Misevic G et al. (2021), Glyconectin Cell Adhesion Epitope, β-d-GlcpNAc3...

ECB-ART-49091

Molecules 2021 Jun 30;2613:. doi: 10.3390/molecules26134012.

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Glyconectin Cell Adhesion Epitope, β-d-GlcpNAc3S-(1→3)-α-l-Fucp, Is Involved in Blastulation of Lytechinus pictus Sea Urchin Embryos.

Misevic G , Checiu I , Popescu O .

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Glycans, as the most peripheral cell surface components, are the primary candidates to mediate the initial steps of cell recognition and adhesion via glycan-glycan binding. This molecular mechanism was quantitatively demonstrated by biochemical and biophysical measurements at the cellular and molecular level for the glyconectin 1 β-d-GlcpNAc3S-(1→3)-α-l-Fucp glycan structure (GN1). The use of adhesion blocking monoclonal antibody Block 2 that specifically recognize this epitope showed that, besides Porifera, human colon carcinoma also express this structure in the apical glycocalyx. Here we report that Block 2 selectively immune-precipitate a Mr 580 × 103 (g580) acidic non-glycosaminoglycan glycan from the total protein-free glycans of Lytechinus pictus sea urchin hatched blastula embryos. Immuno-fluorescence confocal light microscopy and immunogold electron microscopy localized the GN1 structure in the apical lamina glycocalyx attachments of ectodermal cells microvilli, and in the Golgi complex. Biochemical and immune-chemical analyses showed that the g580 glycan is carrying about 200 copies of the GN1 epitope. This highly polyvalent g580 glycan is one of the major components of the glycocalyx structure, maximally expressed at hatched blastula and gastrula. The involvement of g580 GN1 epitope in hatched blastula cell adhesion was demonstrated by: (1) enhancement of cell aggregation by g580 and sponge g200 glycans, (2) inhibition of cell reaggregation by Block 2, (3) dissociation of microvilli from the apical lamina matrix by the loss of its gel-like structure resulting in a change of the blastula embryonal form and consequent inhibition of gastrulation at saturating concentration of Block 2, and (4) aggregation of beads coated with the immune-purified g580 protein-free glycan. These results, together with the previous atomic force microscopy measurements of GN1 binding strength, indicated that this highly polyvalent and calcium ion dependent glycan-glycan binding can provide the force of 40 nanonewtons per single ectodermal cell association of microvilli with the apical lamina, and conservation of glycocalyx gel-like structure. This force can hold the weight of 160,000 cells in sea water, thus it is sufficient to establish, maintain and preserve blastula form after hatching, and prior to the complete formation of further stabilizing basal lamina.

