The Essential Role of N-Glycosylation in Integrin αV and uPAR Interaction in Glioblastoma

doi:10.21203/rs.3.rs-4888759/v1

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The Essential Role of N-Glycosylation in Integrin αV and uPAR Interaction in Glioblastoma

https://doi.org/10.21203/rs.3.rs-4888759/v1

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BACKGROUND

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor in adults, characterized by poor patient survival rates. The glycoproteins Integrin αV (IαV), and the Urokinase-type plasminogen activator receptor (uPAR) are key contributors to tumor malignancy in GBM, and although their interaction is well-described, the role of glycans in this process has been scarcely evaluated. Better understanding this interaction could enhance our knowledge of the disease and lead to potential new therapeutics.

METHODS

We investigated the interaction between IαV and uPAR in human GBM, A172 and LN229, and low-grade glioma, SW1088, cell lines. Expression of these proteins was confirmed via confocal microscopy and co-immunoprecipitation. The role of N-glycosylation was evaluated using the inhibitor Swainsonine (SW) and glycosidase PNGase F. Glycoproteomic analysis by mass spectrometry identified glycosylation sites and differential structures on IαV. The impact of sialic acids and specific glycan structures was assessed using Neuraminidase (NeuA) and lectin binding assays.

RESULTS

The expression of IαV and uPAR, as well as their interaction, was confirmed in GBM cells but not in low-grade glioma cells, even when uPAR was overexpressed. SW and PNGase treatments markedly reduced IαV/uPAR interaction, highlighting the importance of N-glycosylation. Mass spectrometry analysis showed six glycosylation sites on IαV in GBM cells, with complex and hybrid N-glycans, while only oligomannose N-glycans were detected in low-grade glioma cells. NeuA treatment also reduced IαV/uPAR interaction, underscoring the role of sialic acids. Lectin assays suggested β1–6 branched glycans at specific sites are crucial for this interaction. Inhibition of N-glycosylation and sialic acid removal both decreased AKT phosphorylation, indicating a significant role of these glycans in integrin/uPAR signaling.

CONCLUSIONS

Our results demonstrate for the first time the interaction between IαV and uPAR in GBM cells, highlighting the critical role of N-glycosylation, particularly β1–6 branched glycans and sialic acids.

N-glycosylation

Integrin αV

uPAR

glioblastoma

glioma

Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumor affecting adults, accounting for a substantial 45.6% of all malignant primary brain tumors. The age-adjusted annual incidence of GBM increases with age, from 0.15 per 100,000 in children to a peak of 15.03 per 100,000 in patients aged 75–84 years. Regrettably, current therapies do not yield satisfactory outcomes. On average, GBM patients survive for just 12–18 months. However, 25% of them live beyond a year, and 5% survive for more than five years [1]. A better understanding of the biology of GBM is crucial to unravel its complexities and discover new therapeutic targets.

Integrins are heterodimeric proteins constituted by the non-covalent association of both α and β subunits. In vertebrates, 18 α and 8 β different subunits have been described, allowing the assembly and expression of 24 distinct heterodimers [2]. Integrin-ligand pairings can be categorized into four primary classes, determined by the type of molecular interaction involved. In particular, all αV-containing integrins together with two β1 integrins, as well as αIIbβ3, have been described as RGD receptors since they recognize a short peptide sequence present in several extracellular matrix (ECM) proteins such as vitronectin, fibronectin and fibrinogen among others [3]. The role of RGD receptor integrins primarily lies in the regulation of cell behavior in response to the extracellular microenvironment. Altered integrin signaling is known to promote invasive behavior of tumor cells, influence the tumor microenvironment to facilitate angiogenesis [4], participate in the interaction between the ECM and tumor cells [5, 6], promote cancer immune escape [7, 8], regulate stem cell function [9, 10], and take part in the organ-specific targeting of metastatic cancer cells [11]. Expression of αVβ3 and αVβ5 heterodimer has been widely described to be associated with malignant features of GBM [12–15]. αVβ3 was the first integrin to be abundantly detected in high-grade brain tumors [16], with almost 60% positivity reported in GBM samples [17]. Extensive research has underscored its role in sustaining GBM's high proliferation rate, enhancing migratory and invasive capabilities, and promoting angiogenesis [18, 19]. Furthermore, integrin participation in tumor radio- and chemo-resistance has been reported [20, 21].

