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. 2023 Sep 1;108(9):2526-2530.
doi: 10.3324/haematol.2022.281979.

Specific O-glycans in the mechanosensory domain of glycoprotein Ibα are important for its stability and function

Yingchun Wang  1 , Renhao Li  2
Affiliations

Specific O-glycans in the mechanosensory domain of glycoprotein Ibα are important for its stability and function

Yingchun Wang et al. Haematologica. .
No abstract available

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Figures

Figure 1.
Figure 1.
O-glycans in the N-terminal region of the mechanosensory domain are important to GPIbα expression and shedding. (A) The sequence of human GPIbα mechanosensory domain (MSD) includes residues A417-F483. Putative O-glycosylation sites, serine or threonine residues, are colored red. The N-terminal region (A417 to P442), the C-terminal region (V443 to E471), and the Trigger region (S472 to F483) are marked. The ADAM17 cleavage site is denoted by the arrowhead. (B) Overlaid flow cytometry histograms showing surface expression of GPIbα variants in stably transfected CHO cells that expressed wild-type GPIbβ and GPIX. βIX: cells transfected with an empty vector. N∆O, C∆O, and T∆O denote the GPIbα constructs that have mutations of O-glycosylation sites. GPIbα expression was detected by flow cytometry with anti-GPIbα WM23 antibody. (C-E) Bar plots of (C) GPIbα variant, (D) GPIbβ, and (E) GPIX surface expression in noted stably transfected CHO cells. GPIbβ and GPIX was detected with monoclonal antibodies RAM.1 and FMC25, respectively. The mean fluorescence intensity (MFI) was quantified and plotted (mean ± standard deviation, n=3). Comparison of result was performed by one-way ANOVA. *P<0.05; ***P<0.001. (F) Bar plots of GPIbα expression level in each transfected CHO cell treated with vehicle (unfilled bar graph) or GM6001 (color-filled bar graph), quantified by MFI. (G) Representative western blots showing cellular expression of GPIbα in noted stably transfected CHO cells and glycocalicin (GC) released from these cells into the culture media. Cell lysates of denoted cells were resolved in SDS gels and transferred to the PVDF membrane, which was blotted by WM23 or anti-β-actin antibody. Molecular weight markers are labeled on the right. (H) Bar plot of relative GC levels from noted cells after treatment of vehicle (open bar) or GM6001 (filled bar). The GC band was quantitated by densitometry and normalized against that from CHO-WT cells before GM6001 treatment. Data are shown as mean ± standard deviation, n=3. Comparison of results was performed by one-way ANOVA with post hoc tukey test *P<0.05; ***P<0.001.
Figure 2.
Figure 2.
Absence of O-glycans in the N-terminal region increased unfolding of the mechanosensory domain. (A, B) Overlaid flow cytometry histograms showing binding of (A) WM23 antibody and (B) 5G6 antibody in noted stably transfected CHO cells. Each antibody binding level was quantitated by mean fluorescence intensity as described, and in the Online Supplementary Appendix. (C-E) Bar plots showing (C) quantitated WM23 binding level, (D) 5G6 binding level, and (E) ratio of 5G6/WM23 binding in noted variant CHO cells. Data are shown as mean ± standard deviation, n=3. Comparison of results was performed by one-way ANOVA with post hoc tukey test. *P<0.05; ***P<0.001.
Figure 3.
Figure 3.
Differential effects of specific O-glycans in the mechanosensory domain on filopodia formation of transfected CHO cells. (A) Representative confocal microscopy images of noted transfected CHO cells that are adhered to von Willebrand factor (VWF) in the presence of botrocetin and ethylenediaminetetraacetic acid (EDTA). Briefly, a glass slide was coated with human VWF at 10 mg/mL in phosphate-buffered saline (PBS) at 4 °C overnight and blocked with 1% bovine serum albumin in PBS for 1 hour at 22°C. CHO cells stably expressing wild-type and mutant GPIb-IX were resuspended in modified Tyrode’s buffer (134 mM NaCl, 0.34 mM Na2HPO4, 2.9 mM KCl, 1 mM MgCl2, 5 mM glucose, 12 mM NaHCO3, 20 mM HEPES, pH 7.35) containing 5 mM EDTA at the concentration of 1×106 cells/mL. The cells were placed on VWF-coated slides in the presence or absence of 1 mg/mL botrocetin for 30 minutes (min) at 37 °C. The adherent cells on the slide were washed with PBS buffer, fixed with 4% paraformal-dehyde for 10 min, and stained with TRITC-conjugated phalloidin in PBS containing 0.1% Triton X-100 for 30 min. Images were collected on an Olympus Fluo View FV1000 confocal microscope. Images collected in 10-12 viewfields from 2 independent experiments were analyzed using Fiji (ImageJ) and a macro written for quantitative analysis of filopodia. (B-D) Bar plots of (B) number, (C) average surface area, and (D) number of filopodia of adhered CHO cells. Error bars represent mean ± standard deviation. Significance determined by one-way ANOVA with post hoc Tukey test. ***P<0.001.
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