A role of heparan sulphate proteoglycan in the cellular uptake of lipocalins ß-lactoglobulin and allergen Fel d 4

Membrane crosslinking studies indicate close neighbourhood of lipocalins and HSPG binding proteins

Previous studies demonstrated that exogenous animal lipocalins can be internalized by different human cell lines. An example showing uptake and colocalization of bovine BLG and feline Fel d 4 in HeLa cells and CHO cells is depicted in Figure 1. The near perfect colocalization of both proteins in both cell lines, together with the fact that Fel d 4 internalization could be blocked by an excess of BLG (see Supplementary Figure 1), indicated a common uptake mechanism. Therefore, in a first attempt we sought to isolate the corresponding receptor by a crosslinking approach using biotinylated lipocalins which we crosslinked to HeLa cell membranes using SDAD photochemical crosslinker. Then, membranes were solubilized and lipocalin bait-prey complexes were isolated by affinity pull-down and analysed by SDS-PAGE. A silver-stained gel displaying proteins captured by crosslinking lipocalin baits to HeLa membranes is shown in Figure 2. A distinct pattern of protein bands ranging from about 30 to 100 kDa is visible irrespective of lipocalin bait used, although band intensities were weaker for Fel d 4. The most prominent protein bands were excised and identified by mass spectrometry (Table 1). Several heat shock proteins (HSP90, BIP, mtHSP70 and HSP60) and Calnexin, which usually locate to the endoplasmic reticulum, mitochondria and cytosol, were found as well as the cytoskeletal proteins Actin, Vimentin and Emerin. This result was unexpected, but we noticed that a very similar pattern of proteins was identified in an attempt to crosslink lipocalin-type prostaglandin D synthase (PGDS) to NIH3T3 cells (Suk 2012). Later, PGDS was indeed demonstrated to directly interact with cellular and recombinant HSP90 (Binda et al. 2014). Moreover, there is increasing evidence that HSPs can traffic to the cell surface and reinternalize later (Crowe et al. 2017; Okazaki et al. 2000), a mechanism which might be relevant for cellular uptake of PGDS and other lipocalins. To investigate whether BLG and Fel d 4 also directly interact with HSP90, we recombinantly expressed it from E. coli and performed co-affinity precipitation experiments. However, we observed no direct interaction of HSP90 with BLG or Fel d 4 (see Supplementary Figure 2).

Figure 1:

Uptake and colocalization of BLG (a, e) and Fel d 4 (b, f) in HeLa and CHO cells, respectively. Cells were simultaneously incubated with 25 µg/mL Carboxyfluorescein-BLG and 25 µg/mL Hilyte Fluor 594-Fel d 4 in serum-free medium for 2 hours, then fixed in paraformaldehyde and counterstained for nuclei with DAPI (c, g). Merged images (d, h). Scale bar is 10 μm.

Figure 2:

Proteins captured by crosslinking either BLG or Fel d 4 to HeLa membranes. Samples were run on 12 % SDS-PAGE and visualized by mass spectrometry-compatible silver staining. Protein bands indicated by arrows were excised and identified by mass spectrometry (refer to Table 1). Lanes correspond to molecular size marker (M), 200 ng HeLa membrane proteins (1), supernatant, final wash and eluate from BLG (2, 3 and 4) and Fel d 4 (5, 6 and 7) crosslink samples respectively.

Thus, we speculated that crosslinking of lipocalin baits to cell-surface HSP90 might result from indirect interaction. Heat shock proteins (HSPs) usually locate to the endoplasmic reticulum, mitochondria and cytosol, but have also been found on the cell surface (Bzowska et al. 2017; Gonzalez-Gronow et al. 2009; Soltys and Gupta 1996). These cell-surface exposed HSPs may be bound to heparan sulphate, which has been demonstrated for HSP90 (Snigireva et al. 2015), as they often possess heparin binding activity (Harada et al. 2014; Itoh and Tashima 1993; Ménoret and Bell 2000). This assumption was further supported by the high abundance of Calnexin, an endoplasmic reticulum chaperone and lectin, that has been found to associate with cell surface glycoproteins in HeLa cells (Okazaki et al. 2000). In sum, these indications encouraged us to investigate a potential interaction of lipocalins and heparan sulphate proteoglycan (HSPG).

