Glyco

PMCID: PMC7108604

Legend: Gene, Sites, Suger

Section : Isolation of VE-cadherin

Content :

Cultured human endothelial cells were labeled with [6-3H]gluco-samine and subsequently extracted with 1% Triton X-100 in buffer as described in Material and methods
The use of 1% Triton X-100 causes a release of about 70–80% of the cadherin/ catenin complex (Schnittler ) that can be immunoprecipitated from a 8000 × g supernatant using a monoclonal VE-cadherin antibody
Precipitated protein was subjected to SDS-PAGE, and fluorography of the gel displayed one main band at MW of 135 kDa and few weaker labeled bands (Figure 1)
Part of the precipitate was subjected to Western blot analysis using a pan cadherin antibody known to react with a short carboxyterminal sequence of classical cadherins
This antibody stained a single band exactly at the same MW as the main radiolabeled band (data not shown)

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

Section : Isolation of glycopeptides

Content :

Since SDS displayed several additional, radiolabeled protein bands, immunoprecipitated VE-cadherin samples were preparatively separated by SDS-PAGE, and bands containing VE-cadherin were excised (I in Figure 1)
In order to avoid loss of material when eluting the whole glycoprotein , an in-gel proteolytic digestion was performed, after which radioactive glycopeptides could be easily eluted from the gel pieces
From the known amino acid sequence of the glycoprotein (Suzuki ; Breviario et al., 1995), it could be deduced that treatment with trypsin would generate a glycopeptide resistant to peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidase F ( PNGase F) (Tarentino et al., 1985)
Therefore, Asp-N endoproteinase from Pseudomonas fragi mutant was used instead
In total, a glycopeptide preparation was obtained comprising about 105 cpm of incorporated 3H radioactivity
As a control, unstained gel segments (II in Figure 1) were equally treated and eluted
However, no radioactivity could be detected in the respective supernatants

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

Section : Liberation and fractionation of glycans

Content :

Western blot analysis, using a VE-cadherin monoclonal antibody, of endothelial cells treated with PNGase F and endo-β-N-acetylglucosaminidase H (endo H) revealed the presence of both endo H-sensitive and endo H-resistant N-glycans
Treatment with PNGase F resulted in a shift of the VE-cadherin band from about 135 kDa to about 90 kDa (arrow) in agreement with the expected molecular mass calculated from its amino acid sequence (Figure 2, lane 3)
The second band (dotted line) is assumed to represent a degradation product
Incubation with endo H also led to a small but significant shift in the electrophoretic mobility of this glycoprotein (Figure 2, lane 5)
Therefore, isolated glycopeptides were first treated with endo H. Oligosaccharides released were separated from residual glycopeptides by reversephase (RP-) HPLC
Endo H-resistant glycopeptides were incubated with PNGase F
The resulting reaction mixture was again subjected to RP-HPLC to isolate free glycans
Approximately 16% and 73% of total radioactivity were released by the two enzymes
Residual 11% of radioactivity, still eluting in the peptide fraction, could be shown to comprise exclusively N-acetylglucosamine (GlcNAc) and no N-acetylgalactosamine (GalNAc) and are, therefore, assumed to result from the innermost GlcNAc residue (s) remaining bound to the peptide(s) after endo H cleavage (Kobata, 1979)
After reduction, oligosaccharide alditols were fractionated by anion-exchange HPLC
The results revealed that the majority of human VE-cadherin glycans carry negative charges
Complex type species, released by PNGase F , comprised neutral glycans (F0, 7% of radioactivity) in addition to species with one (F1, 49%), two (F2, 38%), or three (F3, 5%) negatively charged residues (Figure 3A)
Oligosaccharides released by endo H, representing oligomannosidic or hybrid-type glycans (Kobata, 1979), similarly carried predominantly (75%) one negative charge (data not shown)
Treatment with sialidase from V.cholerae or mild acid hydrolysis converted all charged species into neutral compounds demonstrating that the negative charge was exclusively conferred by sialic acid
In the case of PNGase F-sensitive oligosaccharide alditols, charged glycans were separated by preparative anion-exchange HPLC and individually digested with α2,3-specific sialidase from Newcastle disease virus
Monosialylated species could not be degraded and, thus, solely contained α2,6-linked sialic acid , whereas di- and trisialylated glycans both contained one (or two) α2,6-linked sialic acid residue (s) in addition to α2,3-bound sialic acid (Figure 3B–D)
For further characterization, all glycans were completely desialylated

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

Section : Characterization of glycans

Content :

