PMID: PMC7108604-2

 

    Legend: Gene, Sites

Title : The structural characterization of the sugar chains of human VE-cadherin was based on chromatographic profiling by anion-exchange HPLC, HPAEC, and gel filtration in combination with exoglycosidase digestions

Abstract :

1. Since the separation systems used relied on different physicochemical parameters, comparison of the chromatographic data with those of oligosaccharide standards with known structures allowed a first structural assignment
2. Although anomeric configurations and linkage positions of the respective monosaccharide units were only unraveled in the case of sialic acid , galactosyl- and, in part, mannosyl residues , structures could be postulated on the basis of the general rules of mammalian glycoprotein-N-glycan architecture (Vliegenthart and Montreuil, 1995; Sharon and Lis, 1997)
3. The results revealed that human VE-cadherin is substituted predominantly (∼40% of total glycans) by sialylated diantennary complex-type glycans in addition to about 28% of sialylated hybrid-type species
4. Higher branched N-glycans, i.e., triantennary and, especially, tetraantennary chains as well as high mannose-type oligosaccharides were less abundant
5. Our data provided no evidence for the presence of oligosaccharides carrying “bisecting” GlcNAc as it has been shown by Nguyen and coworkers for bovine capillary endothelial cell carbohydrates (Nguyen et al., 1992)
6. Since the assignment of glycan structures is solely based on their chromatographic properties, however, the presence of small amounts of (eventually incomplete) bisected oligosaccharides cannot be completely ruled out
7. In conclusion, natural human VE-cadherin appears to be mainly decorated with carbohydrates of restricted branching pattern
8. The high degree of sialylated oligosaccharide structures prompted us to visualize sialic acid residues at the surface of endothelial cells by lectin staining with MAA and SNA
9. Besides labeling of cell surface proteins , interendothelial junctions were strongly stained by these lectins which are specific for α2,3- and α2,6-linked sialic acids
10. The junctional appearance of MAA/SNA-labeling was largely restricted to VE-cadherin (observed by double labeling) whereas PECAM-1 appeared more extendent at the junctions both in vivo and in culture
11. In addition, although endothelial cells are thin reaching seldom more than 3 µm in height, the use of confocal laser microscopy allows a rough localization with a resolution of ∼0.5 µm
12. By this technique, VE-cadherin immunofluorescence as well as MAA/SNA-labeling appeared predominantly at the apical pole of the junctions whereas PECAM-1 was primarily located at the basal pole
13. This is in line with previously published data obtained by immunoelectron microscopy demonstrating a basal localization of PECAM-1 and an apical localization of VE-cadherin within the interendothelial junctions (Ayalon et al., 1994)
14. Furthermore, Ca2 +-depletion experiments showed that the junctional presence of sialic acids as well as the presence of VE-cadherin was reversibly dependent on extracellular Ca2 +-concentration whereas the junctional localization of PECAM-1 remained completely unchanged under all conditions
15. Additionally, it has been shown that VE-cadherin but not N-cadherin is clustered at the intercellular junctions (Salomon ; Navarro )
16. Hence, it may be assumed that junction located sialic acids might be primarily bound to VE-cadherin , which is in agreement with the carbohydrate analyses of purified VE-cadherin
17. The remaining weak MAA/SNA staining after Ca2 +-depletion might be related to Ca2 +-independent molecules such as PECAM-1
18. In highly confluent cultures of human umbilical vein and artery endothelial cells, VE-cadherin appeared in an undisturbed continuous band along the interendothelial junctions
19. At overlapping endothelial cell junctions, a netlike VE-cadherin organization was visualized
20. This network can be considered as extended VE-cadherin clusters and is assumed to considerably increase the interendothelial adhesion properties
21. It has been shown by crystal structural analysis that cadherins are obviously organized as “parallel strand dimers” that interact with “parallel strand dimers” of opposite cells (adhesion dimers ) forming a zipper-like structure (Shapiro ,b)
22. The formation of such a superstructure possibly requires lateral association of the assumed cadherin strand dimers that might be influenced by carbohydrate residues
23. The discussion on the contribution of glycan chains to VE-cadherin function, however, is still contradictory
24. Yoshimura and co-workers (Yoshimura , 1996) provided evidence for a functional role of E-cadherin glycosylation in that murine melanoma B16-hm cells transfected with the ( β1–4)-N-acetylglucosaminyltransferase ( GnT-III ) cDNA showed a higher expression of E-cadherin at cell-cell contacts than control cells
25. Since the presence of bisecting GlcNAc residues is known to block further branching of glycoprotein-N-glycans (Schachter, 1986, 1995; Fujii ), respective glycans can be assumed to remain predominantly in the diantennary state
26. Therefore, the authors conclude that the reduced branching pattern of E-cadherin glycans, induced by ectopically expressed GnT-III , might be responsible for an elevated expression at the cell-cell border
27. This observation is in good agreement with our results revealing mainly diantennary and hybrid-type glycans on natural VE-cadherin
28. On the other hand, it has been observed that E-cadherin containing F9 cells still aggregate after tunicamycin treatment suggesting a glycan independent E-cadherin adhesive function (Shirayoshi )
29. Our data show that sialidase treatment of living endothelial cells caused a significant change in VE-cadherin cellular organization
30. Under these conditions, VE-cadherin still appeared at interendothelial junctions but displayed a scattered immuno-fluorescence pattern including the disappearance of its super-structure
31. In contrast, PECAM-1 underwent no morphological changes
32. Thus, the results described may at least suggest an involvement of sialic acid residues in the structural organization of VE-cadherin
33. The question, as to whether this finding depends, in fact, on sialic acid residues linked to VE-cadherin glycans, remains open since we cannot exclude that removal of sialic acids from the cell surface may cause indirect effects on VE-cadherin organization, as well
34. In conclusion, our results demonstrate that ( 1) VE-cadherin is substituted with oligosaccharide side chains of reduced branching pattern which are highly sialylated, (2) sialic acids present at interendothelial junctions are predominantly bound to Ca2 +-dependent molecules, (3) sialic acids are largely codistributed with VE-cadherin molecules, and ( 4) VE-cadherin superstructural- but not PECAM-1-organization is lost after sialidase treatment
35. From the above results, one might speculate that VE-cadherin glycan chains represent the backbone for the presentation of sialic acids which might be involved in Ca2 +-binding and, thus, in the maintenance of the rod-like VE-cadherin structure and its super-structural organization
36. Further studies are required, however, to definitely prove the influence of carbohydrate substituents on VE-cadherin function

Output (sent_index, trigger, protein, sugar, site):

Output(Part-Of) (sent_index, protein, site):

*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

Protein	NCBI ID	SENTENCE INDEX