Abstract
Cancers display distinctive carbohydrate molecules (glycans) on their surface proteins and lipids. mAb A4, an in-house generated monoclonal IgM antibody, is capable of distinguishing malignant ovarian carcinoma cells from benign ovarian epithelia by binding specifically to cancer cell-associated glycans. However, the structural details of the glycan targets of mAb A4 have been elusive. Here we developed a novel approach of isolating and fractionating glycan molecules released from glycoproteins in cancer cell lysates using HILIC-UPLC, and used them as probes on a microarray for affinity-based identification of the binding targets, allowing full-size, difficult to synthesize, cancer-associated glycans to be directly studied. As a result of this “shotgun” glycomics approach, we corroborate the previously assigned specificity of mAb A4 by showing that mAb A4 binds primarily to large (>15 glucose units), sialylated N-glycans containing the H-type 1 antigen (Fuc-α1,2-Gal-β1,3-GlcNAc). Although mAb A4 was also capable of directly binding to type 1 N-acetyl-lactosamine, this epitope was mostly shielded by sialylation and thus relatively inaccessible to binding. Knowledge of the structure of mAb A4 antigen will facilitate its clinical development as well as its use as a diagnostic biomarker.
Highlights
We have modified the traditional approach to shotgun glycomics[20,21,22] by relying on a single HILIC-UPLC separation step instead of two-dimensional separation with a normal phase chromatography step followed by Porous Graphitic Carbon (PGC)
Western Blot experiments were first carried out to determine if mAb A4 bound a specific class of glycans in IGROV-1 ovarian carcinoma cell lines
Antibody binding was largely abolished by treating IGROV-1 cell lysate with PNGase-F, suggesting that mAb A4 mainly targeted N-glycans
Summary
Cancers display distinctive carbohydrate molecules (glycans) on their surface proteins and lipids. mAb A4, an in-house generated monoclonal IgM antibody, is capable of distinguishing malignant ovarian carcinoma cells from benign ovarian epithelia by binding to cancer cell-associated glycans. Treatment with XMF in combination with Bovine Testis Galactosidase (BTG), a β1-3,4 galactosidase, further reduced binding intensity; treatment with XMF in combination with Streptococcus Pneumonia β-Galactosidase (SPG), a β1-4 galactosidase, did not Taken together, this experiment clearly identifies Fuc-α1,2-Gal-β1,3- as the terminal glycan motif primarily responsible for mAb A4 binding, showing that H-type I antigen was the primary mAb A4 ligand in most. Fractions 37 and 38 both contained sialylated, trior tetra-antennary structures with poly-LacNAc arms terminating in H-type I antigen, which is fully consistent with our shotgun glycan microarray, chemically defined glycan array and live cell binding experimental data
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
