PDGF-D prodomains and variants and fusions thereof

Abstract

The present disclosure relates to a PDGF-D prodomain, or a mutant or fusion thereof. The invention further relates to a method or application of the PDGF-D prodomain or the mutant or the fusion body thereof in inhibiting PDGFR phosphorylation, inhibiting cell proliferation stimulated by PDGF or preventing and / or treating diseases related to PDGFR phosphorylation or cell proliferation stimulated by PDGF or the method or application of the PDGF-D prodomain or the mutant or the fusion body thereof.

Description

Technical Field

[0001] The present application relates to a PDGF-D prodomain or a mutant or fusion thereof. The present application also relates to a method or use of the PDGF-D prodomain or a mutant or fusion thereof for inhibiting PDGFR phosphorylation, inhibiting cell proliferation stimulated by PDGF, or preventing and / or treating diseases related thereto. Background Art

[0002] PDGF-D is a growth factor that regulates vascular development, wound healing, and organogenesis[1-3]. It is highly expressed in the heart, pancreas, and ovaries[2]. Dysfunction of PDGF-D has been associated with progressive kidney disease, cancer, and fibrosis[4-7]. Therefore, it is important to better understand the biological mechanisms of PDGF-D signaling and to develop therapeutic interventions to treat PDGF-D dysfunction.

[0003] PDGF-D belongs to the PDGF / VEGF family of growth factors [2,3]. These growth factors evolved together from a common ancestor of invertebrates and have diverged into two subfamilies (PDGF and VEGF, respectively) in vertebrates [1,8]. All family members share a structurally conserved cysteine ​​knot core that constitutes the mature active form of the growth factor [9]. In addition, they are all involved in the regulation of angiogenesis during development and vascular homeostasis in adults [1].

[0004] Among the PDGF subfamily, PDGF-A and PDGF-B are secreted in active form, diffusing freely in the extracellular space or binding to the extracellular matrix via retention of signal sequences at their C-termini [1]. In contrast, PDGF-C and PDGF-D are synthesized and secreted as potentially inactive complexes called procomplexes, in which the prodomain is covalently linked to the growth factor domain via a peptide bond (Figure S1). Therefore, both the PDGF-C procomplex and the PDGF-D procomplex require proteolytic activation by tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), respectively, to release their growth factor domains [2,3,10-12]. In addition, both procomplexes can also be cleaved and activated by proteases [13,14].

[0005] In addition to the hinge domain identified in the prodomains of PDGF-A and PDGF-B, the prodomains of PDGF-C and PDGF-D contain an additional CUB domain that is used to achieve latency of these two growth factors [3,12]. However, in addition to the known proteolytic activation mechanism, the following questions remain largely unknown: How does the CUB domain interact with the growth factors in the procomplexes of PDGF-C and PDGF-D? How do these interactions affect latency and activation of these two growth factors? Consistent with these mysteries, it has been reported that the PDGF-C and PDGF-D genes can be transcribed into different splice isoforms, with two PDGF-C isoforms and one PDGF-D isoform encoding only their prodomains [15,16]. The biological functions of these splice isoforms are unclear, as conflicting results have been reported [12,16].

[0006] To exert their biological functions, PDGF-D and its homologs are assembled into homo- or heterodimers and recognized by their cognate receptors, including PDGFR-β and PDGFR-α, two receptor tyrosine kinases [1]. Among PDGF growth factor dimers, both PDGF-D homodimers and PDGF-B homodimers are recognized by PDGFR-β and thus share redundant functions in vascular development [3, 17, 18].

[0007] In addition to PDGFR-β, non-classical receptors and co-receptors that recognize PDGF-D have also been identified [7, 19, 20]. NKp44 is a PDGF-D receptor expressed on the surface of natural killer (NK) cells, innate lymphoid cell-1, and innate lymphoid cell-3 [7]. Recognition of PDGF-D by NKp44 can stimulate the innate immunity of NK cells in the tumor microenvironment

[19] . On the other hand, neuropilin-1 is a newly identified co-receptor for PDGF-D

[20] . It also recognizes heparan sulfate and VEGF

[21] . The binding of PDGF-D to neuropilin-1 is involved in cellular interactions between endothelial cells and pericytes

[20] . SUMMARY OF THE INVENTION

[0009] The first aspect of the present application

[0010] In a first aspect, the present application provides a PDGF-D prodomain or a mutant or fusion thereof, wherein the PDGF-D prodomain has a protein sequence of RRDETIQVKGNGYVQSPRFPNSYPRNLLLTWRLHSQENTRIQLVFDNQFGLEEAENDICRYDFVEVEDISETSTIIRGRWCGHKEVPPRIKSRTNQIKITFKSDDYFVAKPGFKIYYSLLEDFQPAAASETNWESVTSSISGVSYNSPSVTDPTLIADALDKKIAEFDTVEDLLKYFNPESWQEDLENMYLDTPRY.

[0011] In another embodiment of the first aspect, the PDGF-D prodomain or variant thereof is in monomeric form or oligomeric form, preferably in oligomeric form.

