Granulin-epithelin precursor interacts with heparan sulfate on liver cancer cells.
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).
Affiliation: Department of Surgery, Centre for Cancer Research and.Show MeSH
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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).
Affiliation: Department of Surgery, Centre for Cancer Research and.