Lipocalins BLG and Fel d 4 bind to heparin

We first tested whether lipocalins potentially possess heparin or heparan sulphate binding affinity at physiological pH by performing heparin affinity chromatography. Despite net negative charge both BLG and Fel d 4 showed specific retention on a heparin sepharose column (Figure 3) indicating the presence of a heparin-binding motif. In contrast, human Lcn-1, which is similar in molecular weight, structure, isoelectric point and number of basic amino acid residues, did not bind. This observation suggested that HSPG may play a role in binding and internalization of lipocalins.

Figure 3:

Heparin-affinity chromatography of Lcn-1, BLG and Fel d 4. 200 µg of each lipocalin were applied to a heparin sepharose column in low salt binding buffer pH 7.2 and eluted by a linear sodium chloride gradient (dashed line). BLG and Fel d 4 were retained and eluted at approximately 100 mM sodium chloride while Lcn-1 did not show any heparin binding.

Lipocalin uptake requires cell surface HSPG in HeLa cells

To verify the above hypothesis, we investigated the effects of different reagents impairing the structure of cellular HSPGs on the internalization of lipocalins. In the first approach, we tested the influence of soluble heparin, a polysaccharide closely related to heparan sulphate, on the endocytosis of BLG (Figure 4). For comparison we used lipocalin-1 (Lcn-1), which is internalized via its specific receptor LIMR (Wojnar et al. 2001). Cellular uptake of BLG and Lcn-1 was investigated in untreated HeLa cells or cells pre-treated with heparin prior to the addition of fluorescently labelled lipocalins. Untreated control cells display a circular, diffuse Lcn-1 fluorescence surrounding the nucleus while a more distinct, punctate, non-overlapping BLG fluorescence pattern indicates a different endocytosis route (Figure 4A). Following treatment with heparin, the Lcn-1 signal remains unchanged, which was also evident from image quantification analysis (Figure 4B). However, the vesicular BLG fluorescence is almost completely lost. A similar effect of heparin treatment was also observed on the endocytosis of Fel d 4 (Figure 5). Overall, image quantification revealed a reduction in both BLG and Fel d 4 uptake of about 75% after heparin treatment (Figure 8).

Figure 4:

(A) Effect of heparin treatment on the internalization of BLG and Lcn-1 in HeLa cells. (a–d) untreated control cells. (e–h) cells treated with 100 µg/mL heparin. Cells were incubated with equal amounts of labelled lipocalins. Scale bar is 10 μm. (B) Quantification of Lcn-1 internalization in untreated and heparin treated cells (normalized mean fluorescence intensity ± 1 SD, analysed by imageJ).

Figure 5:

Effect of heparin treatment on the internalization of Fel d 4 (red) in Hela cells. (a–c) untreated control cells. (d–f) cells treated with 100 µg/mL heparin. Scale bar represents 10 μm.

Figure 6:

(A) Effect of heparinase treatment on the internalization of BLG in Hela cells. (a–c) untreated control cells. Cells pre-treated with 0.5 (d–f), 2.0 (g–i) or 10.0 (j–l) U/mL heparinase for 30 min. Scale bar is 10 μm. (B) Effect of heparinase treatment on the internalization of Fel d 4 in Hela cells. (a–c) untreated control cells. Cells pre-treated with 0.5 (d–f), 2.0 (g–i) or 10.0 (j–l) U/mL heparinase for 30 min. Scale bar represents 10 μm.

Figure 7:

(A) Effect of NaClO₃ treatment on the internalization of BLG in HeLa cells. (a–c) untreated control cells. (d–f) Cells pre-treated with 30 mM NaClO₃ overnight. Scale bar is 10 μm. (B) Effect of NaClO₃ treatment on the internalization of Fel d 4 in HeLa cells. (a–c) Untreated control cells. (d–f) Cells pre-treated with 30 mM NaClO₃ overnight. Scale bar is 10 μm.

