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Characterization of H type 1 and type 1 N -acetyllactosamine glycan epitopes on ovarian cancer specifically recognized by the anti-glycan monoclonal antibody mAb-A4

View Article: PubMed Central - PubMed

ABSTRACT

Cancer-specific glycans of ovarian cancer are promising epitopes for targeting with monoclonal antibodies (mAb). Despite their potential, structural characterization of these glycan epitopes remains a significant challenge in mAb preclinical development. Our group generated the monoclonal antibody mAb-A4 against human embryonic stem cells (hESC), which also bound specifically to N-glycans present on 11 of 19 ovarian cancer (OC) and 8 of 14 breast cancer cell lines tested. Normal cell lines and tissue were unstained by mAb-A4. To characterize the N-linked glycan epitopes on OC cell lines targeted by mAb-A4, we used glycosidases, glycan microarray, siRNA, and advanced high sensitivity matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The mAb-A4 epitopes were found to be Fucα1–2Galβ1–3GlcNAcβ (H type 1) and Galβ1–3GlcNAcβ (type 1 LacNAc). These structures were found to be present on multiple proteins from hESC and OC. Importantly, endo-β-galactosidase coupled with MALDI-MS allowed these two epitopes, for the first time, to be directly identified on the polylactosamines of N-glycans of SKOV3, IGROV1, OV90, and OVCA433. Furthermore, siRNA knockdown of B3GALT5 expression in SKOV3 demonstrated that mAb-A4 binding was dependent on B3GALT5, providing orthogonal evidence of the epitopes' structures. The recognition of oncofetal H type 1 and type 1 LacNAc on OC by mAb-A4 is a novel and promising way to target OC and supports the theory that cancer can acquire stem-like phenotypes. We propose that the orthogonal framework used in this work could be the basis for advancing anti-glycan mAb characterization.

No MeSH data available.


Related in: MedlinePlus

Types of potential mAb-A4 epitopes in the N-glycome of SKOV3. Partial mass spectrum of permethylated desialylated N-glycans from 10 million SKOV3 cells, acquired by MALDI-TOF in positive mode (50% acetonitrile fraction). Intensity was normalized to the m/z 3142 peak. The region from m/z 3700–4250 was magnified 15 times to show the minor peaks. Red boxes indicate outer-arm fucosylated N-glycans that lacked polyLacNAc antennae. Blue boxes indicate outer-arm fucosylated N-glycans with polyLacNAc antennae. Glycan residues shown outside of the brackets indicate ambiguity regarding their placement. Annotations of the polyLacNAc terminus were simplified to show the most probable permutation.
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Figure 5: Types of potential mAb-A4 epitopes in the N-glycome of SKOV3. Partial mass spectrum of permethylated desialylated N-glycans from 10 million SKOV3 cells, acquired by MALDI-TOF in positive mode (50% acetonitrile fraction). Intensity was normalized to the m/z 3142 peak. The region from m/z 3700–4250 was magnified 15 times to show the minor peaks. Red boxes indicate outer-arm fucosylated N-glycans that lacked polyLacNAc antennae. Blue boxes indicate outer-arm fucosylated N-glycans with polyLacNAc antennae. Glycan residues shown outside of the brackets indicate ambiguity regarding their placement. Annotations of the polyLacNAc terminus were simplified to show the most probable permutation.

Mentions: Next, the presence of H type 1 or type 1 LacNAc on ovarian cancer cell lines was investigated. To determine the cellular glycan target, total N-glycans were analyzed on MALDI-TOF as permethylated sodiated adducts in the positive mode (supplemental Fig. 3). Sialylated structures were more abundant than fucosylated structures and hindered identification of fucosylated structures by MS/MS. Therefore, to better identify potentially fucosylated structures, the N-glycans were desialylated (Fig. 5). The desialylated N-glycome consisted of high mannose and core-fucosylated complex-type N-glycans. Bi-, tri-, and tetra-antennary structures were observed. Outer-arm fucosylated glycans were present but were less abundant than non-fucosylated antennae. For example, the biantennary glycan at m/z 2244 was five times more intense than the m/z 2418 species with one antennal fucose (Fig. 5). This pattern was repeated for the tri- and tetra-antennary structures at m/z 2693/2867 and 3142/3316, respectively. This indicated that although there are active outer-arm fucosyltransferases in SKOV3, the fucosylation of antennae does not go to completion. No sialyl Lewis antigens were observed by MS/MS in the non-desialylated N-glycome (supplemental Fig. 3). In other words, antennae were either sialylated or fucosylated, but not both.


