Background The present study aimed to investigate the detailed mode and specific sites for their binding as well as the functional relevance of this binding in the phenotypic proliferation of vascular smooth muscle ce...Background The present study aimed to investigate the detailed mode and specific sites for their binding as well as the functional relevance of this binding in the phenotypic proliferation of vascular smooth muscle cells(SMCs). Methods CREG knocked-down SMCs were employed to evaluate the biological activity of wtCREG and mCREG.Expressions of SMC differentiation markers SM myosin heavy chain(SM-MHC),SM-actin,heavy caldesmon and myocardin were determined by Western blotting using specific antibodies. Cellular growth of SMCs was assessed by bromide dewuridine (BrdU) incorporation and cell cycle analysis on fluorescence-activated cell sorting(FACS).A solid-phase binding assay was used to study the binding of CREG to extracellular domains of M6P/IGF2R.The cellular co-localization of the two recombinant CREGs with M6P/IGF2R was detected on SMC surface by immunoprecipitation and immunofluorescence analysis.Results The molecular weight of wtCREG was around 30 kD while that of the mCREG was～25 kD.Treatment of wtCREG with PNGase F reduced its molecular weight from～30 kD to～25 kD,whereas PNGase F treatment had no effect on the molecular weight of mCREG.Both wtCREG and mCREG proteins enhanced SMC differentiation,inhibited BrdU incorporation,and arrested cell cycle progression when added to the culture medium.In CREG knocked-down SMCs,the amount of CREG detected by immunoblotting in M6P/IGF2R immunoprecipitates was significantly reduced when compared to normal cells.Both recombinant CREGs co-immunoprecipitated with M6P/IGF2R, although slightly reduced amount of the mutant CREG was detected in M6P/IGF2R immunoprecipitates.Immunostaining revealed that His-tagged CREGs co-localized with IGF2R on the cell surface in a glycosylation-independent manner.In vitro binding assay showed that CREGs bound to M6P/ IGF2R extracellular domains 7-10 and 11-13 in a glycosylation -dependent and -independent manner,respectively.Further blocking experiments using soluble M6P/IGF2R fragments and M6P/IGF2R neutralizing antibody indicated that the biological activities of recombinant CREGs in SMC growth and the up-regulation of SMC differentiation markers were all abolished by treatment with the M6P/IGF2R neutralizing antibody. However,although the growth inhibitory effect of wtCREG was nearly abolished by D7-10 or D11-13,the effect of mCREG was only reversed by Dll-13,indicating that the binding to domains 11-13 is required for CREG to modulate the proliferation of SMCs.Conclusions These data suggest that solubleCREG proteins can exert their biological function via binding to the extracellular domains 7-10 and 11-13 of cell surface M6P/IGF2R in both a glycosylation-dependent and -independent manner.展开更多
HER-2/neu oncogene is over-expressed and amplified in patients associated with metastatic breast cancer. An increased level (>15 ng/mL) in the shed extracellular domain (sECD-HER 2/neu) is indicative of the potenti...HER-2/neu oncogene is over-expressed and amplified in patients associated with metastatic breast cancer. An increased level (>15 ng/mL) in the shed extracellular domain (sECD-HER 2/neu) is indicative of the potential presence and as-sociated progression of this disease. A fluo-rescent ELISA incorporating the newly devel-oped ALYGNSA antibody-orientation system revealed a 10-fold increase in sensitivity (≤0.63 ng/mL) of sECD-HER 2/neu when compared to a control standard ELISA kit (≤7.5 ng/mL). This enhanced mode of detection has the potential to not only address breast and other cancers per se but also permit an in depth evaluation of “shed extracellular domains”, in general, and the role of these “proteolytic derived factors” in physiological signalling at normal levels.展开更多
Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.M...Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.展开更多
The extracellular domain(p75ECD)of p75 neurotrophin receptor(p75NTR)antagonizes Aβ neurotoxicity and promotes Aβclearance in Alzheimer’s disease(AD).The impaired shedding of p75ECD is a key pathological process in ...The extracellular domain(p75ECD)of p75 neurotrophin receptor(p75NTR)antagonizes Aβ neurotoxicity and promotes Aβclearance in Alzheimer’s disease(AD).The impaired shedding of p75ECD is a key pathological process in AD,but its regulatory mechanism is largely unknown.This study was designed to investigate the presence and alterations of naturally-occurring autoantibodies against p75ECD(p75ECD-NAbs)in AD patients and their effects on AD pathology.We found that the cerebrospinal fluid(CSF)level of p75ECD-NAbs was increased in AD,and negatively associated with the CSF levels of p75ECD.Transgenic AD mice actively immunized with p75ECD showed a lower level of p75ECD and more severe AD pathology in the brain,as well as worse cognitive functions than the control groups,which were immunized with Re-p75ECD(the reverse sequence of p75ECD)and phosphate-buffered saline,respectively.These findings demonstrate the impact of p75ECD-NAbs on p75NTR/p75ECD imbalance,providing a novel insight into the role of autoimmunity and p75NTR in AD.展开更多