Figure 8:

Quantification of the effect of HSPG-disturbing treatment on the internalization of lipocalins in HeLa cells. (A) fluorescence intensities of BLG. (B) fluorescence intensities of Fel d 4. Fluorescence signals were quantified by ImageJ, normalized to control levels and depicted as mean signal intensity ± 1 SD (obtained from at least 3 cells per image). Original, uncut images, which the presented quantification is based on, are depicted in Supplementary data (Supplementary Figures 3–8).

Next, we treated HeLa cells with different concentrations of heparinase (0.5, 2.0 and 10.0 U/mL) for 30 min prior to the addition of labelled lipocalins. Again, uptake of BLG (Figure 6A) and Fel d 4 (Figure 6B) was markedly reduced. Similar to the results obtained from heparin blocking experiments described above, the amount and intensity of BLG and Fel d 4 fluorescent vesicles was reduced by about 70% in a concentration-dependent manner in comparison to untreated control cells (Figure 8).

Third, we pre-incubated HeLa cells overnight with sodium chlorate, an inhibitor of sulphate transfer onto glycosaminoglycans (GAGs), and then performed uptake experiments. This treatment also resulted in severe reduction of BLG and Fel d 4 uptake (Figures 7A,B). Image quantification demonstrated that the intracellular fluorescence was diminished by about 80% for both proteins (Figure 8).

In sum, these results clearly demonstrated that BLG and Fel d 4 internalization in HeLa cells depends on binding to cell surface heparan sulphate proteoglycan, which is, however, not involved in Lcn-1 uptake.

Lipocalin uptake is inhibited in glycosaminoglycan-deficient CHO cells

Furthermore, we investigated the endocytosis of BLG and Fel d 4 in wild-type and glycosaminoglycan-deficient deletion mutant (pgsA-745) CHO cells (Figure 9). These mutant cells have a defect in xylosyltransferase, the first sugar transfer in GAG synthesis, and do not produce GAG. Wild-type cells display a clear accumulation of fluorescence signal in a perinuclear compartment after 1 hour of incubation with either protein. In contrast, GAG-deletion mutant cells hardly contain any fluorescence signal. This observation again suggests a vital role of GAGs in the endocytosis of BLG and Fel d 4 lipocalins.

Figure 9:

Uptake studies of BLG (green) and Fel d 4 (red) in wt CHO cells (top row) and pgsA-745 mutant CHO cells (bottom row) lacking GAG biosynthesis.Scale bar repreesents 10 μm. In some mutant cells a faint red signal (panel d) is located on the cell surface and might be due to unspecific binding to other cell surface components.

HSPG and BLG colocalize on the plasma membrane of HeLa cells

Finally, we were able to detect colocalization of BLG and heparan sulphate on the plasma membrane of HeLa cells by immunostaining (Figure 10). Cell surface HSPG stained in a punctate pattern, which displayed a high degree of colocalization with BLG fluorescence. Additionally, enrichment of BLG is also observed within a perinuclear compartment while the heparan sulphate antibody used in this study was not internalized but was only detectable on the cell surface. The colocalization of heparan sulphate and BLG signals on the plasma membrane further substantiates our assumption of heparan sulphate requirement for lipocalin internalization. This observation is in agreement with our blocking experiments using heparin, heparinase and sodium chlorate as well as the results obtained from the GAG-deletion mutant CHO line pgsA-745.

Figure 10:

Colocalization of BLG (a) and heparan sulphate (b) on the plasma membrane of a HeLa cell. Nuclei (c) were stained with DAPI post-fixation. Merge of all channels (d). Scale bar is 10 μm.

Cell culture

HeLa, CHO-K1 and glycosaminoglycan (GAG)-deficient CHO-K1 mutant pgsA-745 cells (Esko et al. 1985) were purchased from ATCC. All cell lines were grown in DMEM/Ham's F12 medium (Gibco, Thermo Fisher Science, Austria, Vienna) supplemented with 10% FCS, 2 mM glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin (further abbreviated “growth medium”) and passaged in 2 to 3-day intervals.

