Application Of Superpositively Charged Green Fluorescent Protein In The Detection Of Glycosaminoglycans And Proteoglycans

Proteoglycans(PGs)are biological molecules composed of a core protein substituted with one or more glycosaminoglycan(GAG)chains.GAGs known as mucopolysaccharides,are polyanionic linear heteropolysaccharides composed of repeating disaccharide units.According to the monosaccharide residues,the type of glycosidic bonds,and the number and position of the sulfate groups,GAGs can be classified into hyaluronic(HA),chondroitin sulfate(CS)/dermatan sulfate(DS),keratan sulfate(KS)and heparin(Hep)/heparan sulfate(HS).Among them,HA is the only GAG that does not attach to core protein.PGs are widely expressed on cell surface and in the extracellular matrices.They can interact with many biological molecules such as growth factors and cytokines.PGs are involved in many physiological and pathological processes such as cell proliferation,differentiation,migration and intercellular signal transductions.So PGs have important values of research and application in medical and pharmaceutical fields.PGs are complex and diverse based on the multiple species of core proteins and the various numbers and types of GAG chains.The complexity of GAGs chains provides a critical structural basis for the diversification of the biological functions of PGs.GAGs are often complicated due to the modification of many specific enzymes.These complicated structures provide the functional diversity,but also bring the huge difficulties to the detection.Currently,chemical staining method based on basic dyes,biochemical detection based on specific antibodies against core proteins and specific enzymes for GAGs side chains,and instrumental analysis using High Performance Liquid Chromatography(HPLC),Mass spectrometry(MS)and Nuclear magnetic resonance(NMR)are used for the detection of GAGs/PGs.However,these methods have many limitations,such as the poor biocompatibility which is not suitable for intravital staining;poor sensitivity;poor selectivity;complicated experimental operations which result to long detection time,and the expensive cost for the instruments and maintenance.Thus,one simple,quick,sensitive and high biocompatible method is urgently needed for the detection of GAGs/PGs.Superpositively charged fluorescent protein(ScGFP)is one green fluorescent protein with 36 positive charges developed by genetic mutation.It has important value in biomacromolecular analysis and transmembrane transport due to its low cytotoxicity,high biocompatibility,high stability,and high ability of cell penetration.In the present study,ScGFP was used as one biological probe,combined with GAGs-specific degrading enzymes and specific antibodies against core proteins of PGs,for the analytical detection of GAGs/PGs at the molecular,cellular and organic levels for the first time.Based on the comprehensive optimization of relevant analytical conditions,ScGFP was applied to detect GAGs/PGs in complex biological samples and clinical samples.These studies show that ScGFP is one novel biocompatible fluorescent probe,which has the important value in analysis of GAGs/PGs.The main results are summarized as follows:1.Establishment and application of the homogeneous detection method using graphene and ScGFP for GAGs:The fluorescence of ScGFP was quenched by graphene oxide(GO)through the fluorescence resonance energy transfer in the homogeneous detection system,but the quenching can be inhibited by the presence of GAGs in the solution.The binding of GAGs to ScGFP through charge interaction inhibits the fluorescence quenching of ScGFP by GO.Compared with the negative control group without GAGs,fluorescent recovery was occurred in a dose-dependent manner with the presence of GAGs.Thus,the quantitative analysis of GAGs in the homogeneous detection system can be performed based on the recovery of fluorescent intensity.After establishment of this homogeneous detection method,each reaction condition was firstly optimized.The optimum buffer was 10 mM Tris-HCl,100 mM NaCl,pH 10.0;the optimum reaction sequence was firstly mix ScGFP and GAGs,and then added GO;the optimum final concentration of ScGFP and GO was 2.5 μg/ml and 50 μg/ml;the optimum reaction time was 30 min.After the optimum reaction conditions were determined,different types and concentrations of GAGs were tested in the buffer.Different types of GAGs had the different binding abilities to ScGFP to lead to the different fluorescent recovery capabilities.Hep had the strongest binding ability toScGFP,and the binding constant is 9.35E7/mol;the binding ability of CSA and DS to ScGFP