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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.


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Diagnostic fragments of blood group antigen isomers from polyLacNAc termini from SKOV3.A and B, CID MS/MS of the m/z 739 precursor from SKOV3 and IOSE523. C and D, CID MS/MS of the m/z 913 precursor from SKOV3 and IOSE523. Permethylated and deuteroreduced glycans were analyzed by MALDITOF-TOF in the positive mode. The ratios of intensities of the m/z 503 and 521 peaks are indicated in inset boxes. E, fragmentation diagrams for the type 1 and type 2 LacNAc isomers. F, fragmentation diagrams for Lewis X, Lewis Y, H type 1, and H type 2 LacNAc isomers. Diagnostic ions are shaded according to the isomer of origin: blue, type 1 LacNAc; red, type 2 LacNAc; yellow, Lewis A; and green, Lewis X.
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Figure 9: Diagnostic fragments of blood group antigen isomers from polyLacNAc termini from SKOV3.A and B, CID MS/MS of the m/z 739 precursor from SKOV3 and IOSE523. C and D, CID MS/MS of the m/z 913 precursor from SKOV3 and IOSE523. Permethylated and deuteroreduced glycans were analyzed by MALDITOF-TOF in the positive mode. The ratios of intensities of the m/z 503 and 521 peaks are indicated in inset boxes. E, fragmentation diagrams for the type 1 and type 2 LacNAc isomers. F, fragmentation diagrams for Lewis X, Lewis Y, H type 1, and H type 2 LacNAc isomers. Diagnostic ions are shaded according to the isomer of origin: blue, type 1 LacNAc; red, type 2 LacNAc; yellow, Lewis A; and green, Lewis X.

Mentions: To assist in determination of linkages, schematics illustrating the diagnostic fragment ions for each isomer were made by manually by calculating all the possible fragmentation patterns (Fig. 9). The list of ions with their mathematical derivation can be found in supplemental Table 3. Then, the released termini with potential H type 1 and type 1 LacNAc were fragmented in MS/MS. MS/MS conducted on the m/z 739 precursors from SKOV3 and IOSE523 produced the m/z 486 non-reducing end HexHexNAc fragment, confirming the general GalGlcNAcGal structure (Fig. 9, A and B). Focusing on SKOV3, the strong m/z 503 reducing end HexNAcHex-itol fragment was the elimination of galactose and water, characteristic of substituents at the C-3 position of GlcNAc (34, 35). In contrast, the m/z 521 fragment was very minor in SKOV3, and this fragment is from the glycosidic cleavage of galactose without loss of water from the C-4 position of GlcNAc. The “elimination ratio” of the intensities of the eliminated fragment at m/z 503 to cleaved fragment at m/z 521 was 29.53. Furthermore, the elimination of galactose and water in conjunction with the 0,4XGlcNAc cross-ring fragmentation resulting from a retro-Diels-Alder mechanism produced the m/z 429 fragment that was unique for type 1 (annotated as Z0,4X). The high elimination ratio and the strong Z0,4X fragment indicated that Galβ1–3GlcNAcβ1–4Gal (type 1 LacNAc) was the major species in the m/z 739 precursor from SKOV3 released termini.


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
Diagnostic fragments of blood group antigen isomers from polyLacNAc termini from SKOV3.A and B, CID MS/MS of the m/z 739 precursor from SKOV3 and IOSE523. C and D, CID MS/MS of the m/z 913 precursor from SKOV3 and IOSE523. Permethylated and deuteroreduced glycans were analyzed by MALDITOF-TOF in the positive mode. The ratios of intensities of the m/z 503 and 521 peaks are indicated in inset boxes. E, fragmentation diagrams for the type 1 and type 2 LacNAc isomers. F, fragmentation diagrams for Lewis X, Lewis Y, H type 1, and H type 2 LacNAc isomers. Diagnostic ions are shaded according to the isomer of origin: blue, type 1 LacNAc; red, type 2 LacNAc; yellow, Lewis A; and green, Lewis X.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Diagnostic fragments of blood group antigen isomers from polyLacNAc termini from SKOV3.A and B, CID MS/MS of the m/z 739 precursor from SKOV3 and IOSE523. C and D, CID MS/MS of the m/z 913 precursor from SKOV3 and IOSE523. Permethylated and deuteroreduced glycans were analyzed by MALDITOF-TOF in the positive mode. The ratios of intensities of the m/z 503 and 521 peaks are indicated in inset boxes. E, fragmentation diagrams for the type 1 and type 2 LacNAc isomers. F, fragmentation diagrams for Lewis X, Lewis Y, H type 1, and H type 2 LacNAc isomers. Diagnostic ions are shaded according to the isomer of origin: blue, type 1 LacNAc; red, type 2 LacNAc; yellow, Lewis A; and green, Lewis X.
Mentions: To assist in determination of linkages, schematics illustrating the diagnostic fragment ions for each isomer were made by manually by calculating all the possible fragmentation patterns (Fig. 9). The list of ions with their mathematical derivation can be found in supplemental Table 3. Then, the released termini with potential H type 1 and type 1 LacNAc were fragmented in MS/MS. MS/MS conducted on the m/z 739 precursors from SKOV3 and IOSE523 produced the m/z 486 non-reducing end HexHexNAc fragment, confirming the general GalGlcNAcGal structure (Fig. 9, A and B). Focusing on SKOV3, the strong m/z 503 reducing end HexNAcHex-itol fragment was the elimination of galactose and water, characteristic of substituents at the C-3 position of GlcNAc (34, 35). In contrast, the m/z 521 fragment was very minor in SKOV3, and this fragment is from the glycosidic cleavage of galactose without loss of water from the C-4 position of GlcNAc. The “elimination ratio” of the intensities of the eliminated fragment at m/z 503 to cleaved fragment at m/z 521 was 29.53. Furthermore, the elimination of galactose and water in conjunction with the 0,4XGlcNAc cross-ring fragmentation resulting from a retro-Diels-Alder mechanism produced the m/z 429 fragment that was unique for type 1 (annotated as Z0,4X). The high elimination ratio and the strong Z0,4X fragment indicated that Galβ1–3GlcNAcβ1–4Gal (type 1 LacNAc) was the major species in the m/z 739 precursor from SKOV3 released termini.

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