[0012] In another embodiment of the first aspect, the mutant comprises 1, 2, 3, 4, 5 or 6 amino acid substitutions, deletions or insertions in the protein sequence of the PDGF-D prodomain. In another embodiment of the first aspect, the mutant comprises one or more of the mutations 161-DKK / AAA-163, E37R, E71R and R93E relative to the PDGF-D prodomain.

[0013] In another embodiment of the first aspect, the fusion comprises a multivalent protein in addition to the PDGF-D prodomain. In another embodiment of the first aspect, the multivalent protein is fused to the N-terminus of the PDGF-D prodomain. In another embodiment of the first aspect, the multivalent protein is a GCN4 protein.

[0014] The second aspect of the present application

[0015] In a second aspect, the present application provides a method or use of the PDGF-D prodomain or a mutant or fusion thereof as defined in the first aspect (including each embodiment of the first aspect) for inhibiting PDGFR phosphorylation.

[0016] In another embodiment of the second aspect, the use comprises the use of the PDGF-D prodomain or a mutant or fusion thereof as defined in the first aspect (including each embodiment of the first aspect) in the preparation of a kit or a drug for inhibiting PDGFR phosphorylation.

[0017] In another embodiment of the second aspect, the PDGFR is PDGFR-β. In another embodiment of the second aspect, the phosphorylation is mediated by PDGF-B or PDGF-D. In another embodiment of the second aspect, the PDGF-D is in an activated form.

[0018] The third aspect of the present application

[0019] In a third aspect, the present application provides a method or use of the PDGF-D prodomain or a mutant or fusion thereof as defined in the first aspect (including each embodiment of the first aspect) for inhibiting cell proliferation stimulated by PDGF.

[0020] In another embodiment of the third aspect, the use includes the use of the PDGF-D prodomain or its mutant or fusion as defined in the first aspect (including each embodiment of the first aspect) in the preparation of a kit or drug for inhibiting cell proliferation stimulated by PDGF.

[0021] In another embodiment of the third aspect, the cell proliferation is stimulated by PDGF-B or PDGF-D. In another embodiment of the third aspect, the PDGF-D is in an activated form.

[0022] In another embodiment of the third aspect, the cell is a cell expressing PDGFR. In another embodiment of the third aspect, the cell is a kidney cell (e.g., BHK-21) or a fibroblast (e.g., NIH 3T3). In another embodiment of the third aspect, the PDGFR is PDGFR-β.

[0023] In another embodiment of the third aspect, when the PDGFR is PDGFR-β, the cell proliferation is stimulated by PDGF-B and PDGF-D, and when the PDGFR is not PDGFR-β, the cell proliferation is stimulated by PDGF-D.

[0024] The fourth aspect of the present application

[0025] In a fourth aspect, the present application provides a method or use of the PDGF-D prodomain or its mutant or fusion as defined in the first aspect (including each embodiment of the first aspect) for preventing and / or treating diseases associated with PDGFR phosphorylation or cell proliferation stimulated by PDGF.

[0026] In another embodiment of the fourth aspect, the use includes the use of the PDGF-D prodomain or its mutant or fusion as defined in the first aspect (including each embodiment of the first aspect) in the preparation of a kit or medicament for preventing and / or treating diseases associated with PDGFR phosphorylation or cell proliferation stimulated by PDGF.

[0027] In another embodiment of the fourth aspect, the PDGFR phosphorylation is as defined in the second aspect (including various embodiments of the second aspect). In another embodiment of the fourth aspect, the cell proliferation is as defined in the third aspect (including various embodiments of the third aspect).

[0028] In another embodiment of the fourth aspect, the disease associated with PDGFR phosphorylation or cell proliferation stimulated by PDGF includes atherosclerosis, fibrosis, and tumors (eg, malignant tumors).

[0029] Without departing from the subject matter or scope of the present application, the present application may be specifically implemented in any other form. The present application encompasses any and all combinations of the above aspects and embodiments. It should be understood that any embodiment may be combined with any other one or more embodiments to describe another embodiment. It should also be understood that a single element from any embodiment may be combined with any and all other elements from any other one or more embodiments to describe another embodiment.

[0030] The fifth aspect of the present application

[0031] In a fifth aspect, the present application provides a mutant PDGF-D, wherein one or more mutations are present in the CUB domain of WT PDGF-D, the uPA cleavage site of WT PDGF-D, or both. In a fifth aspect, the present application also provides a method or use of the mutant PDGF-D for stimulating cell proliferation.

[0032] In another embodiment of the fifth aspect, the one or more mutations are selected from the group consisting of E87R, E121R and R134E mutations on the CUB domain of WT PDGF-D. In another embodiment of the fifth aspect, the one or more mutations are mutations in which the fragment "RGRS" in WT PDGF-D is replaced by the fragment "GAGA". In another embodiment of the fifth aspect, the cell proliferation is intended for cell culture. In another embodiment of the fifth aspect, the cell is a NK cell.

[0033] In another embodiment of the fifth aspect, the use includes use of the mutant PDGF-D in the preparation of a kit or a medicament for stimulating cell proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 .PDGF-D prodomain inhibits PDGFR-β phosphorylation stimulated by PDGF-B.