The association between integrin and urokinase-type plasminogen activator receptor (uPAR) has been described in several tissues and tumors modulating signaling and cell behavior. uPAR is a glycosylated single-chain protein GPI-anchored with a molecular weight ranging from 50 kD to 60 kD. It exerts control over the plasminogen activation system, an extracellular proteolytic cascade, through its interaction with the serine protease urokinase-type plasminogen activator (uPA) and its inactive zymogen form, pro-uPA [22]. In addition to regulating extracellular plasmin activity, uPAR is a signaling receptor. It is reported that signaling through uPAR activates the Ras-mitogen-activated protein kinase (MAPK) pathway, the Tyr kinases focal adhesion kinase (FAK), and the Rho family small GTPase Rac. Furthermore, FAK signaling can activate the PI3K-Akt/PKB [23–26], participating in the modulation of cell motility, invasion, proliferation and survival. Since uPAR lacks a transmembrane domain, it requires cooperation with transmembrane receptors to drive intracellular signaling. The expression of uPAR has been highlighted in several cancers as a key actor in malignant behavior as well as being proposed as a bad prognostic marker [27]. In particular, the overexpression of uPAR in GBM is involved in epithelial-mesenchymal transition and its association with poor prognosis have been reported [28, 29]. Although several membrane proteins have been identified as possible co-receptors of uPAR, substantial evidence points to integrins as the main and most significant co-receptors of uPAR signaling [30, 31]. In this regard, numerous pieces of evidence confirm that the interaction between integrins and uPAR is mainly established between the β-propeller region of the α chain and uPAR Domain III [32, 33].

Even though both are glycosylated proteins, very little evidence provides information on the involvement of glycans in this interaction [34]. Despite significant advances in understanding the role of glycans in several types of tumors, the impact of glycosylation and the potential of glycans as therapeutic targets still merit further investigation [35]. Aberrant glycosylation involves changes in the glycosylation patterns of normal cell progenitors and occurs alongside the multistep process of malignant transformation [36]. These altered glycosylation profiles are linked to cellular characteristics that facilitate tumor progression, including adhesion to the extracellular matrix, migration, invasion, inhibition of apoptosis, host immunoregulation, and resistance to chemotherapy [37].

In this study we demonstrated the interaction between integrin αV (IαV) and uPAR in human GBM cells and identified a differential pattern of glycans in the low-grade counterpart. In addition, we present compelling evidence of the significant role played by N-glycosylation in this interaction.

Cell lines

Human low-grade glioma cell line derived from a diffuse astrocytoma (grade II) SW1088 and human high-grade glioma cell line (grade IV) A172, obtained from American Type Culture Collection (ATCC, USA), were grown in Roswell Park Memorial Institute (RPMI) 1640 Medium (Sigma-Aldrich, USA). Human GBM cell line (grade IV) LN229, kindly provided by Dr. Marianela Candolfi, Institute of Biomedical Research (CONICET), was grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich, USA). Cell lines were supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, USA) and 80 µg/ml of gentamicin (Northia, Argentina). The cells were maintained at 37°C in a humidified atmosphere containing 5% CO2, routinely subculture was performed using Trypsin-EDTA solution (Gibco, USA) following standard procedures. Monthly screening for Mycoplasma contamination was conducted using 4',6-diamino-2-phenylindole (DAPI) staining (Vector, USA) and visualization under a fluorescence microscope.