Metabotropic glutamate receptor 7, coupled with a chemical neurotransmitter L-glutamate, plays an important role in the development of many psychiatric and neurological disorders. To study the biological and genetic m...Metabotropic glutamate receptor 7, coupled with a chemical neurotransmitter L-glutamate, plays an important role in the development of many psychiatric and neurological disorders. To study the biological and genetic mechanism of the mGluR7-related diseases, a physical map covering the full-length mGluR7 genomic sequence has been constructed through seed clone screening and fingerprinting database searching. These BAC clones in the physical map have been sequenced with shotgun strategy and assembled by Phred-Phrap-Consed software; the error rate of the final genoniic sequence is less than 0.01%. mGluR7 spans 880 kb genoniic region, the GC content and repeat content of mGluR7 genoniic sequence are 38% and 37.5% respectively. mGluR7 has a typical 'house-keeping' promoter and consists of 11 exons, with introns ranging from 6 kb to 285 kb. mGluR7a and mGluR7b are two known alternatively splicing variants. Comparing the genomic structures of extracellular domains of mGluR family, their genomic structures can展开更多
Receptor for Advanced Glycation End-products(RAGE) binds to a number of ligand families to display important roles in hyperglycemia, senescence, inflammation, neurodegeneration and cancer. It is reported that RAGE reg...Receptor for Advanced Glycation End-products(RAGE) binds to a number of ligand families to display important roles in hyperglycemia, senescence, inflammation, neurodegeneration and cancer. It is reported that RAGE regulates the related biological processes via homo-dimerization by the transmembrane(TM) domain, and evidence further shows that the intracellular domain of RAGE has an influence on the dimerization activity of RAGE. In this study, we explored the underlying interaction mechanism of RAGE TM domains by multiscale coarse-grained(CG) dynamic simulations. Two switching packing modes of the TM dimeric conformations were observed. Through a series of site-directed mutations, we further emphasized the key roles of the A342xxxG346xxG349xxxT353xxL356xxxV360motif in the left-handed configuration and the L345xxxG349xxG352xxxL356motif in the right-handed configuration. In addition, we revealed that the juxtamembrane(JM) domain within JM-A375 can determine the RAGE TM dimeric structure. Overall, we provide the molecular insights into the switching dimerization of RAGE TM domains, as well as the regulation from the JM domains mediated by the anionic lipids.展开更多
Objective Leucine-rich repeats and immunoglobulin-like domains 1(LRIG1) is a newly identified human gene that inhibits the epidermal growth factor receptor(EGFR), which on combining with a ligand, can drive tumor grow...Objective Leucine-rich repeats and immunoglobulin-like domains 1(LRIG1) is a newly identified human gene that inhibits the epidermal growth factor receptor(EGFR), which on combining with a ligand, can drive tumor growth. This study investigated the interaction between human LRIG1 and EGFR and attempted to delineate the functions of as well as the mechanisms used by the extracellular(ECD) and cytoplasmic(CPD) domains of the human LRIG1 protein to downregulate human EGFR signaling activity.Methods Two constructed chimeric eukaryotic expression vectors, pIRES2-EGFP-3XFLAG-LRIG1-ET and p3FLAG-LRIG1-TC, encoding the extracellular and transmembrane regions(LRIG1-ET) and the transmembrane and cytoplasmic regions(LRIG1-TC), respectively, and the plasmid p3XFLAG-CMV-9-LRIG1 encoding full-length LRIG1(LRIG1-FL) were transfected into the human glioma cell line U251 or primary astrocytoma cells by using liposomes. The number and affinity of cell surface EGFR on transfected cells was determined by ^(125)I-EGF binding assay. Results The dissociation constant(KD) values for EGFR were higher, and the maximum increase was observed in the cells transfected into LRIG1-ET(1.36 folds). The number of maximal binding sites(Bmax) of the receptors was decreased in all transfected cells; the maximum decrease was noted in the cells transfected into LRIG1-FL(40.05%).Conclusion Both the ECD and CPD of LRIG1 are important to negate EGFR signaling. The ECD may interfere with the binding between EGFR and its ligand and facilitate the functions of CPD. The CPD may, when brought in proximity to EGFR, enhance receptor degradation. These two mechanisms can contribute to the downregulation of EGFR-mediated signaling by LRIG1.展开更多