[0035] A) The main structure of the PDGF-D prodomain (aa.51-246), hinge (aa.197-246) and PDGF-D DKK / AAA Schematic representation of the prodomain (aa.51-246,211-DKK / AAA-213). An MBP tag was introduced at the N-terminus of these proteins, followed by a His×6 tag, a Flag tag, and a 3C cleavage site.

[0036] B) Purification of the PDGF-D prodomain with MBP as a tag. The affinity-purified MBP-prodomain is loaded onto a Hitrap Q column balanced with buffer A (20mM Tris-HCl, pH 8.0). The bound protein is graded with a linear gradient of NaCl from 0M to 1M concentration. The two fractions containing the MBP-prodomain are collected separately and then purified with a Superdex G200 column balanced with 20mMTris-HCl (pH 8.0) and 150mM NaCl. Non-reducing SDS-PAGE electrophoresis and Coomassie brilliant blue staining are performed on the graded samples from the gel filtration column.

[0037] C) Purification of the hinge domain of PDGF-D tagged with MBP. The affinity-purified MBP-hinge was purified using a Hitrap Q column and then a Superdex G200 column. Gel filtration chromatograms and Coomassie blue staining of fractionated samples are shown.

[0038] D) Purified PDGF-D prodomain inhibits PDGFR-β phosphorylation stimulated by PDGF-B and PDGF-D. BHK-21 / PDGFR-β cells were starved in DMEM medium for 20 hours and then treated with 4 nM purified PDGF-B or PDGF-D-stimulated prodomains. RGRS / GAGA Conditioned medium of transiently transfected BHK-21 cells was stimulated for 15 minutes. While stimulating, 64 nM purified PDGF-DMBP-prodomain oligomers or monomers were added to inhibit stimulated PDGFR-β phosphorylation. After treatment, cells were collected, lysed and analyzed by Western blotting. The expression and phosphorylation of PDGFR-β were detected with anti-protein C, pY740 and 4G10 antibodies, respectively.

[0039] E) Comparison of the inhibitory activity of oligomeric and monomeric PDGF-D MBP-prodomains on PDGFR-β phosphorylation stimulated by PDGF-B. BHK-21 / PDGFR-β cells were starved for 20 hours in DMEM medium and then stimulated for 15 minutes with 4 nM purified PDGF-B in the presence and absence of 64 nM purified MBP-prodomain oligomers or monomers. Expression and phosphorylation of PDGFR-β were detected by Western blotting using anti-protein C and 4G10 antibodies, respectively.

[0040] F) PDGF-DMBP-prodomain inhibits the PDGFR-β phosphorylation stimulated by PDGF-B in a dose-dependent manner.BHK-21 / PDGFR-β cells were starved in DMEM culture medium for 20 hours, then stimulated with 4nM purified PDGF-B for 15 minutes.Meanwhile, PDGF-DMBP-prodomain oligomers were diluted with 2 times of serial dilutions and added at specified concentrations to inhibit PDGFR-β phosphorylation.The relative phosphorylation level of PDGFR-β was quantitatively determined by Western blot results and as shown in the figure.

[0041] G and H) In the MBP pull-down analysis, the PDGF-D prodomain is directly combined with PDGF-D, but not with PDGFR-β. The PDGF-D prodomain or control protein with MBP as a label is incubated separately with the lysate (left) of BHK-21 / PDGFR-β cells or the conditioned medium (right) of BHK-21 cells with full-length PDGF-D from transient transfection. Both PDGFR-β and full-length PDGF-D are labeled with protein C tags. The mixture is then incubated with MBP resin, and then washed repeatedly to remove unbound protein. Anti-protein C and anti-MBP antibodies are then used to analyze the protein input and binding by Western blotting.

[0042] I) In a dose-dependent experiment, PDGF-D MBP hinge barely inhibited PDGFR-β phosphorylation stimulated by PDGF-B. The experiment was performed as in panel G.

[0043] J) Purified PDGF-D DKK / AAA MBP-prodomain also inhibited PDGFR-β phosphorylation stimulated by PDGF-B and PDGF-D. The experiment was performed as in panel D.

[0044] K) Mutations in the binding loop of the PDGF-D CUB domain have an effect on the expression of WT PDGF-D or PDGF-D RGRS / GAGAThe biosynthesis of the mutants was barely affected. Equal amounts of plasmids encoding WT PDGF-D or the indicated mutants were transiently transfected into BHK-21 cells individually. The PDGF-D protein expressed in the conditioned medium was detected by Western blotting using anti-protein C antibody.

[0045] L) Mutations in the predicted growth factor binding loop of the CUB domain enhance PDGF-D RGRS / GAGA Activity in stimulating PDGFR-β phosphorylation, while the same mutation has a minor effect on the activity of WT PDGF-D. BHK-21 / PDGF-β cells were stimulated with conditioned medium from cells transfected with WT and mutated PDGF-D. Expression and phosphorylation of PDGFR-β were detected by Western blotting using anti-protein C, pY740 and 4G10 antibodies, respectively.

[0046] Figure 2 .PDGF-D MBP-prodomain DKK / AAA of purification.