Glycosylation inhibition and removal

Cells were cultured in their respective medium and incubated with 100 nM Swainsonine (SW) (Sigma-Aldrich, USA), 5 UI/ml Peptide-N-Glycosidase F (PNGase) (New England Biolabs, USA), or 1 UI/ml of Neuraminidase A (NeuA) (New England Biolabs, USA) for 24 h. Control cells were treated with an equivalent volume of phosphate buffered saline (PBS).

Lectin interference

Cell monolayers cultured without FBS were incubated with 20 µg/ml of the lectins Phytohemagglutinin-L (PHA-L) or Concanavalin A (ConA) (Vector Labs, USA) for 1 h at 37°C. Following this incubation, protein co-immunoprecipitation and immunofluorescence assays were conducted.

Plasmid DNA transfection

For overexpression of the uPAR protein, cells were grown to approximately 90% confluence and transfected with uPAR-plasmid (HG10925, Sino Biological, USA) using Lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer's instructions. Immunofluorescence or western blot (WB) assays were conducted 48 h post-transfection.

Immunofluorescence

A total of 2x10⁵ (for A172) or 5x10⁵ (for LN229 or SW1088) cells were seeded on coverslips (JSHD, China) in 6-well plates. Cells were washed with PBS and fixed for 10 min with 4% formalin in PBS at room temperature. Following fixation, cells were blocked for 30 min with 1% FBS in PBS. Subsequently, samples were incubated overnight at 4°C with primary antibodies against IαV (1:200, Ab179475, Abcam, United Kingdom). After washing with PBS, cells were incubated with FITC-conjugated secondary antibodies (1:200, Ab6717, Abcam, United Kingdom) in PBS for 1 h at room temperature. Following three washes with PBS, cells were incubated with primary antibodies against uPAR (1:50, sc-376494, Santa Cruz Biotechnology, USA) and Alexa 594-conjugated secondary antibodies (1:200, A11005, Life Technologies, USA), as it was previously mentioned. Finally, coverslips were incubated with 0.1 µg/ml of DAPI (Sigma-Aldrich, USA) for 15 minutes at room temperature and mounted on glass slides with 80% glycerol in PBS.

Confocal microscopy

Confocal microscopy (CM) was performed using an inverted Leica TCS SP8 spectral scanning confocal microscope (Leica Microsystem, Germany) equipped with specific lasers for the fluorochromes Alexa Fluor-594 (exc: 561nm), FITC (exc: 488 nm) and DAPI (exc: 405 nm). Observation was conducted with 60X objective, and the images were captured by sequential scanning configuring different detection channels for each fluorochrome using LasX software to digitize images with a resolution of 1024 x 1024 pixels.

Colocalization analysis of proteins was performed using Pearson's correlation coefficient analysis, which was implemented through the Fiji image processing package of ImageJ software. The Pearson's correlation coefficient values were interpreted as follows: a value of + 1 indicated perfect positive correlation, values greater than 0.5 denoted positive interaction, 0 represented no correlation, and − 1 indicated perfect negative correlation.

Protein co-immunoprecipitation

Protein co-immunoprecipitation (Co-IP) was conducted using the PierceTM Direct IP Kit (Invitrogen, USA) following the manufacturer's protocol. Briefly, cells at 90% of confluence were lysed with Lysis Buffer containing protease inhibitor cocktail (Sigma-Aldrich, USA) at 4°C and centrifuged at 13,000 x g for 10 min. The supernatant was incubated overnight at 4°C on an orbital shaker with agarose columns to which 5ug anti-IαV (Ab179475, Abcam, United Kingdom) or 4ug anti-uPAR (Ab22168, Abcam, United Kingdom) antibodies were attached. The proteins retained in the column were washed and eluted using Elution Buffer to obtain the desired fraction. Immunoprecipitated proteins were analyzed by WB.