文摘Background The present study aimed to investigate the detailed mode and specific sites for their binding as well as the functional relevance of this binding in the phenotypic proliferation of vascular smooth muscle cells(SMCs). Methods CREG knocked-down SMCs were employed to evaluate the biological activity of wtCREG and mCREG.Expressions of SMC differentiation markers SM myosin heavy chain(SM-MHC),SM-actin,heavy caldesmon and myocardin were determined by Western blotting using specific antibodies. Cellular growth of SMCs was assessed by bromide dewuridine (BrdU) incorporation and cell cycle analysis on fluorescence-activated cell sorting(FACS).A solid-phase binding assay was used to study the binding of CREG to extracellular domains of M6P/IGF2R.The cellular co-localization of the two recombinant CREGs with M6P/IGF2R was detected on SMC surface by immunoprecipitation and immunofluorescence analysis.Results The molecular weight of wtCREG was around 30 kD while that of the mCREG was～25 kD.Treatment of wtCREG with PNGase F reduced its molecular weight from～30 kD to～25 kD,whereas PNGase F treatment had no effect on the molecular weight of mCREG.Both wtCREG and mCREG proteins enhanced SMC differentiation,inhibited BrdU incorporation,and arrested cell cycle progression when added to the culture medium.In CREG knocked-down SMCs,the amount of CREG detected by immunoblotting in M6P/IGF2R immunoprecipitates was significantly reduced when compared to normal cells.Both recombinant CREGs co-immunoprecipitated with M6P/IGF2R, although slightly reduced amount of the mutant CREG was detected in M6P/IGF2R immunoprecipitates.Immunostaining revealed that His-tagged CREGs co-localized with IGF2R on the cell surface in a glycosylation-independent manner.In vitro binding assay showed that CREGs bound to M6P/ IGF2R extracellular domains 7-10 and 11-13 in a glycosylation -dependent and -independent manner,respectively.Further blocking experiments using soluble M6P/IGF2R fragments and M6P/IGF2R neutralizing antibody indicated that the biological activities of recombinant CREGs in SMC growth and the up-regulation of SMC differentiation markers were all abolished by treatment with the M6P/IGF2R neutralizing antibody. However,although the growth inhibitory effect of wtCREG was nearly abolished by D7-10 or D11-13,the effect of mCREG was only reversed by Dll-13,indicating that the binding to domains 11-13 is required for CREG to modulate the proliferation of SMCs.Conclusions These data suggest that solubleCREG proteins can exert their biological function via binding to the extracellular domains 7-10 and 11-13 of cell surface M6P/IGF2R in both a glycosylation-dependent and -independent manner.
文摘HER-2/neu oncogene is over-expressed and amplified in patients associated with metastatic breast cancer. An increased level (>15 ng/mL) in the shed extracellular domain (sECD-HER 2/neu) is indicative of the potential presence and as-sociated progression of this disease. A fluo-rescent ELISA incorporating the newly devel-oped ALYGNSA antibody-orientation system revealed a 10-fold increase in sensitivity (≤0.63 ng/mL) of sECD-HER 2/neu when compared to a control standard ELISA kit (≤7.5 ng/mL). This enhanced mode of detection has the potential to not only address breast and other cancers per se but also permit an in depth evaluation of “shed extracellular domains”, in general, and the role of these “proteolytic derived factors” in physiological signalling at normal levels.
基金Nanjing Science and Technology Plan Project(No.ZX20200009)Jiangsu Province Postgraduate Research and Practice Innovation Program(No.SJCX22-0895)。
文摘Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.
基金supported by the National Natural Science Foundation of China(81870860).
文摘The extracellular domain(p75ECD)of p75 neurotrophin receptor(p75NTR)antagonizes Aβ neurotoxicity and promotes Aβclearance in Alzheimer’s disease(AD).The impaired shedding of p75ECD is a key pathological process in AD,but its regulatory mechanism is largely unknown.This study was designed to investigate the presence and alterations of naturally-occurring autoantibodies against p75ECD(p75ECD-NAbs)in AD patients and their effects on AD pathology.We found that the cerebrospinal fluid(CSF)level of p75ECD-NAbs was increased in AD,and negatively associated with the CSF levels of p75ECD.Transgenic AD mice actively immunized with p75ECD showed a lower level of p75ECD and more severe AD pathology in the brain,as well as worse cognitive functions than the control groups,which were immunized with Re-p75ECD(the reverse sequence of p75ECD)and phosphate-buffered saline,respectively.These findings demonstrate the impact of p75ECD-NAbs on p75NTR/p75ECD imbalance,providing a novel insight into the role of autoimmunity and p75NTR in AD.