[0047] The affinity purified MBP-prodomain DKK / AAA The protein was loaded onto a Hitrap Q column equilibrated with buffer A (20 mM Tris-HCl, pH 8.0). The bound protein was fractionated using a linear gradient of NaCl from 0 M to 1 M. DKK / AAA The two fractions were collected separately and then purified using a Superdex G200 column equilibrated with 20 mM Tris-HCl (pH 8.0), 150 mM NaCl. The fractionated samples from the gel filtration column were subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining.

[0048] Figure 3 .PDGF-D prodomain fused to GCN4 inhibits PDGFR-β phosphorylation mediated by PDGF-B.

[0049] A) Schematic representation of the main structure of the GCN4-PDGF-D prodomain chimera. By adding GCN4 at the N-terminus of the PDGF-D MBP-prodomain, the prodomain will be linked to the multivalent protein via the tetramer of GCN4.

[0050] B) Purification of PDGF-D MBP-GCN4-prodomain chimera. Affinity-purified MBP-GCN4-prodomain was purified using an anion exchange column and Superose 6 gel filtration column in sequence. Gel filtration chromatograms of fractionated samples and non-reducing SDS-PAG results are shown.

[0051] D) Purified MBP-GCN4-prodomain inhibits PDGFR-β phosphorylation stimulated by PDGF-B in a dose-dependent manner. BHK-21 / PDGFR-β cells were starved in DMEM medium for 20 hours and then stimulated with 4nM purified PDGF-B for 15 minutes. At the same time, MBP-GCN4-prodomain was diluted in 2-fold serial dilutions and added at specified concentrations to inhibit PDGFR-β phosphorylation. The expression and phosphorylation of PDGFR-β were detected with protein C and 4G10 antibodies, respectively.

[0052] E) The relative phosphorylation level of PDGFR-β was quantified from the Western blot results in the figure.

[0053] Figure 4 .The PDGF-D prodomain differentially inhibits proliferation of NIH 3T3 cells and BHK-21 / PDGFR-β cells stimulated by PDGF-B and PDGF-D.

[0054] A and B) NIH 3T3 (A) and BHK-21 / PDGFR-β (B) cells were cultured at 3×10 4 The cells were inoculated into 96-well plates at a density of 10 cells / well. The inoculated cells were starved in DMEM for 20 hours. Then, 2.5 nM purified PDGF-B or PDGF-D or PDGF-D RGRS / GAGA Conditioned medium of transfected BHK-21 cells stimulates cells. While stimulating, oligomer or monomer PDGF-D prodomain processing cells were used at a concentration of 64nM. Cell density was determined using the cell proliferation assay described in the method. Bar graphs represent mean ± SD of three biological replicates. Statistical significance was determined as described in the method.

[0055] C and D) PDGF-D DKK / AAA Inhibitory activity of the prodomain on proliferation of NIH 3T3 (C) or BHK-21 / PDGFR-β (D) cells stimulated by PDGF-B and PDGF-D. The experiment was performed as in panel A. Example

[0056] Material

[0057] Recombinant human PDGF-B was purchased from Arco (China, catalog number DDB-H4112). Plasmids encoding phosphotyrosine antibodies 4G10 and Flag antibodies were constructed according to the literature. These antibodies were affinity purified from transformed BL21 (DE3) cells and transfected HEK293 cells, respectively. PDGFR-βpY740 phosphotyrosine antibody was purchased from Abmart (China, catalog number T55673). Protein C antibody was from Genscript Biotech. (China, catalog number A01774). Goat anti-rabbit and goat anti-mouse antibodies were from Proteintech (China, catalog number SA00001-2 / SW067A00160). DMEM was from Biological Industries (catalog number zc013-06-1005-57-1ACS). DMSO and fetal bovine serum (FBS) were from Sangon Biotech (China, catalog number E600001). Puromycin and MTT (3-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide) were from Solarbio (China, catalog number A8020). PVDF membrane was purchased from Thermo Fisher (catalog number 88518).

[0058] General Procedures

[0059] Plasmid construction, protein expression and purification

[0060] The full-length PDGF-D with a protein C tag at the C-terminus was cloned into the pTT5 vector. A variety of PDGF-D mutants were generated from the recombinant plasmid using site-directed mutagenesis mediated by DpnI (New England Biolabs, catalog number R0176S). All constructs used in this study were confirmed by DNA sequencing.

[0061] The PDGF-D prodomain (aa 51-246), hinge (aa 197-246), PDGF-D DKK / AAA The genes of the prodomain and GCN4 prodomain were individually subcloned into the pET vector. In this vector, the sequence encoding the MBP tag followed by the His×6 tag, the Flag tag and the 3C protease cleavage site was inserted at the 5' end of the subcloned gene. These plasmids were transformed into Rosetta gami2 E. coli cells to express the recombinant protein.