Since the majority of endo H-sensitive glycans carried one sialic acid residue , they could be assumed to represent predominantly hybrid-type species
This was corroborated by high-pH anion-exchange chromatography (HPAEC) of the desialylated glycans (Figure 4A)
Only species H3 (14% of radioactivity within this fraction) coeluted with an oligomannosidic standard oligosaccharide (OM9), whereas the elution volumes of the two major components, H1 and H2 (62% and 24%), did not correspond to those of oligomannosidic standards, but to two hybrid-type standard glycans, MIII and MIV (Geyer et al., 1984), respectively (for detailed structures, see Figure 4 caption)
To further prove their identity, half the amount of endo H-sensitive compounds obtained was digested with α-mannosidase from jack beans
Rechromatography of the resulting products by HPAEC demonstrated that two products were formed eluting at the positions of authentic ManGlcNAcOH and GalGlcNAcMan2GlcNAcOH (Figure 4B)
Digestion of the second half of the sample with β-galactosidase (Figure 4C) resulted in a shift of the elution volumes of the two hybrid-type species corresponding to the loss of one galactosyl residue each whereas the elution position of the oligomannosidic glycan H3 remained unchanged
Therefore, it may be assumed that the two major oligosaccharide alditols, H1 and H2, represent hybrid-type species with one N-acetyllacto-samine antenna and two or three a-linked mannosyl residues
From their sensitivity towards endo H, it may be further concluded that the terminal mannosyl residue present in H1 glycans is a1,3-linked (Kobata, 1979)
The minor component H3, on the other hand, represented an oligomannosidic glycan with nine Man residues
Neutral complex type glycans, obtained after individual desialylation of isocharged species, were chromatographically characterized by HPAEC (Figure 5) and gel filtration using a Bio-Gel P-4 column (data not shown)
Although determination of chromatographic parameters does not allow a structural assignment a priori, comparison with the elution volumes of a set of appropriate oligosaccharide standards in at least two different chromatographic systems gives reliable results (Liedtke )
The glucose units obtained from internal calibration with isomaltooligosaccharides were compared with those of authentic fucosylated di-, two isomers of tri-, tetrα-, and bisected diantennary standard oligosaccharide alditols
The majority of glycans thus identified (Figure 5A–D; fractions F01, F11 , F13, F21, F31, F32) represented fucosylated species with two, three, or four N-acetyllactosamine antennae in addition to a few species not coeluting with any of these standard glycans (fractions F02, F12 )
This assumption was supported by gel filtration data which suggested the presence of incomplete triantennary species lacking one (or two) galactosyl residue(s)
To further substantiate these assignments, the four fractions of complex type glycans as well as the respective oligosaccharide standards were treated with β-galactosidase from D.pneumoniae
Resulting products were again analyzed by HPAEC and, in part, also by gel filtration
As shown in Figure 5E–H, compounds identified as fucosylated di-, tri-, and tetraantennary species coeluted with the respective agalacto oligosaccharide standards after β-galactosidase treatment
Species F02 and F12 , formerly eluting at 4.62 and 4.44 glucose units (Figure 5A,B), now coeluted with triantennary agalacto oligosaccharide standards (Figure 5E,F) and thus may be assumed to comprise fucosylated triantennary species with one (or two) incomplete antenna(e)
Differentiation between the possible isomeric forms, however, was not possible
The elution position of species with 2.9 glucose units (Figure 5A) did not change after digestion with β-galactosidase , indicating the presence of incomplete species without any galactosyl residue
Because of their elution properties, it may be further concluded that they were also lacking, to a certain extent, outer GlcNAc residues
Since these glycans represented only minor constituents, they were not further analyzed
The structural conclusions, drawn from the analytical data described, are summarized in Table I

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

Section : Distribution of sialic acids and junctional cell adhesion molecules in endothelial cells in situ and in culture

Content :

Based on the glycosylation data described above, the localization of sialic acids and Ca2 +-dependent VE-cadherin was studied in endothelial cells of the human umbilical vein and arteries in situ as well as in culture (Figures 6, 7)
As a control, Ca2 +-independent PECAM-1 was also localized (Figure 8)
Digoxigenin labeled MAA- and SNA-lectins, used together or individually (MAA binds to α2,3-linked and SNA binds to α2,6-linked sialic acids), stained the intercellular junctions as well as surface proteins of endothelial cells in situ (Figure 6) and in culture (Figures 7, 8)
Sialic acids, found at interendothelial junctions, largely colocalized with VE-cadherin in a not interrupted continuous pattern in endothelial cells of human umbilical vein and arteries in situ (Figure 6) and in culture (Figure 7)
In addition, at overlapping junctional areas of adjacent highly confluent endothelial cells a netlike distribution of VE-cadherin was observed (Figure 7A,B,D,E) that, again, largely colocalized with MAA/SNA-staining (Figure 7B1,D1,E1)
This netlike structure has not been described before and represents a highly ordered VE-cadherin organization only observable in confluent endothelial cell cultures
Therefore, we termed it VE-cadherin superstructure
Double labeling of VE-cadherin and catenins also displayed a codistribution of this highly organized structure (not shown)
PECAM-1 , labeled with a monoclonal antibody, partially colo-calized with MAA/SNA but to a lesser extent than VE-cadherin (Figure 8C,D)
This was further confirmed by serial optical z-sections using confocal laser microscopy
Whereas sialic acids and VE-cadherin were primarily localized within the apical area of intercellular junctions, PECAM-1 appears to reside predominantly at the basal side (data not shown)