SDS PAGE and Western Blot

Monolayers containing 5x10⁵ cells were lysed with RIPA buffer supplemented with protease and phosphatase inhibitors (Sigma-Aldrich, USA). Protein lysates were clarified, and protein concentrations were normalized using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Immunoprecipitated proteins or 30 ug of total cell lysates were subjected to electrophoresis in polyacrylamide gels, followed by semi-dry transfer to 0.45µm polyvinylidene difluoride membranes (GE Healthcare, USA). Membranes were blocked with low-fat milk and then incubated overnight at 4°C with primary antibodies against IαV (1:5000, Ab179475, Abcam, United Kingdom), uPAR (1:100, sc-376494, Santa Cruz Biotechnology, USA), AKT (1:500, AF1775, R&D Systems, USA), phospho-AKT (pAKT) (1:500, PACO02670, Assay Genie, Ireland) and β-tubulin (1:5000, 556321, BD Bioscience, USA). After washing, membranes were incubated for an hour with secondary antibodies anti-rabbit (170–6515, BioRad, USA) or anti-mouse (170–6516, BioRad, USA) immunoglobulins HRP conjugated. Finally, the membranes were revealed using a bioluminescence kit (Bio-Lumina, PBL, Argentina) and membrane images were captured using the C-DiGit® Blot Scanner (Li-Cor, USA).

Protein concentration

For mass spectrometry assays, immunoprecipitations were concentrated using Vivaspin® 500 Centrifugal Concentrators (Sartorius AG, Germany) following the manufacturer's instructions. Immunoprecipitation products were centrifuged at 12000 x g for 2 h, until reaching a final volume between 50 µl. Concentrated samples were subjected to N-glycan analysis by mass spectrometry.

Mass Spectrometry

Mass spectrometry (MS) was conducted on IαV from cell lines cultures. After the electrophoretic run, the gels were fixed for 3 h in a solution containing 50% methanol and 2% phosphoric acid. Following three washes, the gels were incubated for an hour in a balancing solution composed of 33% methanol, 17% ammonium sulfate, and 3% phosphoric acid. Next, Coomassie Brilliant Blue G 250 (Sigma Aldrich, USA) was added at a final concentration of 0.06%, and the gels were incubated overnight. After three additional washes with bidistilled water under agitation, the bands corresponding to the proteins of interest were excised.

Protein digestion and MS analysis were performed at the Proteomics Service of CEQUIBIEM at the University of Buenos Aires/CONICET. In brief, the excised bands were sequentially washed with 50 mM ammonium bicarbonate (AB), 25 mM AB, 50% acetonitrile (ACN), and 100% ACN. They were then reduced and alkylated with 10 mM dithiothreitol and 20 mM iodoacetamide. In-gel digestion with 100 ng of trypsin (Promega, USA) in 25 mM AB was carried out overnight at 37°C. The resulting peptides were recovered by elution with 50% ACN/0.5% Trifluoroacetic Acid and concentrated by rapid vacuum drying. Finally, the samples were resuspended in 15 µL of water containing 0.1% formic acid. The peptide digestion products were analyzed by nanoLC-MS/MS using a nanoHPLC EASY-nLC 1000 (Thermo Fisher Scientific, USA) coupled to a QExactive mass spectrometer. A C18 pre-column (Acclaim PepMap 3 µm, 100 Å, 75 µm x 20 mm) was used for peptide desalination, followed by a 75-minute gradient of H2O: ACN at a flow rate of 33 nL/min with an Easy Spray C18 column (2 mm x 150 mm). The data-dependent MS2 method was employed to fragment the most intense peaks of each cycle. Raw data from the MS analysis were processed using Proteome Discoverer software, version 2.1.1.21 (Thermo Fisher Scientific, USA). The glycan structures found were corroborated using the GlyConnect platform and graphical representation were obtained from GlyTouCan repository.