[0062] E. coli cells were cultured in LB medium to an OD of 600The cells were then induced with 100 μM IPTG at 20-30°C for 12 hours to express the recombinant protein. The cultured cells were harvested by centrifugation, lysed by French Press in a buffer containing 20 mM Tris-HCl (pH 8.0), 5 mM imidazole (pH 8.0), 300 mM NaCl, and clarified by centrifugation. The clarified lysate was loaded onto 3 mL Ni 2+ -NTA column, the column was then washed with 20 column volumes (CV) of a washing buffer containing 20 mM Tris-HCl (pH 8.0), 40 mM imidazole (pH 8.0), 300 mM NaCl, and eluted with 20 mM Tris-HCl (pH 8.0), 300 mM imidazole (pH 8.0), 300 mM NaCl. The yield and purity of the purified protein were analyzed by SDS-PAGE electrophoresis and UV absorbance.

[0063] After affinity purification, the recombinant protein was further purified by anion exchange chromatography (Hitrap Q column) and gel filtration chromatography. In anion exchange chromatography, the column was balanced with buffer A (20 mM Tris-HCl, pH 8.0), and the bound protein was fractionated with a linear gradient of NaCl from 0 M to 1 M concentration. In gel filtration chromatography, Superdex G200 or Superose 6 columns were used and balanced with a buffer containing 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl.

[0064] The proteins involved in this application include PDGF-D, PDGF-D RGRS / GAGA , MBP-prodomain, MBP-prodomain DKK / AAA , MBP-hinge and MBP-GCN4-prodomain, having the sequences shown below:

[0065] PDGF-D:

[0066] MHRLIFVYTLICANFCSCRDTSATPQSASIKALRNANLRRDESNHLTDLYRRDETIQVKGNGYVQSPRFPNSYPRNLLLTWRLHSQENTRIQLVFDNQFGLEEAENDICRYDFVEVEDISETSTIIRGRWCGHKEVPPRIKSRTNQIKITFKSDDYFVAKPGFKIYYSLLEDFQPAAASETNWESVTSSISGVSYNSPSVTDPTLIADALDKKIAEFDTVEDLLKYFNPESWQEDLENMYLDTPRYRGRSYHDRKSKVDLDRLNDDAKRYSCTPRNYSVNIREELKLANVVFFPRCLLVQRCGGNCGCGTVNWRSCTCNSGKTVKKYHEVLQFEPGHIKRRGRAKTMALVDIQLDHHERCDCICSSRPPR

[0067] PDGF-D RGRS / GAGA :

[0068] MHRLIFVYTLICANFCSCRDTSATPQSASIKALRNANLRRDESNHLTDLYRRDETIQVKGNGYVQSPRFPNSYPRNLLLTWRLHSQENTRIQLVFDNQFGLEEAENDICRYDFVEVEDISETSTIIRGRWCGHKEVPPRIKSRTNQIKITFKSDDYFVAKPGFKIYYSLLEDFQPAAASETNWESVTSSISGVSYNSPSVTDPTLIADALDKKIAEFDTVEDLLKYFNPESWQEDLENMYLDTPRYGAGAYHDRKSKVDLDRLNDDAKRYSCTPRNYSVNIREELKLANVVFFPRCLLVQRCGGNCGCGTVNWRSCTCNSGKTVKKYHEVLQFEPGHIKRRGRAKTMALVDIQLDHHERCDCICSSRPPR

[0069] MBP - pre - domain:

[0070] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSHMHHHHHHDYKDHDGDYKDHDIDYKDDDDKLEVLFQGPGSRRDETIQVKGNGYVQSPRFPNSYPRNLLLTWRLHSQENTRIQLVFDNQFGLEEAENDICRYDFVEVEDISETSTIIRGRWCGHKEVPPRIKSRTNQIKITFKSDDYFVAKPGFKIYYSLLEDFQPAAASETNWESVTSSISGVSYNSPSVTDPTLIADALDKKIAEFDTVEDLLKYFNPESWQEDLENMYLDTPRY

[0071] MBP - pre - domain DKK / AAA :

[0072] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSHMHHHHHHDYKDHDGDYKDHDIDYKDDDDKLEVLFQGPGSRRDETIQVKGNGYVQSPRFPNSYPRNLLLTWRLHSQENTRIQLVFDNQFGLEEAENDICRYDFVEVEDISETSTIIRGRWCGHKEVPPRIKSRTNQIKITFKSDDYFVAKPGFKIYYSLLEDFQPAAASETNWESVTSSISGVSYNSPSVTDPTLIADALAAAIAEFDTVEDLLKYFNPESWQEDLENMYLDTPRY

[0073] MBP - Hinge:

[0074] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSHMHHHHHHDYKDHDGDYKDHDIDYKDDDDKLEVLFQGPGSSPSVTDPTLIADALDKKIAEFDTVEDLLKYFNPESWQEDLENMYLDTPRY

[0075] MBP-GCN4 pro-domain:

[0076] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWP LIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAA TMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSHMHHHHHHDYKDHDGDYKDHDIDYKDDDDKLEVLFQGPSGRMKQLEDKVEELLSKNYHLENEVARLKKLCGERGRRDETIQVKGNGYVQSPRFPNSYPRNLLLTWRLHSQ ENTRIQLVFDNQFGLEEAENDICRYDFVEVEDISETSTIIRGRWCGHKEVPPRIKSRTNQIKITFKSDDYFVAKPGFKIYYSLLEDFQPAAASETNWESVTSSISGVSYNSPSVTDPTLIADALDKKIAEFDTVEDLLKYFNPESWQEDLENMYLDTPRY