Characterization of H type 1 and type 1 N -acetyllactosamine glycan epitopes on ovarian cancer specifically recognized by the anti-glycan monoclonal antibody mAb-A4
Types of potential mAb-A4 epitopes in the N-glycome of SKOV3. Partial mass spectrum of permethylated desialylated N-glycans from 10 million SKOV3 cells, acquired by MALDI-TOF in positive mode (50% acetonitrile fraction). Intensity was normalized to the m/z 3142 peak. The region from m/z 3700–4250 was magnified 15 times to show the minor peaks. Red boxes indicate outer-arm fucosylated N-glycans that lacked polyLacNAc antennae. Blue boxes indicate outer-arm fucosylated N-glycans with polyLacNAc antennae. Glycan residues shown outside of the brackets indicate ambiguity regarding their placement. Annotations of the polyLacNAc terminus were simplified to show the most probable permutation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5391748&req=5

Figure 5: Types of potential mAb-A4 epitopes in the N-glycome of SKOV3. Partial mass spectrum of permethylated desialylated N-glycans from 10 million SKOV3 cells, acquired by MALDI-TOF in positive mode (50% acetonitrile fraction). Intensity was normalized to the m/z 3142 peak. The region from m/z 3700–4250 was magnified 15 times to show the minor peaks. Red boxes indicate outer-arm fucosylated N-glycans that lacked polyLacNAc antennae. Blue boxes indicate outer-arm fucosylated N-glycans with polyLacNAc antennae. Glycan residues shown outside of the brackets indicate ambiguity regarding their placement. Annotations of the polyLacNAc terminus were simplified to show the most probable permutation.
Mentions: Next, the presence of H type 1 or type 1 LacNAc on ovarian cancer cell lines was investigated. To determine the cellular glycan target, total N-glycans were analyzed on MALDI-TOF as permethylated sodiated adducts in the positive mode (supplemental Fig. 3). Sialylated structures were more abundant than fucosylated structures and hindered identification of fucosylated structures by MS/MS. Therefore, to better identify potentially fucosylated structures, the N-glycans were desialylated (Fig. 5). The desialylated N-glycome consisted of high mannose and core-fucosylated complex-type N-glycans. Bi-, tri-, and tetra-antennary structures were observed. Outer-arm fucosylated glycans were present but were less abundant than non-fucosylated antennae. For example, the biantennary glycan at m/z 2244 was five times more intense than the m/z 2418 species with one antennal fucose (Fig. 5). This pattern was repeated for the tri- and tetra-antennary structures at m/z 2693/2867 and 3142/3316, respectively. This indicated that although there are active outer-arm fucosyltransferases in SKOV3, the fucosylation of antennae does not go to completion. No sialyl Lewis antigens were observed by MS/MS in the non-desialylated N-glycome (supplemental Fig. 3). In other words, antennae were either sialylated or fucosylated, but not both.

View Article: PubMed Central - PubMed

ABSTRACT

Cancer-specific glycans of ovarian cancer are promising epitopes for targeting with monoclonal antibodies (mAb). Despite their potential, structural characterization of these glycan epitopes remains a significant challenge in mAb preclinical development. Our group generated the monoclonal antibody mAb-A4 against human embryonic stem cells (hESC), which also bound specifically to N-glycans present on 11 of 19 ovarian cancer (OC) and 8 of 14 breast cancer cell lines tested. Normal cell lines and tissue were unstained by mAb-A4. To characterize the N-linked glycan epitopes on OC cell lines targeted by mAb-A4, we used glycosidases, glycan microarray, siRNA, and advanced high sensitivity matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The mAb-A4 epitopes were found to be Fucα1–2Galβ1–3GlcNAcβ (H type 1) and Galβ1–3GlcNAcβ (type 1 LacNAc). These structures were found to be present on multiple proteins from hESC and OC. Importantly, endo-β-galactosidase coupled with MALDI-MS allowed these two epitopes, for the first time, to be directly identified on the polylactosamines of N-glycans of SKOV3, IGROV1, OV90, and OVCA433. Furthermore, siRNA knockdown of B3GALT5 expression in SKOV3 demonstrated that mAb-A4 binding was dependent on B3GALT5, providing orthogonal evidence of the epitopes' structures. The recognition of oncofetal H type 1 and type 1 LacNAc on OC by mAb-A4 is a novel and promising way to target OC and supports the theory that cancer can acquire stem-like phenotypes. We propose that the orthogonal framework used in this work could be the basis for advancing anti-glycan mAb characterization.

No MeSH data available.


Related in: MedlinePlus