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Granulin-epithelin precursor interacts with heparan sulfate on liver cancer cells.

Yip CW, Cheung PF, Leung IC, Wong NC, Cheng CK, Fan ST, Cheung ST - Carcinogenesis (2014)

Bottom Line: Suppression of the HS polymerase exostosin-1 reduced the rGEP binding and rGEP-mediated signaling transduction.Suppression of a specific HS proteoglycan, glypican-3, also showed a partial reduction of rGEP binding and an inhibition on rGEP-mediated activation of AKT.Furthermore, glypican-3 was shown to correlate with the expressions of GEP in clinical samples (Spearman's ρ = 0.363, P = 0.001).

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Centre for Cancer Research and.

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Related in: MedlinePlus

Mapping of heparin-binding domain in GEP and the contribution of the heparin-binding domain in HCC cell surface binding. (A) Schematic diagram of GEP protein, rGEP and derivatives and three granulin subunits. The seven and half granulins are shown as grey boxes in the GEP protein. Open arrows represent the N-glycosylation sites identified previously. The locations of rGEP, the deletion mutants (N492, C101, C77 and C51), the derivatives (rGEP-2A, rGEP-3A and rGEP-5A) and three granulin proteins corresponding to the GEP protein are shown. (B and C) Immunoblotting after heparin sepharose chromatography. The rGEP, deletion mutants, derivatives and granulin proteins were collected from media of the transfected COS-1. These media (P) were applied to heparin sepharose for incubation. The flow through (FT) was collected after 30min incubation. The sepharose was washed by Tris buffer for twice (W1 and W2). Elution buffer containing 0.125–2M NaCl was applied sequentially to the sepharose. The recombinant proteins were then detected by anti-His antibody in the immunoblotting. (D) Purified rGEP-3A and rGEP derivatives N492 and C101 were analyzed in SDS–PAGE. Protein was stained by Coomassie blue (CB) or detected by anti-His antibody in western blot (WB). (E) Polymerization of rGEP is independent of heparin and heparin-binding domain. Different amounts of heparin (0–100 µg/ml) were incubated with the recombinant protein at room temperature with or without DSS cross-linking. The polymerization status of rGEP (lanes A–F) and rGEP-3A (lanes G-L) was assessed by SDS–PAGE analysis and immunoblotting detected by anti-His antibody. (F) Indicated amounts of purified rGEP, rGEP-3A, N492 and C101 were incubated with detached Hep3B to compare their binding ability. The binding was then detected by FITC-anti-His antibody. In the case of HS depletion, cells were incubated with heparinase III in lyase buffer at 37°C for an hour before cell detachment. The histogram shows the fluorescent signal of the cells when incubate with 0.8 µg rGEP, N492 and C101. The shaded area represents isotypic control. The bar chart shows the geometric mean fluorescent intensity (MFI) of the cells when incubated with different amount of proteins.
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Figure 4: Mapping of heparin-binding domain in GEP and the contribution of the heparin-binding domain in HCC cell surface binding. (A) Schematic diagram of GEP protein, rGEP and derivatives and three granulin subunits. The seven and half granulins are shown as grey boxes in the GEP protein. Open arrows represent the N-glycosylation sites identified previously. The locations of rGEP, the deletion mutants (N492, C101, C77 and C51), the derivatives (rGEP-2A, rGEP-3A and rGEP-5A) and three granulin proteins corresponding to the GEP protein are shown. (B and C) Immunoblotting after heparin sepharose chromatography. The rGEP, deletion mutants, derivatives and granulin proteins were collected from media of the transfected COS-1. These media (P) were applied to heparin sepharose for incubation. The flow through (FT) was collected after 30min incubation. The sepharose was washed by Tris buffer for twice (W1 and W2). Elution buffer containing 0.125–2M NaCl was applied sequentially to the sepharose. The recombinant proteins were then detected by anti-His antibody in the immunoblotting. (D) Purified rGEP-3A and rGEP derivatives N492 and C101 were analyzed in SDS–PAGE. Protein was stained by Coomassie blue (CB) or detected by anti-His antibody in western blot (WB). (E) Polymerization of rGEP is independent of heparin and heparin-binding domain. Different amounts of heparin (0–100 µg/ml) were incubated with the recombinant protein at room temperature with or without DSS cross-linking. The polymerization status of rGEP (lanes A–F) and rGEP-3A (lanes G-L) was assessed by SDS–PAGE analysis and immunoblotting detected by anti-His antibody. (F) Indicated amounts of purified rGEP, rGEP-3A, N492 and C101 were incubated with detached Hep3B to compare their binding ability. The binding was then detected by FITC-anti-His antibody. In the case of HS depletion, cells were incubated with heparinase III in lyase buffer at 37°C for an hour before cell detachment. The histogram shows the fluorescent signal of the cells when incubate with 0.8 µg rGEP, N492 and C101. The shaded area represents isotypic control. The bar chart shows the geometric mean fluorescent intensity (MFI) of the cells when incubated with different amount of proteins.