[0077] Phosphorylation assay

[0078] BHK-21 / PDGFR-β cells stably transfected with PDGFR-β were established as described and maintained in DMEM supplemented with 10% FBS and 10 μg / mL puromycin

[33] . Before the experiment, these cells were cultured at 6 × 10 5 Cells were seeded at a density of 10 cells / well in 12-well plates and starved in DMEM at 37°C in 5% CO2 for 24 hours. The indicated concentrations of PDGF-D prodomain, hinge, PDGF-D DKK / AAA Cells were treated with the prodomain for 15 min. At the same time, cells were treated with or without 4 nM PDGF-B or PDGF-D from cells transiently transfected with PDGF-D or PDGF-D RGRS / GAGAThe cells were then lysed with RIPA (0.1% SDS, 150 mM NaCl, 1% Triton X-100, 10 mM EDTA, 50 mM Tris-HCl, pH 7.5, 2 mM PMSF, 1 mM Na3VO4); and the lysates were subjected to SDS-PAGE electrophoresis. The expression and phosphorylation of PDGFR-β were detected by Western blotting using protein C antibody, pY740 phosphorylated PDGFR-β antibody, and 4G10 antibody, respectively.

[0079] MBP pull-down assay

[0080] Recombinant MBP-prodomain or MBP-control protein was expressed in Rosetta gami2 E. coli cells. PDGFR-β or PDGF-D with protein C tag was expressed in BHK-21 cells as described. 650 μg of MBP-prodomain or MBP-control protein was incubated overnight at 4°C in a binding buffer containing 150 mM NaCl, 1% Triton X-100, 10 mM EDTA, 50 mM Tris-HCl, pH 7.5, 2 mM PMSF and 1 mM Na3VO4. The mixture was then incubated for 4 hours with MBP beads (Smart Lifesciences, SA026GC01). After sufficient washing, the bound proteins were analyzed by SDS-PAGE and Western blotting using anti-protein C antibody and anti-MBP antibody. To analyze the binding of the prodomain to PDGF-D growth factor, 25 ml of conditioned medium from BHK-21 cells transiently transfected with full-length PDGF-D was incubated with 650 μg of MBP-prodomain or MBP-control protein and the binding assay was performed similarly to PDGFR-β.

[0081] Cell proliferation assay

[0082] NIH 3T3 cells or BHK-21 / PDGFR-β cells were cultured at 3×10 4 The cells were seeded at a density of 10 cells / well in a 96-well plate. The seeded cells were starved in DMEM for 20 hours and then treated with or without 2.5 nM concentration of purified PDGF-B or cells transiently transfected with PDGF-D or PDGF-D. RGRS / GAGAThe cells were treated with conditioned medium of BHK-21 cells. At the same time, these cells were treated with or without 64nM concentration of purified PDGF-D prodomain oligomers or monomers. The treated cells were cultured for another 48 hours at 37°C. Then 100 μL of MTT at a concentration of 0.5 mg / mL was added to each well of the plate. The plate was incubated at RT for 4 hours, and then 100 μL of DMSO was added to each well to dissolve the formazan. The plate was shaken at RT for 10 minutes. The cell density in each well was determined by measuring the optical density at 570 nm using an EnSpireMultilabel reader.

[0083] Example 1. The PDGF-D prodomain and its mutants are used to inhibit PDGFR-β phosphorylation mediated by PDGF-B and PDGF-D.

[0084] We expressed and purified the wild-type (WT) PDGF-D prodomain (aa 51-246), hinge (aa 197-246), and prodomain mutants (PDGF-D DKK / AAA )( Figure 1 A), and subsequently tested their inhibitory activity on PDGFR-β phosphorylation stimulated by PDGF-B or PDGF-D.

[0085] Use Ni 2+ The MBP-tagged WT PDGF-D prodomain (hereinafter referred to as MBP-prodomain) was affinity purified by -NTA column and subjected to reducing and non-reducing SDS-PAGE electrophoresis. As shown by the non-reducing SDS-PAGE results, the MBP-prodomain was detected as a 68.5 kDa protein and did not form a disulfide-linked dimer ( Figure 1 B). The affinity purified MBP-prodomain was further purified by anion exchange chromatography (Hitrap Q) ( Figure 1 B). Two different fractions containing the MBP-prodomain eluted from the column at conductivities of 27 mS and 34 mS, respectively. These fractions were analyzed separately by gel filtration chromatography using a Superdex G200 column. Figure 1 As shown in Figure B, the first fraction eluted from the Hitrap Q column contains a large amount of monomeric MBP-prodomain, while the second fraction from the Q column has more oligomeric MBP-prodomain ( Figure 1 B) When we purified PDGF-D tagged with MBP DKK / AAA Similar results were obtained when the prodomain mutants were used ( Figure 2 ). In addition, following the same protocol, we purified the hinge domain of PDGF-D tagged with MBP. It was monodisperse in gel filtration chromatography with an apparent molecular weight of 51.4 kDa ( Figure 1 C).