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

Section : Effect of extracellular Ca2 +-depletion on sialic acid distribution

Content :

To investigate whether junctional staining with MAA/SNA was mediated by sialic acids linked to Ca2 +-dependent molecules, Ca2 +-depletion experiments were performed
Incubation of cell monolayers with 3 mM EGTA for 30 min (leading to an extracellular Ca2 +-concentration of <10–7 M) caused a complete absence of VE-cadherin and an almost complete disappearance of the MAA/SNA staining from interendothelial junctions (Figure 7C,C1) whereas PECAM-1 remained unchanged (Figure 8A,B)
A very weak junctional staining of sialic acids, leftover after Ca2 +-depletion (Figure 7C1, 8A), might be caused by the remaining PECAM-1 and/or additional Ca2 +-independent components
After recalcification (1.8 mM of extracellular Ca2 +), VE-cadherin and MAA/SNA staining completely reappeared at the junctions within 30 min (Figure 7D,D1,E,E1)
These findings are in line with previous observations showing a reappearance of VE-cadherin within 5 min after recalcification (Ayalon et al., 1994; Dejana ; Schnittler )
They further indicate that sialic acids located at the interendothelial junctions are predominantly bound to Ca2 +-dependent molecules, presumably VE-cadherin
Preabsorption of MAA/SNA lectins with sialylated pig stomach type III mucin completely abolished the lectin staining in all experimental approaches

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

Section : Effect of sialic acid removal on VE-cadherin organization

Content :

Treatment of cultured endothelial cells with sialidase caused a complete loss of sialic acids from both the intercellular junctions and cell surface proteins without a loss of monolayer integrity
This was revealed by double-staining of sialidase treated cells with MAA/SNA-lectins and anti- PECAM-1 antibodies (Figure 8E,F)
Under these conditions, VE-cadherin changed its continuous junctional distribution to a largely scattered morphology but was still localized at interendothelial junctions
Importantly, the VE-cadherin superstructure largely disappeared showing small protein clusters of various sizes indicating a loss of lateral adhesion between VE-cadherin molecules (Figure 7A,A1)
Similar results were obtained when sialidase treatment was carried out in the presence of protease inhibitors ruling out that this observation might be due to contaminant proteolytic activities of the enzyme used (data not shown)
In contrast, PECAM-1-immunostaining was completely maintained after sialidase treatment (Figure 8F) and demonstrated an intact endothelial cell monolayer in which the cells remained attached to each other

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

Section : Western blot analysis of human VE-cadherin

Content :

HUVEC extracts were subjected to SDS gel electrophoresis and Western blotting before (lanes 2, 4, and 6) and after treatment with PNGase F (lane 3) or endo H (lane 5)
VE-cadherin bands were visualized by specific antibodies
Lane 1, mass marker proteins
The arrow indicates the molecular mass calculated from its amino acid sequence (lane 3)
The dotted line indicates an assumed degradation product (lane 3)

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

Section : Fractionation of PNGase F-released oligosaccharides from human VE-cadherin by anion-exchange HPLC

Content :

(A), oligosaccharide alditols obtained after treatment of endo H-resistant glycopeptides with PNGase F , RP-HPLC and reduction were subjected to anion-exchange HPLC using a Mikropak AX-10 column (4.6 × 250 mm) and a linear gradient of 0–300 mM potassium phosphate, pH 4 .4, within 60 min
Fractions (400 µl) were collected at 1 ml/min and monitored for radioactivity
Fractions (F0–F3) were pooled as indicated by brackets
(B–D) Rechromatography of F1- (B), F2- (C), and F3- (D) derived glycans after treatment with α2,3-specific sialidase from Newcastle disease virus under similar conditions
Numbers (0–4) with arrows indicate the elution volumes of standard oligosaccharides with 0–4 sialic acid residues
* in (C) and (D), released sialic acid

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

Section : Chromatographic profile of desialylated endo H-sensitive glycans from human VE-cadherin

Content :

