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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

N-Glycan dependence of mAb-A4's binding to HES-3 and SKOV3.A, SDS-PAGE Western blot of the mAb-A4 antigen immunoprecipitated from SKOV3 and digested with no enzyme (lanes 1 and 5), sialidase A (lanes 2, 3, 6, and 7), PNGaseF (lanes 3, 4, 7, and 8), or subjected to on-blot NaOH β-elimination (lanes 5–8), and immunoblotted (IB) with mAb-A4. B, SDS-PAGE Western blot of HES-3 lysate treated with no enzyme (lanes 1 and 3) or PNGaseF (lanes 2 and 4) and subjected to on-blot β-elimination (lanes 3 and 4), and immunoblotted with mAb-84. C, effect of 72 h of tunicamycin treatment on binding of mAb-A4, basigin (BSG), and biotinylated R. solanacearum lectin (RSL). Histograms show negative control (filled) overlaid with sample (black line). Dashed vertical lines show the mean fluorescence of the DMSO control without tunicamycin. D, effect of tunicamycin on the mean fluorescence of mAb-A4 binding to SKOV3. E, effect of tunicamycin on the cytotoxicity of mAb-A4 against SKOV3, as measured by relative viability through PI exclusion. F, effect of tunicamycin on the cytotoxicity of mAb-A4 against SKOV3. Contour plots of forward scatter versus PI uptake of FACS negative control (left), DMSO control (middle), and tunicamycin-treated (right). Data were representative of three biological replicates over successive passages. Error bars indicate one S.D.
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Figure 2: N-Glycan dependence of mAb-A4's binding to HES-3 and SKOV3.A, SDS-PAGE Western blot of the mAb-A4 antigen immunoprecipitated from SKOV3 and digested with no enzyme (lanes 1 and 5), sialidase A (lanes 2, 3, 6, and 7), PNGaseF (lanes 3, 4, 7, and 8), or subjected to on-blot NaOH β-elimination (lanes 5–8), and immunoblotted (IB) with mAb-A4. B, SDS-PAGE Western blot of HES-3 lysate treated with no enzyme (lanes 1 and 3) or PNGaseF (lanes 2 and 4) and subjected to on-blot β-elimination (lanes 3 and 4), and immunoblotted with mAb-84. C, effect of 72 h of tunicamycin treatment on binding of mAb-A4, basigin (BSG), and biotinylated R. solanacearum lectin (RSL). Histograms show negative control (filled) overlaid with sample (black line). Dashed vertical lines show the mean fluorescence of the DMSO control without tunicamycin. D, effect of tunicamycin on the mean fluorescence of mAb-A4 binding to SKOV3. E, effect of tunicamycin on the cytotoxicity of mAb-A4 against SKOV3, as measured by relative viability through PI exclusion. F, effect of tunicamycin on the cytotoxicity of mAb-A4 against SKOV3. Contour plots of forward scatter versus PI uptake of FACS negative control (left), DMSO control (middle), and tunicamycin-treated (right). Data were representative of three biological replicates over successive passages. Error bars indicate one S.D.

Mentions: The mAb-A4 antigen in SKOV3 was found by immunoprecipitation (IP) followed by Western blotting to be a smear from 40 to 191 kDa with more intense regions at 51, 60, and 100 kDa (Fig. 2A, lanes 1 and 5). This staining of IP product was similar to that of SKOV3 whole cell lysate (Fig. 3E, lane 4), suggesting that IP with mAb-A4 successfully enriched its antigen from the cell lysate. The presence of the smear suggested that the mAb-A4 antigen was a glycoprotein.


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
N-Glycan dependence of mAb-A4's binding to HES-3 and SKOV3.A, SDS-PAGE Western blot of the mAb-A4 antigen immunoprecipitated from SKOV3 and digested with no enzyme (lanes 1 and 5), sialidase A (lanes 2, 3, 6, and 7), PNGaseF (lanes 3, 4, 7, and 8), or subjected to on-blot NaOH β-elimination (lanes 5–8), and immunoblotted (IB) with mAb-A4. B, SDS-PAGE Western blot of HES-3 lysate treated with no enzyme (lanes 1 and 3) or PNGaseF (lanes 2 and 4) and subjected to on-blot β-elimination (lanes 3 and 4), and immunoblotted with mAb-84. C, effect of 72 h of tunicamycin treatment on binding of mAb-A4, basigin (BSG), and biotinylated R. solanacearum lectin (RSL). Histograms show negative control (filled) overlaid with sample (black line). Dashed vertical lines show the mean fluorescence of the DMSO control without tunicamycin. D, effect of tunicamycin on the mean fluorescence of mAb-A4 binding to SKOV3. E, effect of tunicamycin on the cytotoxicity of mAb-A4 against SKOV3, as measured by relative viability through PI exclusion. F, effect of tunicamycin on the cytotoxicity of mAb-A4 against SKOV3. Contour plots of forward scatter versus PI uptake of FACS negative control (left), DMSO control (middle), and tunicamycin-treated (right). Data were representative of three biological replicates over successive passages. Error bars indicate one S.D.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5391748&req=5

Figure 2: N-Glycan dependence of mAb-A4's binding to HES-3 and SKOV3.A, SDS-PAGE Western blot of the mAb-A4 antigen immunoprecipitated from SKOV3 and digested with no enzyme (lanes 1 and 5), sialidase A (lanes 2, 3, 6, and 7), PNGaseF (lanes 3, 4, 7, and 8), or subjected to on-blot NaOH β-elimination (lanes 5–8), and immunoblotted (IB) with mAb-A4. B, SDS-PAGE Western blot of HES-3 lysate treated with no enzyme (lanes 1 and 3) or PNGaseF (lanes 2 and 4) and subjected to on-blot β-elimination (lanes 3 and 4), and immunoblotted with mAb-84. C, effect of 72 h of tunicamycin treatment on binding of mAb-A4, basigin (BSG), and biotinylated R. solanacearum lectin (RSL). Histograms show negative control (filled) overlaid with sample (black line). Dashed vertical lines show the mean fluorescence of the DMSO control without tunicamycin. D, effect of tunicamycin on the mean fluorescence of mAb-A4 binding to SKOV3. E, effect of tunicamycin on the cytotoxicity of mAb-A4 against SKOV3, as measured by relative viability through PI exclusion. F, effect of tunicamycin on the cytotoxicity of mAb-A4 against SKOV3. Contour plots of forward scatter versus PI uptake of FACS negative control (left), DMSO control (middle), and tunicamycin-treated (right). Data were representative of three biological replicates over successive passages. Error bars indicate one S.D.
Mentions: The mAb-A4 antigen in SKOV3 was found by immunoprecipitation (IP) followed by Western blotting to be a smear from 40 to 191 kDa with more intense regions at 51, 60, and 100 kDa (Fig. 2A, lanes 1 and 5). This staining of IP product was similar to that of SKOV3 whole cell lysate (Fig. 3E, lane 4), suggesting that IP with mAb-A4 successfully enriched its antigen from the cell lysate. The presence of the smear suggested that the mAb-A4 antigen was a glycoprotein.

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