[0086] We compared the inhibitory activities of these purified proteins. MBP-prodomain inhibited PDGFR-β autophosphorylation stimulated by PDGF-B as well as PDGF-D in a dose-dependent manner. In particular, oligomeric MBP-prodomain was more effective than monomeric MBP-prodomain in inhibiting PDGFR-β phosphorylation ( Figure 1 D, 1E). As the concentration of oligomeric MBP-prodomain increased, the phosphorylation level of PDGFR-β stimulated by 64 nM PDGF-B gradually decreased to 60% of that in samples not treated with the prodomain ( Figure 1 F, 1G).

[0087] To understand the potential mechanism of the inhibitory activity of the PDGF-D prodomain, we performed pull-down assays to investigate whether the prodomain could directly bind to the receptor PDGFR-β or the growth factor PDGF-D. Purified MBP-prodomain or MBP-control protein was incubated separately with lysates of BHK-21 / PDGF-β cells or conditioned medium from BHK-21 cells transiently transfected with PDGF-D. After incubation with MBP resin and extensive washing, the bound proteins were eluted and detected by Western blotting. Figure 1 As shown in H, the MBP-prodomain specifically pulls down only PDGF-D but not PDGFR-β. Combined with the inhibitory activity of the prodomain, our results suggest that the prodomain binds back to PDGF-D, thereby preventing the growth factor from stimulating PDGFR-β phosphorylation.

[0088] We then investigated how the hinge and CUB domains of PDGF-D affect the inhibitory activity of the prodomain. Figure 1 As shown in Figure 1, the hinge itself has very low inhibitory activity on PDGFR-β phosphorylation stimulated by PDGF-B. Inhibition was detected only at a hinge concentration of 1 μM. On the other hand, introduction of the mutation DKK / AAA at the hinge of the prodomain also impaired the inhibition of PDGFR-β phosphorylation stimulated by PDGF-B and PDGF-D by the MBP-prodomain ( Figure 1 J) Together, these data suggest that the hinge and CUB domains act together to synergistically inhibit PDGFR-β recognition and stimulation by growth factors.

[0089] We mutated three conserved residues, one from each of the three loops on the CUB domain that are postulated to be involved in binding to the PDGF-D growth factor, to analyze their effects on the biosynthesis and activity of the growth factor. All of these mutations had no effect on the biosynthesis and activity of wild-type PDGF-D or uncleavable latent PDGF-D.RGRS / GAGA The biosynthesis of the mutants was almost unaffected ( Figure 1 K). However, E87R, E121R, and R134E mutations in the CUB domain enhanced the expression of PDGF-D RGRS / GAGA activity in stimulating PDGFR-β phosphorylation, whereas the same mutations introduced in WT PDGF-D had only a minor effect on stimulating PDGFR-β phosphorylation ( Figure 1 L). This result suggests that interfering with the interaction between the CUB domain and the growth factor domain can partially attenuate the inhibitory effect of the prodomain.

[0090] Example 2. PDGF-D prodomain fused to GCN4 is used to inhibit PDGFR-β phosphorylation mediated by PDGF-B.

[0091] Since the oligomeric PDGF-D prodomain is more potent than the monomeric prodomain in inhibiting growth factor activity, we tested whether fusing the prodomain to a multivalent protein could enhance the inhibitory activity of the prodomain.

[0092] We fused the GCN4 protein to the N-terminus of the prodomain ( Figure 3 A), followed by purification of the chimera using affinity chromatography and anion exchange chromatography. We then analyzed the samples by gel filtration chromatography using a Superose 6 column ( Figure 3 BE). Figure 3 As shown in BE, the PDGF-D prodomain fused to GCN4 eluted from the column as a single but broad peak, indicating that these samples were conformationally heterogeneous.

[0093] In a PDGFR-β phosphorylation assay, we found that GCN4-prodomain fused at the N-terminus inhibited PDGFR-β phosphorylation stimulated by PDGF-B in a dose-dependent manner ( Figure 3 D, 3E, S2). However, the potency of the chimera was lower than that of the oligomeric MBP-prodomain. At a chimera concentration of 64 nM, the phosphorylation level of PDGFR-β was only reduced by 16%, while at the same concentration, the oligomeric MBP-prodomain reduced the phosphorylation of PDGFR-β by 40% ( Figure 1 G, 3E).

[0094] Example 3. Use of PDGF-D prodomain and its mutants to inhibit proliferation of NIH 3T3 and BHK-21 / PDGFR-β cells stimulated by PDGF-B and PDGF-D

[0095] To further explore the function of the PDGF-D prodomain, we examined its inhibitory activity on fibroblast proliferation. To our surprise, we found that the PDGF-D prodomain exhibited different inhibitory effects on cell proliferation mediated by PDGF-B and PDGF-D.