Biotin and SDAD-labelling of lipocalin baits

Lipocalins beta-Lactoglobulin (BLG) and Fel d 4 were simultaneously labelled with Sulfo-NHS-SS-Diazirine (SDAD) photoactivatable crosslinker and Sulfo-NHS-LC-Biotin (both from Thermo Scientific, Austria, Vienna). Labelling efficiency was measured as biotin incorporation by a HABA-biotinylation-assay (Thermo Scientific) at 1-2 biotins per protein molecule. SDAD-labelling was estimated to be equally efficient due to similar molecular weight and identical reaction chemistry. Both StrepTactin-binding and crosslinking activity were successfully tested (not shown).

HeLa membrane preparation

HeLa cells grown to confluency were washed in PBS and incubated in serum-free growth medium for another 2 hours. Then, cells were washed in ice-cold PBS and harvested using a cell scraper. Cells were collected by centrifugation and the pellet resuspended in cold lysis buffer (250 mM sucrose, 1 mM EDTA, 10 mM HEPES pH 7.4 supplemented with protease inhibitors). Cells were lysed mechanically by glass beads in a mixer mill (Retsch MM 400, Germany, Haan) set to 25 Hz for 3 min. Membranous compounds were pelleted by ultracentrifugation at 100 000 g at 4 °C for 1 hour. The pellet was resuspended in buffer (250 mM sucrose, 1 mM MgCl₂, 10 mM HEPES pH 7.4), layered onto sucrose density gradient ranging from 28-43% and centrifuged in a swinging bucket rotor overnight at 100 000 g. Plasma membrane fraction was collected and protein concentration was determined by Bradford assay.

Isolation and identification of crosslinked proteins from HeLa membranes

Ten µg of labelled lipocalin bait were combined with 100 µg of HeLa cell membranes in 20 mM sodium phosphate pH 7.4. After 1 hour of incubation at room temperature, crosslinking was induced by UV irradiation at 365 nm for 5 min. Thereafter, the complexes were solubilized by boiling in 1 % sodium dodecylsulphate (SDS) and diluted 10-fold in wash buffer (50 mM Tris pH 7.4, 500 mM urea, 500 mM NaCl, 1% Triton X-100). Lipocalin bait-prey complexes were pulled down on StrepTactin sepharose beads (IBA Lifesciences, Germany, Göttingen), extensively washed and finally eluted by reductive cleavage of SDAD’s disulphide bond with 50 mM dithiothreitol (DTT). Supernatants were mixed with 5× Laemmli sample buffer and boiled for 5 min at 95°C. These eluates were then run on a 12% polyacrylamide gel and visualized by mass spectrometry-compatible silver staining. Proteins bands were excised and identified by mass spectrometry (see supplementary information).

Production and purification of lipocalins and HSP90

Recombinant Lcn-1 was produced as described previously (Holzfeind and Redl 1994). A codon-optimized Fel d 4 gene was synthesized (GenScript, USA, Piscataway Township), cloned into Qiagen’s pQE70 expression plasmid, produced and purified in M15 E. coli strain according to the manufacturer’s instructions (QIAexpressionist). BLG used in this study originated from bovine milk (L7880, Sigma-Aldrich, Austria, Vienna) and was further purified by gel filtration. The gene encoding HSP90 was amplified from HeLa cDNA (attaching an N-terminal Strep-tag via PCR), cloned into the pET21d+ expression plasmid and expressed in Rosetta(DE3) cells.

Lipocalin-HSP90 co-affinity precipitation experiments

Recombinant Strep-tagged HSP90 was directly bound to magnetic StrepTactin beads (MagStrep “type 3” XT beads, IBA Lifesciences) from clarified E. coli lysate. The HSP90-loaded beads were washed extensively in PBS before BLG or Fel d 4 were added at a final concentration of 25 µg/mL in PBS buffer. The reactions were incubated for 1 hour at room to allow binding of lipocalins to HSP90. Afterwards, beads were washed in PBS thrice before bound HSP90 was eluted in 50 mM D-biotin in TBS (100 mM Tris pH 8.0, 150 mM NaCl). Samples collected from supernatant post-incubation, final washing step and eluate were analysed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-Page). Two control experiments were done applying each protein (HSP90 or lipocalin) alone to StrepTactin beads.