基金This work was supported by the "863" Program of the MOSC (Grant No. 863-J19) the Creative Program of the Chinese Academy of Sciences (Grant No. KSCX1-D4).
文摘Metabotropic glutamate receptor 7, coupled with a chemical neurotransmitter L-glutamate, plays an important role in the development of many psychiatric and neurological disorders. To study the biological and genetic mechanism of the mGluR7-related diseases, a physical map covering the full-length mGluR7 genomic sequence has been constructed through seed clone screening and fingerprinting database searching. These BAC clones in the physical map have been sequenced with shotgun strategy and assembled by Phred-Phrap-Consed software; the error rate of the final genoniic sequence is less than 0.01%. mGluR7 spans 880 kb genoniic region, the GC content and repeat content of mGluR7 genoniic sequence are 38% and 37.5% respectively. mGluR7 has a typical 'house-keeping' promoter and consists of 11 exons, with introns ranging from 6 kb to 285 kb. mGluR7a and mGluR7b are two known alternatively splicing variants. Comparing the genomic structures of extracellular domains of mGluR family, their genomic structures can
基金supported by the National Natural Science Foundation of China (Nos. 21672019, 21372026, 21402006)the Fundamental Research Funds for the Central Universities (No. XK1701)partly supported by CHEMCLOUDCOMPUTING
文摘Receptor for Advanced Glycation End-products(RAGE) binds to a number of ligand families to display important roles in hyperglycemia, senescence, inflammation, neurodegeneration and cancer. It is reported that RAGE regulates the related biological processes via homo-dimerization by the transmembrane(TM) domain, and evidence further shows that the intracellular domain of RAGE has an influence on the dimerization activity of RAGE. In this study, we explored the underlying interaction mechanism of RAGE TM domains by multiscale coarse-grained(CG) dynamic simulations. Two switching packing modes of the TM dimeric conformations were observed. Through a series of site-directed mutations, we further emphasized the key roles of the A342xxxG346xxG349xxxT353xxL356xxxV360motif in the left-handed configuration and the L345xxxG349xxG352xxxL356motif in the right-handed configuration. In addition, we revealed that the juxtamembrane(JM) domain within JM-A375 can determine the RAGE TM dimeric structure. Overall, we provide the molecular insights into the switching dimerization of RAGE TM domains, as well as the regulation from the JM domains mediated by the anionic lipids.
基金Supported by the grants of the National Natural Science Foundation of China(No.30973073 and 81172402)
文摘Objective Leucine-rich repeats and immunoglobulin-like domains 1(LRIG1) is a newly identified human gene that inhibits the epidermal growth factor receptor(EGFR), which on combining with a ligand, can drive tumor growth. This study investigated the interaction between human LRIG1 and EGFR and attempted to delineate the functions of as well as the mechanisms used by the extracellular(ECD) and cytoplasmic(CPD) domains of the human LRIG1 protein to downregulate human EGFR signaling activity.Methods Two constructed chimeric eukaryotic expression vectors, pIRES2-EGFP-3XFLAG-LRIG1-ET and p3FLAG-LRIG1-TC, encoding the extracellular and transmembrane regions(LRIG1-ET) and the transmembrane and cytoplasmic regions(LRIG1-TC), respectively, and the plasmid p3XFLAG-CMV-9-LRIG1 encoding full-length LRIG1(LRIG1-FL) were transfected into the human glioma cell line U251 or primary astrocytoma cells by using liposomes. The number and affinity of cell surface EGFR on transfected cells was determined by ^(125)I-EGF binding assay. Results The dissociation constant(KD) values for EGFR were higher, and the maximum increase was observed in the cells transfected into LRIG1-ET(1.36 folds). The number of maximal binding sites(Bmax) of the receptors was decreased in all transfected cells; the maximum decrease was noted in the cells transfected into LRIG1-FL(40.05%).Conclusion Both the ECD and CPD of LRIG1 are important to negate EGFR signaling. The ECD may interfere with the binding between EGFR and its ligand and facilitate the functions of CPD. The CPD may, when brought in proximity to EGFR, enhance receptor degradation. These two mechanisms can contribute to the downregulation of EGFR-mediated signaling by LRIG1.