Statistical analysis

Statistical analysis was performed using Prism 8 software (GraphPad, Inc., USA). Data is presented as mean values ± standard deviation. Prior to statistical testing, normality of the data was assessed. For comparisons involving two independent samples, the Mann–Whitney test was employed. Multiple comparisons between experimental groups were analyzed using ANOVA followed by Tukey's post-test. Significance thresholds were set at *p < 0.05, **p < 0.01, and ***p < 0.001.

To begin to explore the interaction between uPAR and IαV, we first examined the expression of the glycoproteins in A172 and LN229 human GBM cell lines as well as SW1088 human glioma low grade by WB. Our results showed that these proteins are expressed in both GBM cell lines, although in SW1088 uPAR expression couldn't be detected (Fig. 1A). In order to evaluate the interaction between these two glycoproteins, we first performed CM image analysis. Pearson´s coefficient analysis revealed a high colocalization rate of these two glycoproteins in both GBM cell lines (Fig. 1B). In the same lane, Co-IP confirmed this interaction in both cell lines either when IP was performed on IαV or uPAR (Fig. 1C). Since uPAR was not detected in the SW1088 cell line, PLAUR gene transfection was performed to induce uPAR expression in these cells, which were then termed uSW1088 (Fig. 1A). Noteworthily, although uPAR expression was confirmed in uSW1088 cells, no molecular interaction between this protein and IαV was detected (Fig. 1B and 1C).

Given that both uPAR and IαV are glycosylated proteins with multiple N-glycosylation sites, we questioned to what extent this type of glycosylation is involved in their interaction. SW specifically inhibits alpha-mannosidase II, an essential enzyme for the maturation of N-linked glycoproteins. PNGase is an enzyme that cleaves the N-linked glycans from glycoproteins by targeting the glycosidic bond between the asparagine residue of the protein and the first N-acetylglucosamine of the glycan. Both treatments induced a significant decrease in IαV/uPAR interaction. As shown by CM analysis, Pearson´s colocalization coefficient showed a reduction of nearly 50% between control and SW or PNGase treated cells (Fig. 2A). In the same line, when Co-IP assays were performed no IαV/uPAR interaction was detected by WB in treated cells (Fig. 2B).

The amino acid sequence of IαV, stored in the UniProtKB/Swiss-Prot repository, predicted the existence of 12 potential N-glycosylation sites (Uniprot ID: P06756). Upon MS analysis of the IαV obtained from the GBM cell line LN229, 6 of these 12 sites were detected to be N-glycosylated (Supplementary Table 1). To map and characterize all the potential glycan structure of each specific glycosite, the m/z observed values obtained from MS-based glycoproteomic strategy were used. Main glycan structures for each position are depicted in Fig. 3A. The results showed that position Asn704 contains only complex/hybrid N-glycans, while only oligomannose N-glycans are present at position Asn945. In addition, at positions Asn74, Asn554, and Asn874 mostly complex/hybrid type N-glycans were present, with a lower abundance of core or oligomannose structures. On the other hand, at Asn615, a higher percentage of oligomannose type N-glycans was detected. As for the presence of sialic acid, only the glycans in Asn945 showed no presence of sialic acid, whereas glycans with N-Glycolylneuraminic acid (NeuGc), N-Acetylneuraminic acid (NeuAc) or both were observed in all other glycosites. Conversely, the analysis of IαV glycosylation in the low-grade SW1088 cell line revealed glycan structures at only two glycosylation sites, Asn74 and Asn874, where only oligomannose structures were present. No sialic acid expression was detected in the SW1088 cell line. Relative expression given the microheterogeneity of N-glycans associated with each glycosite of the protein is shown in Fig. 3B.

To identify the participation of sialic acids in the interaction between IαV and uPAR, cultured cells were treated with NeuA, an enzyme that removes the terminal sialic acids of glycan structures. As shown in Fig. 4, NeuA treatment decreases IαV/uPAR colocalization analyzed by CM and co-IP assay in both GBM cell lines.

