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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

Fragments from four precursors potentially carrying H type 1 from SKOV3 desialylated permethylated N-glycans.A, MS/MS spectrum of m/z 3286 precursor. B, MS/MS spectrum of m/z 3490 precursor. C, MS/MS spectrum of m/z 4215 precursor. D, MS/MS spectrum of m/z 4664 precursor. Spectra were acquired by MALDI-TOF-TOF MS/MS using collision-induced dissociation at 1 kV. Small schematic annotation of peaks from the left indicated fucosylated LacNAc ions and from the right (dotted arrows) indicated their loss from the precursor ion. Glycan schematics indicate the major precursor ion, with solid arrows and numbers indicating observed fragmentation points and the resulting m/z of fragments. Note that which branch the antennae were on could not be assigned, but the schematics indicate a likely arrangement to annotate the points of fragmentation.
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Figure 6: Fragments from four precursors potentially carrying H type 1 from SKOV3 desialylated permethylated N-glycans.A, MS/MS spectrum of m/z 3286 precursor. B, MS/MS spectrum of m/z 3490 precursor. C, MS/MS spectrum of m/z 4215 precursor. D, MS/MS spectrum of m/z 4664 precursor. Spectra were acquired by MALDI-TOF-TOF MS/MS using collision-induced dissociation at 1 kV. Small schematic annotation of peaks from the left indicated fucosylated LacNAc ions and from the right (dotted arrows) indicated their loss from the precursor ion. Glycan schematics indicate the major precursor ion, with solid arrows and numbers indicating observed fragmentation points and the resulting m/z of fragments. Note that which branch the antennae were on could not be assigned, but the schematics indicate a likely arrangement to annotate the points of fragmentation.

Mentions: From the comprehensive MS/MS analysis done on many of the peaks, four representative glycans (m/z 3286, 3490, 4215, and 4664) were chosen to demonstrate the pool of potential H type 1 candidates. This pool was classified as short (Fig. 6, A and B) and extended (Fig. 6, C and D) based on the presence of fucosylated polyLacNAc antennae.


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
Fragments from four precursors potentially carrying H type 1 from SKOV3 desialylated permethylated N-glycans.A, MS/MS spectrum of m/z 3286 precursor. B, MS/MS spectrum of m/z 3490 precursor. C, MS/MS spectrum of m/z 4215 precursor. D, MS/MS spectrum of m/z 4664 precursor. Spectra were acquired by MALDI-TOF-TOF MS/MS using collision-induced dissociation at 1 kV. Small schematic annotation of peaks from the left indicated fucosylated LacNAc ions and from the right (dotted arrows) indicated their loss from the precursor ion. Glycan schematics indicate the major precursor ion, with solid arrows and numbers indicating observed fragmentation points and the resulting m/z of fragments. Note that which branch the antennae were on could not be assigned, but the schematics indicate a likely arrangement to annotate the points of fragmentation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Fragments from four precursors potentially carrying H type 1 from SKOV3 desialylated permethylated N-glycans.A, MS/MS spectrum of m/z 3286 precursor. B, MS/MS spectrum of m/z 3490 precursor. C, MS/MS spectrum of m/z 4215 precursor. D, MS/MS spectrum of m/z 4664 precursor. Spectra were acquired by MALDI-TOF-TOF MS/MS using collision-induced dissociation at 1 kV. Small schematic annotation of peaks from the left indicated fucosylated LacNAc ions and from the right (dotted arrows) indicated their loss from the precursor ion. Glycan schematics indicate the major precursor ion, with solid arrows and numbers indicating observed fragmentation points and the resulting m/z of fragments. Note that which branch the antennae were on could not be assigned, but the schematics indicate a likely arrangement to annotate the points of fragmentation.
Mentions: From the comprehensive MS/MS analysis done on many of the peaks, four representative glycans (m/z 3286, 3490, 4215, and 4664) were chosen to demonstrate the pool of potential H type 1 candidates. This pool was classified as short (Fig. 6, A and B) and extended (Fig. 6, C and D) based on the presence of fucosylated polyLacNAc antennae.

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