Mentions: Previous study on heparin-binding domain (24) indicated its constitution of basic residues such as Lys, Arg and His. The C-terminal region of GEP is relatively rich of these basic residues and therefore four truncated mutants of GEP either lacking the C-terminus (N492) or covering different lengths of the C-terminus (C101, C77 and C51) were expressed from stable clones of transfected COS-1 cells (Figure 4A). Heparin sepharose chromatography showed that rGEP, C101, C77 and C51 could bind to the heparin and eluted with 0.5 M sodium chloride, whereas N492 did not bind to the heparin sepharose specifically (Figure 4B). This result showed a direct interaction between heparin and rGEP and implicated the heparin-binding domain of GEP located at the C-terminal 51 residues. From these 51 residues, two regions RRH(555-557) and RR(574-575) were suspected as the amino acids contributing to the binding. Therefore, three rGEP derivatives, rGEP-2A, rGEP-3A and rGEP-5A, were generated by alanine mutation. Our results showed that the alanine mutation of RRH(555-557) alone efficiently abolished the rGEP binding to heparin column, whereas the mutation of RR(574-575) showed no effect (Figure 4C). This indicated the RRH(555-557) is the determining domain in GEP for heparin binding. On the other hand, among the three tested granulin subunits only granulin E bind to the heparin column, implicating a different binding property of the full length GEP from most of the granulin subunits (Figure 4B).


Granulin-epithelin precursor interacts with heparan sulfate on liver cancer cells.

Yip CW, Cheung PF, Leung IC, Wong NC, Cheng CK, Fan ST, Cheung ST - Carcinogenesis (2014)

Mapping of heparin-binding domain in GEP and the contribution of the heparin-binding domain in HCC cell surface binding. (A) Schematic diagram of GEP protein, rGEP and derivatives and three granulin subunits. The seven and half granulins are shown as grey boxes in the GEP protein. Open arrows represent the N-glycosylation sites identified previously. The locations of rGEP, the deletion mutants (N492, C101, C77 and C51), the derivatives (rGEP-2A, rGEP-3A and rGEP-5A) and three granulin proteins corresponding to the GEP protein are shown. (B and C) Immunoblotting after heparin sepharose chromatography. The rGEP, deletion mutants, derivatives and granulin proteins were collected from media of the transfected COS-1. These media (P) were applied to heparin sepharose for incubation. The flow through (FT) was collected after 30min incubation. The sepharose was washed by Tris buffer for twice (W1 and W2). Elution buffer containing 0.125–2M NaCl was applied sequentially to the sepharose. The recombinant proteins were then detected by anti-His antibody in the immunoblotting. (D) Purified rGEP-3A and rGEP derivatives N492 and C101 were analyzed in SDS–PAGE. Protein was stained by Coomassie blue (CB) or detected by anti-His antibody in western blot (WB). (E) Polymerization of rGEP is independent of heparin and heparin-binding domain. Different amounts of heparin (0–100 µg/ml) were incubated with the recombinant protein at room temperature with or without DSS cross-linking. The polymerization status of rGEP (lanes A–F) and rGEP-3A (lanes G-L) was assessed by SDS–PAGE analysis and immunoblotting detected by anti-His antibody. (F) Indicated amounts of purified rGEP, rGEP-3A, N492 and C101 were incubated with detached Hep3B to compare their binding ability. The binding was then detected by FITC-anti-His antibody. In the case of HS depletion, cells were incubated with heparinase III in lyase buffer at 37°C for an hour before cell detachment. The histogram shows the fluorescent signal of the cells when incubate with 0.8 µg rGEP, N492 and C101. The shaded area represents isotypic control. The bar chart shows the geometric mean fluorescent intensity (MFI) of the cells when incubated with different amount of proteins.
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Related In: Results  -  Collection