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	Figure 1. Expression of the GN1 epitope during L. pictus sea urchin development. (A) immunodot assay of total sea urchin glycans isolated from six developmental stages with the Block 2 monoclonal antibody. Then, 0.5 μL of glycans of two-fold dilutions starting with the concentration of 1 μg/μL water were spotted onto a DEAE-nitrocellulose paper. After brief drying DEAE-nitrocellulose was immunodecorated with the Block 2 monoclonal antibody and stained with anti-mouse peroxidase conjugated antibodies as described in Material and Methods. (B) polyacrylamide gel electrophoresis on 5–20% gradient gels of 25 μg sea urchin total protein-free glycans prepared from six different developmental stages, and 2.5 μg of immunopurified g580 sea urchin glycan from hatched blastula stage glycans were performed as described under Material and Methods. Gels were stained with Alcian blue for detecting acidic glycans. (C) immuno-detection of the GN1 epitope by the Block 2 monoclonal antibody in sea urchin glycans after gel electrophoresis as in (A), and electro-blotting. Glycosaminoglycans, heparin of Mr = 11 × 103 D, chondroitin sulfate of Mr = 20 × 103 and Mr = 100 × 103 and hyaluronic acid of Mr = 225 × 103 D, and sponge glyconectins g200 of Mr = 200 × 103 D and g6 of Mr = 6 × 103 were used as molecular weight standards.
	Figure 2. Structure of the GN1 cell adhesion epitope and molecular model of glycocalyx and part of microvilli. (A) Glyconectin 1 (GN1) glycan epitope structure. (B) M. prolifera sponge glyconectin glycan. (C) L. pictus sea urchin glyconectin glycan. (D) L. pictus apical lamina glyconectins self-assembly in glycocalyx. (E) L. pictus apical lamina glyconectin glycocalyx.
	Figure 3. Immuno-histological localization of the GN1 glycan adhesion epitope in L. pictus hatched blastula. (A–C) immunofluorescence confocal microscopy of L. pictus hatched blastula stained with the ant-GN1 Block 2 monoclonal antibody. Whole mounts of blastula L. pictus embryos were stained with 100 μg/mL Block 2 as described in Materials and Methods. (A–C) parfocal series of compost images from fluorescence (upper image) and transmitted light (lower image) were taken with Zeiss 63× objective every 0.6 μm in z-axis starting from the apical part of ectodermal cells. Image in z-axis at distance from scan beginning (A) 0.0 μm (B) 1.2 μm, (C) 2.4 μm, (D) three-dimensional reconstruction of the embryo part obtained from images scanned with 63× objective, (E) image of the whole embryo, (F–H) immunogold electron microscopical localization of the glycan adhesion epitope in L. pictus hatched blastula, (F) apical lamina with microvilli, (G) part of microvilli, and (H) Golgi complex. Arrows mark Golgi.
	Figure 4. Model of hatched blastula apical lamina glycocalyx association with microvilli by the g580 glyconectin acidic glycan.
	Figure 5. Hatched blastula cell reaggregation. (A) promotion of sea urchin hatched blastula cell aggregation by 15 μg of the g580 glycan in the volume of 100 μL FSW containing 105 cells was performed using gentle rotation at 16 °C for 10 min, (B) aggregation promotion by 15 μg of the sponge g200 glycan under the same condition as in (A), (C) not treated culture of 105 dissociated hatched blastula cells, and (D) inhibition of sea urchin hatched blastula cell reaggregation by 15 μg of the anti-GN1 Fab fragments of the Block 2 monoclonal antibody was performed as in (A,B), but for 1 h.
	Figure 6. Intermolecular glycan–glycan binding of g580 glyconectin glycans demonstrated by adhesion of g580 coated beads. Aggregation of 100 μL 2% bead suspension: (A) g580 glyconectin glycans coated beads in the absence of physiological calcium ions (CMF-ASW—buffered with 10 mM Tris pH 7.2), (B) g580 glyconectin glycans coated beads in the presence of physiological calcium ions (SWT—buffered with 10 mM Tris pH 7.2), (C) g580 glyconectin glycans coated beads in the presence of physiological calcium ions (SWT—buffered with 10 mM Tris pH 7.2) with Fab monovalent fragments of the anti-GN1 Block 2 monoclonal antibodies (100 μg/mL), and (D) non coated beads in the presence of physiological calcium ions (SWT—buffered with 10 mM Tris pH 7.2).
	Figure 7. Alterations of embryo integrity and inhibition of gastrulation by the anti GN1 Block 2 monoclonal antibody. Hatched blastula embryos were grown in 100 μL FSW to which different concentrations of purified monoclonal antibodies were added. Inhibition of gastrulation and shape changes were scored during 24 h incubation at 16 °C with the (A) 10 μg Block 2, (B) 5 μg Block 2, (C) 2.5 μg Block 2, (D) 1.25 μg Block 2, (E) 10 μg control mouse C-16 monoclonal antibody, and (F) control untreated embryos. Arrows mark archenteron.

References [+] :

Adelson, On the ultrastructure of hyalin, a cell adhesion protein of the sea urchin embryo extracellular matrix. 1992, Pubmed, Echinobase