(A) Oligosaccharide alditols obtained after treatment of proteolytic glycopeptides with endo H, RP-HPLC, reduction and enzymatic desialylation were separated by HPAEC on a CarboPak PA-100 column (4 × 250 mm) using a gradient of 10–30 mM sodium acetate in 80 mM NaOH within 70 min
Fractions (380 µl) were collected at 1 ml/min and monitored for radioactivity
(B) Desialylated endo H-sensitive glycans after treatment with α-mannosidase ; (C) desialylated endo H-sensitive glycans after treatment with β-galactosidase from D.pneumoniae
Numbers (OM5-9) with arrows indicate the elution volumes of oligomannosidic oligosaccharide standard alditols Man5-9GlcNAcOH; MIII, MIV, elution volumes of hybrid-type oligosaccharide standard alditols Galβ4GlcNAcβ2Manα3[Manα3Manα6]Manβ4GlcNAcOH and Galβ4GlcNAcβ2Manα3[Manα3(Manα6)Manα6]Manβ4GlcNAcOH, respectively; I, II, elution volumes of Manβ4GlcNAcOH and Galβ4GlcNAcβ2Manα3Manβ4GlcNAcOH.
* in (C), unidentified product

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

Section : Chromatographic profiles of neutral and desialylated complex-type glycans from human VE-cadherin

Content :

Oligosaccharide fractions obtained after preparative anion-exchange HPLC ((A), F0, (B), F1, (C), F2, (D), F3) were enzymatically desialylated and separated by HPAEC under the same conditions as in Figure 4
(E-H) The same fractions as in (A–D) after treatment with β-galactosidase from D.pneumoniae
Numbers (IM3-6) with arrows indicate the elution volumes of isomaltooligosaccharides with 3–6 glucose units; 2, 2b, 3, 3′, 4, elution volumes of fucosylated diantennary, bisected diantennary, 2,4-branched (3) and 2,6-branched (3′) isomers of triantennary and tetraantennary oligosaccharide standard alditols; 2g, 3g, 4g, elution volumes of the respective agalacto oligosaccharide standards (after degalactosylation, the two triantennary isomers and the bisected diantennary species coelute at 3g)

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

Section : Structures proposed for the major oligosaccharide fractions obtained from human VE-cadherin Structures were deduced from the analytical results in accordance with the general rules of mammalian glycoprotein-N-glycan architecture (Vliegenthart and Montreuil, 1995; Sharon and Lis, 1997)

Content :

The molar ratios were roughly estimated from the distribution of 3H radioactivity incorporated into GlcNAc assuming a uniform labeling
aLinkage position of sialic acid not assigned
bBoth sialylation variants possible

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

Section : Colocalization of sialic acids, VE-cadherin and filamentous actin in endothelial cells of intact human umbilical veins in situ using confocal laser microscopy

Content :

Double staining of sialic acids (labeled by MAA/SNA) and VE-cadherin (labeled by a monoclonal antibody) (A, B) as well as sialic acids and actin filaments (stained by FITC-labeled phalloidin ) (C, D)
Both, VE-cadherin and the junctional located actin filaments display a codistribution with sialic acids (arrows in A and B as well as in C and D point to the very same cells)
Scale bar, 20 µm

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

Section : 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

Content :

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
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)
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
Higher branched N-glycans, i.e., triantennary and, especially, tetraantennary chains as well as high mannose-type oligosaccharides were less abundant
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)
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
In conclusion, natural human VE-cadherin appears to be mainly decorated with carbohydrates of restricted branching pattern
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
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
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
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
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
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)
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
Additionally, it has been shown that VE-cadherin but not N-cadherin is clustered at the intercellular junctions (Salomon ; Navarro )
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
The remaining weak MAA/SNA staining after Ca2 +-depletion might be related to Ca2 +-independent molecules such as PECAM-1
In highly confluent cultures of human umbilical vein and artery endothelial cells, VE-cadherin appeared in an undisturbed continuous band along the interendothelial junctions
At overlapping endothelial cell junctions, a netlike VE-cadherin organization was visualized
This network can be considered as extended VE-cadherin clusters and is assumed to considerably increase the interendothelial adhesion properties
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)
The formation of such a superstructure possibly requires lateral association of the assumed cadherin strand dimers that might be influenced by carbohydrate residues
The discussion on the contribution of glycan chains to VE-cadherin function, however, is still contradictory
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
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
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
This observation is in good agreement with our results revealing mainly diantennary and hybrid-type glycans on natural VE-cadherin
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 )
Our data show that sialidase treatment of living endothelial cells caused a significant change in VE-cadherin cellular organization
Under these conditions, VE-cadherin still appeared at interendothelial junctions but displayed a scattered immuno-fluorescence pattern including the disappearance of its super-structure
In contrast, PECAM-1 underwent no morphological changes
Thus, the results described may at least suggest an involvement of sialic acid residues in the structural organization of VE-cadherin
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
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
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
Further studies are required, however, to definitely prove the influence of carbohydrate substituents on VE-cadherin function

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

Protein	NCBI ID	SENTENCE INDEX