To further our analysis, the interference of glycan-mediated protein interaction was evaluated using lectins. The PHA-L lectin mainly binds to β1–6 branched N-glycans which were detected in N74, N554 and N874 glycosites of IαV of GBM cell line. ConA recognizes oligomannose-type N-glycans that were detected at the N945 glycosylation site in both glioma and GBM cell lines, and also with a lower abundance at the N74, N554, N615 and N874 glycosylation sites of the IαV from GBM cell line. As expected from the glycosylation profile of GBM cells, incubation with PHA-L lectin strongly inhibited the interaction between IαV and uPAR. However, no effect was observed after ConA incubation (Fig. 5) indicating the participation of β1–6 branched N-glycans in IαV/uPAR interaction.

Finally, to assess the impact of glycans on IαV/uPAR-associated signaling pathways, AKT phosphorylation was evaluated in response to SW and NeuA. Both treatments resulted in a decreased AKT phosphorylation in LN229 and A172 cell lines. Interestingly, upregulation of uPAR expression in these cell lines by transfection of the PLAUR gene, resulting in the cells named uLN229 and uA172, increased AKT phosphorylation, suggesting the active involvement of uPAR in this signaling pathway. in the same line, either SW or NeuA treatment strongly, in these cells inhibited AKT phosphorylation (Fig. 6).

The interaction between the uPAR and integrins is highly relevant in the progression of many types of cancer, being these proteins key players in processes such as cell migration, invasion and metastasis. uPAR, a glycosylphosphatidylinositol-anchored cell surface receptor, binds to uPA, activating proteolytic cascades that degrade the ECM, facilitating tumor cell invasion. In parallel, uPAR can trigger intracellular signaling, thereby modulating physiological processes such as wound healing, immune responses and stem cell mobilization, as well as pathological events including inflammation and tumor progression [38, 39]. Integrins and integrin-dependent processes play crucial roles in nearly every phase of cancer progression, actively participating in signaling pathways that promote cell survival, proliferation, and metastasis [40, 41]. Both cell membrane receptors are identified as key contributors to tumor malignancy in GBM.

Interaction between integrin and uPAR has been shown in some other tumors. For instance, in gastrointestinal cancers, the interplay between αVβ6 and uPAR are implicated in the regulation of downstream signaling following uPA binding [30, 32] and also in the induction of MMP9 secretion, thus facilitating the degradation of multiples ECM components [42, 43]. In breast cancer, Annis et al. demonstrated the participation of integrin αVβ3 in tumor invasion via activation of SRC/MAPK signaling and FRA-1 phosphorylation [44].

Although in GBM some reports suggest an interaction between integrins and uPAR, no concrete evidence in this regard has been published up to now. Among the few reports on this are the results published by Veeravalli et al. where they demonstrated a down-regulation of the expression of several integrins such as α1, α2, α6, α7, α9 and αV as well as β1 and β3 in response to the reduction of uPAR mRNA level [45]. Their research underscores the critical roles of integrins, uPAR and MMP9 in glioma tumor biology, and proposes a possible interaction model between them [46].

Under the knowledge that IαV and uPAR are relevant antigens associated with poor clinical prognosis in human GBM, we focused our efforts on understanding this interaction. Employing two high-grade GBM cell lines that express both proteins, the results showed an interaction between them, demonstrated by Co-IP assays and CM. Since no uPAR expression was detected in the low-grade glioma cell line, we attempted to induce its expression by transfection of the uPAR gene sequence, termed PLAUR. Interestingly, no IαV/uPAR interaction was observed in these cells, suggesting that the mere presence of the protein in the low-grade glioma cell is not sufficient to establish a positive interaction. This implies that an additional factor participates in the interaction between these two proteins.

Glycosylation plays a significant role in protein interaction, especially in cell membrane proteins, where a broad spectrum of glycan structures is observed.

