray at 20 and 200 μg/mL. Galectin-2 was also screened at 2 μg/mL (not shown here; Supplementary data, File 2), but showed no binding. (B) A list of the top five structures bound by galectin-2. These five structures were bound at both 20 and 200 μg/mL. The fifth structure shown in this table was bound in a dose-independent manner, suggesting this was a nonspecific binder. Note that additional structures were bound at 200 μg/mL galectin-2 only (Supplementary data, File 2), but are not shown here. (C) Proposed glycan-binding motif of galectin-2 based on manual inspection of the structures in (B) and glycopattern analysis. This structure represents the Histo-Blood Group Antigen A Type 2 (i.e. Blood Group A2) determinant. This figure is available in black and white in print and in color at Glycobiology online.

Fig. 3.

Summary of defined HMG microarray binding by galectins. One concentration of each biotinylated galectin screened on the HM-SGM-v2 shotgun microarray (left panels) along with the same concentration of galectin screened in the presence of 0.1 M lactose (right panels). Streptavidin-Cy5 was used for detection. Error bars represent the standard deviation of binding to four technical replicates printed on the array after removing the highest and lowest RFU value of six total technical replicates. Refer to Supplementary data, File 3 for the total data from these screenings at all concentrations of all galectins in the presence and absence of 0.1 M lactose. This figure is available in black and white in print and in color at Glycobiology online.

Fig. 4.

Summary of inhibition of galectin binding to the defined HMG microarray by free HMGs. Biotinylated galectin-7 (A) and biotinylated galectin-4 (B) were screened on the defined HMG microarray in the presence or absence of 50 μM 2′-FL, 500 μM 2′-FL, 5 mM 2′-FL or 5 mM 3-FL. Streptavidin-Cy5 was used for detection. Error bars represent the standard deviation of binding to four technical replicates printed on the array after removing the highest and lowest RFU value of six total technical replicates. The y-axis is set to the same scale for all graphs. Similar results were seen for 50 μM, 500 μM and 5 mM LNT, LNnT and LNFPI as 2′-FL but are not shown here (refer to Supplementary data, Figure S4, for the total inhibition data for hGal-4 and hGal-7). This figure is available in black and white in print and in color at Glycobiology online.

Fig. 5.

ITC thermograms for galectin-7 with HMGs and curve-fitting results after subtraction of buffer-HMG titration data. Thermograms are 28 μM hGal-7 with 8.6 mM lactose (top left and top center), 9.1 mM 2′-FL (top right), 9.1 mM LNnT (middle left), 4.6 mM LNnT (center), 4.28 mM LNT (middle right), 4.11 mM LNFPI (bottom left) and 4.89 mM 3-FL (bottom center). One-Site Model curve-fitting results are shown in the box below each thermogram.