[0096] like Figure 4 As shown in A, both purified PDGF-B and conditioned medium from cells transfected with PDGF-D stimulated the proliferation of NIH 3T3 cells. In contrast, conditioned medium from cells transfected with uPA cleavage site mutant PDGF-D stimulated the proliferation of NIH 3T3 cells. RGRS / GAGA Conditioned medium from transfected cells failed to stimulate proliferation of NIH 3T3 cells. Addition of PDGF-D MBP-prodomain, either monomeric or oligomeric, inhibited the proliferation of untreated, PDGF-D-treated or PDGF-D-treated cells. RGRS / GAGA This suggests that the PDGF-D prodomain can inhibit endogenous as well as autocrine PDGF-D signaling.

[0097] However, PDGF-D MBP-prodomain failed to inhibit the proliferation of NIH 3T3 cells treated with PDGF-B ( Figure 4 A). To investigate whether this difference is related to PDGFR-β signaling, we stably transfected PDGFR-β into BHK-21 cells, which have low endogenous PDGFR-β expression. In BHK-21 cells transfected with PDGFR-β, PDGF-D MBP-prodomain inhibited cell proliferation mediated by PDGF-B ( Figure 4 B).

[0098] As expected, the DKK at the hinge of PDGF-D is a cleavage site for the protease PLpro (a papain-like protease)

[28] . Therefore, we investigated whether sequential cleavage of the prodomain is essential for its inhibitory function and whether mutation of this cleavage site could restore its inhibitory activity. Figure 4 C, PDGF-D DKK / AAA The prodomain still failed to inhibit the proliferation of NIH 3T3 cells stimulated with PDGF-B, but it inhibited the proliferation of NIH 3T3 cells treated with PDGF-D. DAK / AAA The prodomain inhibited the proliferation of BHK-21 / PDGFR-β cells stimulated with PDGF-B and PDGF-D ( Figure 4 D).

[0099] These results suggest that the additional factors expressed by NIH 3T3 cells are attributed to the desensitization of the PDGF-D prodomain to PDGF-B stimulation.

[0100] In summary, PDGD-D plays an important role in vascular development, cancer, and innate immunity

[29] . In this study, we investigated the inhibitory effect of PDGF-D MBP-prodomain on PDGF-B and PDGF-D signaling.

[0101] Based on alphafold2 predictions and known biology [1-3,22], we expected that the CUB and hinge domains of PDGF-D bind on opposite sides of the growth factor, thereby synergistically preventing growth factor activation. We provided several PDGF-D MBP-prodomain mutants and chimeras and compared their inhibitory activity against PDGFR-β phosphorylation stimulated by PDGF-B or PDGF-D. Our biochemical data indicate that the prodomain can bind back to the growth factor, thereby preventing stimulation of PDGFR-β. In addition, we found that three residues on three different loops of the CUB domain are involved in inhibitory interactions with growth factors. The PDGF-D growth factor is recognized by multiple receptors, including PDGFR-β, NKp44, and the co-receptor neuropilin-1 [7,19,20].

[0102] In our study, we found that purified PDGF-D MBP-prodomain formed oligomers as well as monomers. The oligomeric MBP-prodomain was more effective than the monomer in inhibiting PDGFR-β phosphorylation and fibroblast proliferation.

[0103] Both PDGF-B and PDGF-D are cognate ligands of PDGFR-β and share redundant functions in certain biological and pathological contexts

[32] . In our study, we found that the PDGF-D MBP-prodomain cross-inhibited PDGF-B and PDGF-D signaling in PDGFR-β phosphorylation and BHK-21 cell proliferation. However, when we analyzed the inhibitory effect of the PDGF-D MBP-prodomain on NIH 3T3 cell proliferation, different inhibitory activities against PDGF-B and PDGF-D signaling were observed. The PDGF-DMBP-prodomain failed to inhibit the proliferation of NIH 3T3 cells stimulated with PDGF-B, but was able to inhibit the proliferation of NIH 3T3 cells stimulated with PDGF-D.

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Claims

1. A PDGF-D prodomain or a mutant or fusion thereof, wherein the PDGF-D prodomain has a protein sequence of RRDETIQVKGNGYVQSPRFPNSYPRNLLLTWRLHSQENTRIQLVFDNQFGLEEAENDICRYDFVEVEDISETSTIIRGRWCGHKEVPPRIKSRTNQIKITFKSDDYFVAKPGFKIYYSLLEDFQPAAASETNWESVTSSISGVSYNSPSVTDPTLIADALDKKIAEFDTVEDLLKYFNPESWQEDLENMYLDTPRY.

2. A method or use of the PDGF-D prodomain or a mutant or fusion thereof according to claim 1 for inhibiting PDGFR phosphorylation.

3. A method or use of the PDGF-D prodomain or a mutant or fusion thereof according to claim 1 for inhibiting cell proliferation stimulated by PDGF.

4. A method or use of the PDGF-D prodomain or a mutant or fusion thereof according to claim 1 for preventing and / or treating diseases associated with PDGFR phosphorylation or cell proliferation stimulated by PDGF.

5. A mutant PDGF-D, wherein one or more mutations are present in the CUB domain of WT PDGF-D, the uPA cleavage site of WT PDGF-D, or both.

6. A method or use of mutated PDGF-D for stimulating cell proliferation.

Images