Aberrant glycosylation refers to the particular glycophenotype of cancer cells, which involves modification of the glycosylation profile of normal cell progenitors and is concomitant with the multistage process of malignant transformation [36]. The glycophenotype of tumor cells may be based primarily on N- or O-glycans, depending on the biology of the tumor. While in Neuroblastoma the glycophenotype is mostly expressed based on O-glycans [47], the glycosylation profile found in GBM is primarily expressed by N-glycans [48]. An altered glycosylation profile has been associated with cellular features that promote tumor progression, such as adhesion to the extracellular matrix, migration, invasion, inhibition of apoptosis, host immunoregulation and resistance to chemotherapy [49].

The glycosylation of integrins has been demonstrated as a relevant player in cell adhesion, migration, and survival in tumor cells [50–52]. αβ integrin dimers have more than 20 potential N-linked glycosylation sites, as reported [50]. Additionally, integrin αV specifically has 12 putative N-glycosylation sites. The presence of these N-glycan core structures has been demonstrated as crucial for several reasons: they are essential for integrin heterodimerization, stabilization of the conformation, expression at the cell membrane and interaction with ligands [53, 54].

The structural association between integrins and uPAR is extensively reported. Simon et al. and Zhang et al. described that the surface loop of the β-propeller domain of integrin α-chain in α3β1 and αMβ2 heterodimers functionally associates with uPAR [55, 56]. Subsequently, Chaurasia et al. further showed that integrin α5β1 interacts with the domain III region of uPAR [57]. Also, Ahn et al. suggested that the interaction with uPAR requires the expression of the complete αβ heterodimer (e.g. αVβ6) rather than individual subunits [33]. Additional insights through docking simulations of integrin αVβ3 and uPAR showed that the β-propeller region of αV from αVβ3 interacts with domains II and III of uPAR [58].

Although Integrin/uPAR interaction is well described, the role of glycans in it has been scarcely evaluated. To the best of our knowledge, only one report shows that treatment of melanoma cells with SW inhibits the interaction of αV and α3 with uPAR, suggesting an important role of N-glycosylation in this interaction [34]. In the same line, our results demonstrate that N-glycosylation plays a key role in integrin αV/uPAR interaction since treatment with both SW and PNGase inhibits it as demonstrated by Co-IP and CM. Of the 12 reported potential glycosylated positions of IαV, the evaluation in the GBM cell line LN229 showed six glycosylated positions (N74, N554, N615, N704, N874 and N945) and only two in the low-grade glioma SW1088 (N74 and N874). Interestingly, the glycan profiles between these cells showed substantial differences. The glycosylation profile of IαV obtained from the GBM cell line exhibited a high proportion of complex and hybrid glycans, with a limited presence of oligomannose-type glycans, except at position N945, which showed 100% of oligomannose. In contrast, IαV from low-grade glioma glycosylation was characterized exclusively by oligomannose-type glycans at the two glycosylated positions. As mentioned before, the β-propeller domain of integrin is, according to the literature, involved in the interaction with uPAR with N74 being the only glycosylation site within this domain. Although position N74 of IαV is glycosylated in both cell lines, the glycan profiles differ significantly: in the GBM cell line predominantly exhibits complex-type glycans, while the low-grade glioma cell line primarily shows oligomannose-type glycans. To identify the participating glycan structures, we performed treatments with NeuA which severely affects the IαV/uPAR interaction in both GBM cell lines A172 and LN229, indicating a significant involvement of sialic acids which were found in all glycosylated positions. Moreover, incubation with PHA-L that binds to β1–6 branched N-glycans also inhibits the IαV/uPAR interaction. Conversely incubation with ConA that recognizes oligomannose structures does not inhibits the IαV/uPAR interaction. Our results postulate that β1–6 branched glycans could be present at positions N74, N554, and N874. The first site is located within the β-propeller domain, the second adjacent to it, and the third near the transmembrane domain, forming part of Ig Domain 3. Considering the role of the integrin β-propeller domain in contacting uPAR, the glycan at N554 is unlikely to participate in the interaction, and the involvement of N874 is even less expected. Therefore, we speculate that the predicted branched β1–6 glycan at N74 plays an important role in this interaction. Further studies should be conducted to evaluate the participation of the glycosite N554.

