Mining the "glycocode"--exploring the spatial distribution of glycans in gastrointestinal mucin using force spectroscopy

Abstract

Mucins are the main components of the gastrointestinal mucus layer. Mucin glycosylation is critical to most intermolecular and intercellular interactions. However, due to the highly complex and heterogeneous mucin glycan structures, the encoded biological information remains largely encrypted. Here we have developed a methodology based on force spectroscopy to identify biologically accessible glycoepitopes in purified porcine gastric mucin (pPGM) and purified porcine jejunal mucin (pPJM). The binding specificity of lectins Ricinus communis agglutinin I (RCA), peanut (Arachis hypogaea) agglutinin (PNA), Maackia amurensis lectin II (MALII), and Ulex europaeus agglutinin I (UEA) was utilized in force spectroscopy measurements to quantify the affinity and spatial distribution of their cognate sugars at the molecular scale. Binding energy of 4, 1.6, and 26 aJ was determined on pPGM for RCA, PNA, and UEA. Binding was abolished by competition with free ligands, demonstrating the validity of the affinity data. The distributions of the nearest binding site separations estimated the number of binding sites in a 200-nm mucin segment to be 4 for RCA, PNA, and UEA, and 1.8 for MALII. Binding site separations were affected by partial defucosylation of pPGM. Furthermore, we showed that this new approach can resolve differences between gastric and jejunum mucins.

Keywords: adhesion; atomic force microscopy; lectin-carbohydrate interaction; molecular recognition.

Figure 1.

Preparation of mucin for AFM imaging. A) Native pPGM. B) NaCl-dialyzed pPGM. Bright features in the dialyzed sample correspond to chain crossover points.

Figure 2.

AFM topography images of lectins (arrows) bound to pPGM. A) UEA (composite image from two adjacent scans). B) RCA. C) PNA.

Figure 3.

Schematic diagram illustrating the force spectroscopy protocol used in this study. A lectin-functionalized AFM tip is retracted away from the surface of a glass slide coated with tethered mucin molecules. The experiment is carried out under PBS solution. Inset: a more detailed view of the interaction between the lectin and glycan side-chains. Labels A–G represent the sugar composition, but not the sequence. Images are not drawn to scale.

Figure 4.

Example force-distance spectrum of a lectin (UEA)-functionalized AFM tip probing a pPGM-coated glass slide in PBS (A) and following addition of the cognate sugar (Fuc; B). Gray trace, approach; black trace, retraction.

Figure 5.

Interaction energy plots obtained using lectin-functionalized AFM tips to probe pPGM in PBS (gray) and following the addition of the relevant cognate sugars (black). A) UEA. B) RCA. C) PNA.

Figure 6.

Example force spectra obtained by probing a pPGM-coated glass slide at 3 discrete locations with an RCA-functionalized AFM tip in PBS. Arbitrary offsets in the y axis have been added to the blue and black datasets for clarity of presentation.

Figure 7.

Fitted γ probability density function and observed density distribution of the nearest neighbor binding site separations measured with lectin-functionalized AFM tips probing mucins in PBS. A, B) UEA on pPGM (A) and pPJM (B). C, D) RCA on pPGM (C) and pPJM (D). E, F) PNA on pPGM (E) and pPJM (F). G, H) MALII on pPGM (G) and pPJM (H). Panel H includes insufficient binding events to allow fitting.

Figure 8.

Comparison of γ density distribution functions fitted to the binding site separations observed with lectin-functionalized AFM tips probing pPGM or pPJM. Green trace, UEA on pPGM; yellow trace, UEA on pPJM; red trace, RCA on pPGM; olive green trace, RCA on pPJM; blue trace, PNA on pPGM; purple trace, PNA on pPJM; turquoise trace, MALII on pPGM. Same letter indicates distributions that are not significantly different according to an F test.

Figure 9.

Comparison of the γ density distribution function fitted to the observed binding site separations. A) UEA-functionalized AFM tip probing: green trace, pPGM; dark brown trace, 40 U fucosidase-treated pPGM; light brown trace, 100 U fucosidase-treated pPGM. B) PNA-functionalized AFM tip probing: blue trace, pPGM; light brown trace, 100 U fucosidase-treated pPGM. Same letter indicates distributions that are not significantly different according to an F test.

Figure 10.

Observed frequencies (black) and predicted mass function (gray) for the number of binding sites in a mucin segment of 200 nm length for RCA-functionalized AFM tips probing pPGM.