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Figure 4: Mapping of heparin-binding domain in GEP and the contribution of the heparin-binding domain in HCC cell surface binding. (A) Schematic diagram of GEP protein, rGEP and derivatives and three granulin subunits. The seven and half granulins are shown as grey boxes in the GEP protein. Open arrows represent the N-glycosylation sites identified previously. The locations of rGEP, the deletion mutants (N492, C101, C77 and C51), the derivatives (rGEP-2A, rGEP-3A and rGEP-5A) and three granulin proteins corresponding to the GEP protein are shown. (B and C) Immunoblotting after heparin sepharose chromatography. The rGEP, deletion mutants, derivatives and granulin proteins were collected from media of the transfected COS-1. These media (P) were applied to heparin sepharose for incubation. The flow through (FT) was collected after 30min incubation. The sepharose was washed by Tris buffer for twice (W1 and W2). Elution buffer containing 0.125–2M NaCl was applied sequentially to the sepharose. The recombinant proteins were then detected by anti-His antibody in the immunoblotting. (D) Purified rGEP-3A and rGEP derivatives N492 and C101 were analyzed in SDS–PAGE. Protein was stained by Coomassie blue (CB) or detected by anti-His antibody in western blot (WB). (E) Polymerization of rGEP is independent of heparin and heparin-binding domain. Different amounts of heparin (0–100 µg/ml) were incubated with the recombinant protein at room temperature with or without DSS cross-linking. The polymerization status of rGEP (lanes A–F) and rGEP-3A (lanes G-L) was assessed by SDS–PAGE analysis and immunoblotting detected by anti-His antibody. (F) Indicated amounts of purified rGEP, rGEP-3A, N492 and C101 were incubated with detached Hep3B to compare their binding ability. The binding was then detected by FITC-anti-His antibody. In the case of HS depletion, cells were incubated with heparinase III in lyase buffer at 37°C for an hour before cell detachment. The histogram shows the fluorescent signal of the cells when incubate with 0.8 µg rGEP, N492 and C101. The shaded area represents isotypic control. The bar chart shows the geometric mean fluorescent intensity (MFI) of the cells when incubated with different amount of proteins.
Mentions: Previous study on heparin-binding domain (24) indicated its constitution of basic residues such as Lys, Arg and His. The C-terminal region of GEP is relatively rich of these basic residues and therefore four truncated mutants of GEP either lacking the C-terminus (N492) or covering different lengths of the C-terminus (C101, C77 and C51) were expressed from stable clones of transfected COS-1 cells (Figure 4A). Heparin sepharose chromatography showed that rGEP, C101, C77 and C51 could bind to the heparin and eluted with 0.5 M sodium chloride, whereas N492 did not bind to the heparin sepharose specifically (Figure 4B). This result showed a direct interaction between heparin and rGEP and implicated the heparin-binding domain of GEP located at the C-terminal 51 residues. From these 51 residues, two regions RRH(555-557) and RR(574-575) were suspected as the amino acids contributing to the binding. Therefore, three rGEP derivatives, rGEP-2A, rGEP-3A and rGEP-5A, were generated by alanine mutation. Our results showed that the alanine mutation of RRH(555-557) alone efficiently abolished the rGEP binding to heparin column, whereas the mutation of RR(574-575) showed no effect (Figure 4C). This indicated the RRH(555-557) is the determining domain in GEP for heparin binding. On the other hand, among the three tested granulin subunits only granulin E bind to the heparin column, implicating a different binding property of the full length GEP from most of the granulin subunits (Figure 4B).

Bottom Line: Suppression of the HS polymerase exostosin-1 reduced the rGEP binding and rGEP-mediated signaling transduction.Suppression of a specific HS proteoglycan, glypican-3, also showed a partial reduction of rGEP binding and an inhibition on rGEP-mediated activation of AKT.Furthermore, glypican-3 was shown to correlate with the expressions of GEP in clinical samples (Spearman's ρ = 0.363, P = 0.001).

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Centre for Cancer Research and.

Show MeSH
Related in: MedlinePlus