To assess the impact of integrin-related glycosylation on cell response, we evaluated the phosphorylation of AKT, a known Integrin/uPAR-derived signal transducer [59, 60]. Treatment with SW in both GBM cell lines inhibits AKT phosphorylation by more than 50%, indicating that N-glycans are involved in AKT activating signaling. Similar results were observed after treatment with NeuA, pointing to the presence of sialic acid as a relevant player in this process. We performed the same experiments in cells transfected with PLAUR, showing an increase in the phosphorylation of AKT most likely due to overexpression of uPAR in both cell lines and an inhibition of it as a consequence of SW and NeuA treatment.

In conclusion, our findings demonstrate for the first time the interaction of IαV and uPAR in GBM cells and the major role played by N-glycans in it, suggesting an essential participation of β1–6 branched N-glycans as well as sialic acids, both structures found at glycosylation position N74 of IαV within the β-propeller domain. This work provides novel and valuable insights into the interaction between IαV and uPAR, two significant proteins in GBM tumor biology, highlighting the crucial role of glycosylation.

Our findings demonstrate, for the first time, the interaction between αV integrin and uPAR in GBM cells, provide relevant information on glycan motifs in high- and low-grade glioma, and highlight the essential role of N-glycosylation in this binding, in particular β1–6 branched glycans and sialic acids.

AB: Ammonium bicarbonate

ACN: Acetonitrile

CM: Confocal microscopy

Co-IP: Co-Immunoprecipitation

ConA: Concanavalin A

DAPI: 4′,6-diamidino-2-phenylindole

DMEM: Dulbecco's Modified Eagle's Medium

ECM: Extracellular Matrix

FAK: Focal adhesion kinase

FBS: Fetal bovine serum

GBM: Glioblastoma

IαV: Integrin αV

MAPK: Mitogen-activated protein kinase

MS: Mass spectrometry

NeuA: Neuraminidase A

NeuAc: N-Acetylneuraminic acid

NeuGc: N-Glycolylneuraminic acid

pAKT: Phospho-AKT

PBS: Phosphate buffered saline

PHA-L: Phytohemagglutinin-L

PNGase: Peptide-N-Glycosidase F

RPMI: Roswell Park Memorial Institute

SW: Swainsonine

uPA: Urokinase-type plasminogen activator

uPAR: Urokinase-type plasminogen activator receptor

WB: Western blot

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

All data generated or analyzed during this study are included in this published article [and its supplementary information files].

Competing interests

The authors declare that they have no competing interests.

Funding

Universidad Nacional de Quilmes, Argentina. Award Number: EXPTE 2283/22

Instituto Nacional del Cáncer, Argentina. Award Number: EXPTE 827-492/23

Authors' contributions

G.M.F., V.I.S. and M.R.G. contributed to the conception and design of the experiments, the analysis and interpretation of data and in the process of manuscript writing. G.M.F., H.A.C., A.C.N., J.O.C., S.R., and C.A.G. worked in development of methodology and data acquisition. All authors read and approved the final manuscript.

Acknowledgements

G.M.F and S.R. are research fellows of CONICET.

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No competing interests reported.

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The Essential Role of N-Glycosylation in Integrin αV and uPAR Interaction in Glioblastoma

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Background

Methods

Cell lines

Glycosylation inhibition and removal

Lectin interference

Plasmid DNA transfection

Immunofluorescence

Confocal microscopy

Protein co-immunoprecipitation

SDS PAGE and Western Blot

Protein concentration

Mass Spectrometry

Statistical analysis

Results

Discussion

Conclusions

Abbreviations

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References

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