Heparin affinity chromatography

Lipocalins (Lcn-1, BLG and Fel d 4) in low salt buffer (buffer A: 20 mM sodium phosphate pH 7.4) were applied to a 2 mL heparin sepharose column (Heparin Sepharose 6 FastFlow, GE Healthcare, Austria, Vienna) connected to an ÄKTA prime chromatography system (Amersham Pharmacia Biotech, UK, Little Chalfont). Analysis was performed at a flow rate of 1 mL/min. Salt concentration was measured by conductivity and protein concentration by UV absorbance at 280 nm. Samples were loaded and washed under low salt conditions (16 min at 0% buffer B: 20 mM sodium phosphate pH 7.4, 500 mM sodium chloride) and eluted by a linear salt gradient (0 to 100% B over 8 min).

Fluorescence labelling of Lipocalins

BLG was labelled with an 8-fold molar excess of NHS-Carboxyfluorescein (Sigma) in PBS for 1 hour at room temperature. Lcn-1 and Fel d 4 were labelled with a 5-fold molar excess of Hilyte Fluor 594, SE (Anaspec, Germany, Göttingen) in PBS for 1 hour at room temperature. Then, coupling reactions were quenched and protein-dye conjugates separated from unreacted dye by diafiltration. Degree of labelling was about 1.5 and 1 dye molecules for BLG and Lcn-1/Fel d 4 labelling reactions respectively.

Fluorescence microscopy

One day prior to experiments, 2.5×10⁴ HeLa or Chinese hamster ovary (CHO) cells were settled in 4-well chamber slides (Nunc Lab-Tek, Thermo Scientific) in 500 µl of growth medium. The next morning, cells were washed in PBS+ (PBS supplemented with 1 mM each of MgCl₂ and CaCl₂), incubated in serum free medium and treated as described below. Afterwards, samples were washed, fixed in 4% paraformaldehyde, counterstained with DAPI and imaged on an Axioplan fluorescence microscope (Carl Zeiss, Germany, Jena) equipped with an AcioCam MR3 camera and filter sets 365/445, 470/540 and 565/620. All image processing and analysis was done on Axiovision (Carl Zeiss), Microsoft PowerPoint (Microsoft Corp.) and by ImageJ (imagej.net).

Heparin, heparinase and sodium chlorate treatment of HeLa cells

HeLa cells were treated with PBS (control), 100 µg/mL heparin (from porcine intestinal mucosa, Sigma), 0.5, 2.0 or 10.0 U/mL heparinase I/III blend (Sigma) in serum-free growth medium for 30 min before 25 µg/mL (equalling 1.25 µM) fluorescently labelled BLG or Fel d 4 were added for 2 hours.

For chlorate experiments, HeLa cells were cultured overnight in growth medium without (control) or with 30 mM NaClO₃. The next morning, cells were washed with PBS and incubated in serum-free growth medium for 30 min before labelled lipocalins were added as indicated above.

CHO K1 wild-type and GAG-deficient pgsA-745 mutant cells

CHO K1 wild-type (control) and GAG-deficient pgsA-745 mutant cells were incubated with 25 µg/mL (equalling 1.25 µM) fluorescently labelled BLG or Fel d 4 in serum-free medium for 2 hours and then processed for fluorescence microscopy.

Colocalization of cell surface heparan sulphate and BLG

HeLa cells were simultaneously incubated with Carboxyfluorescein-BLG (25 µg/mL equalling 1.25 µM) and 1 µg of biotinylated heparan sulphate antibody (amsbio F58-10E4, AMS Biotechnology Ltd, UK, Abingdon) coupled to AlexaFluor594-Streptavidin (Thermo Scientific) for 1 hour in serum-free growth medium. Afterwards, samples were washed, fixed and counterstained with DAPI. Imaging was performed as described above.