was second,and the binding constant were 4.53E7/mol and 4.54E7/mol,respectively;but the binding ability of HA was too weak to calculate the binding constant.From the results we also found that the detected fluorescence intensity gradually increased with the increasing concentration of GAGs in the detection system.By preparing the standard curve,the concentration of GAGs in the solution can be analyzed quickly.On these bases,the method was further applied to detect Hep in serum.The result showed that this homogeneous detection method was still effective for the detection of GAGs in complex biological samples and was a simple and reliable method for detecting Hep in serum.This was important in the clinical monitoring of the concentration of Hep in the blood and was significant in reducing complications such as bleeding caused by Hep overdose.Finally,the homogeneous detection method was used to detect other GAGs impurities and oversulfated chondroitin sulfate(OSCS)contaminants in Hep.The results showed that the detection method was insensitive to the detection of other GAGs in Hep,but was sensitive and specific to the detection of OSCS with the minimum of 10-6(w/w)%.Thus,it is an effective method for monitoring the quality of Hep drugs.Compared with the traditional methods,the new homogeneous detection method was simple,sensitive and fast,and had important application value in the analysis and quality control of GAGs samples.2.Visualization of GAGs on the surface of living cells and in mouse organs by ScGFP:According to the high biocompatibility,low cytotoxicity and high stability,ScGFP was used as fluorescent probe to visually detect the type and express of GAGs in living cells and in vivo organs of mice.The results showed that the type and express of GAGs in different kinds of cells were different.The express of CS/DS on A549 cells surface was higher than Hep/HS,while the express of Hep/HS on HEK 293T cells surface was higher than CS/DS.These results was consistent with the corresponding results from the GAG disaccharide composition assay.In addition,ScGFP was inj ected into the mice through the tail vein to observe the distribution of GAGs in the organs of the mice.Through visual detection,ScGFP was detained in various organs for a long time.The cleanup of ScGFP in mice was relatively slow and the fluorescent intensity was still significant 8h after injection.After treatment with GAGs-specific enzymes,the organ staining was significantly reduced and the effect of treatment with Hepases was much weaker than that with CSase ABC.These results indicated that the organ staining in mice was due to the binding of ScGFP to GAGs,and CS/DS had more important role than HS/Hep in the binding of ScGFP.The above results indicated that it is simple and effective to detect GAGs in living cells and organs by using ScGFP as fluorescent probe.Therefore,it was shown for the first time that ScGFP was an effective probe which had the high biocompatibility for the visualization and detection of GAGs in many physiological and pathological processes.3.Establishment of homogeneous detection method of PGs and its application in detection of HCC:Two high affinity monoclonal antibodies(mAbs)against different epitopes of the core protein of Glypican-3(GPC3)were obtained by antigen preparation,mouse immunization,cell fusion,hybridoma screening,and purification.Then a simple,sensitive and efficient homogeneous detection method of GPC3 was established by using the specific binding between antigen and antibody and the electrostatic interaction between ScGFP and HS chains.The principle of the method is that two different monoclonal antibodies coupled to carboxyl graphene can recognize and capture different antigenic epitopes of core protein of GPC3 to form a sandwich structure;ScGFP can bind to the HS chains of GPC3 to locate in the middle of the sandwich structure which resulted in a greater degree of fluorescence quenching.Finally,the amount of GPC3 in the system was detected by the degree of fluorescence quenching.After the homogeneous detection method was established,the reaction conditions were optimized.The optimum addition order of reagents was mixing mAb1-graphene and GPC3 firstly,then adding ScGFP and mAb2-graphene in sequence.This order allowed ScGFP to be located in the middle of the sandwich structure which lead the greatest extent of fluorescence quenching to increase the sensitivity of the detection.In addition,other optimization conditions were as follows:carboxyl graphene was the optimum quenching reagent;the final concentration of mAb coupled on carboxyl graphene was 0.01 mg/ml;the optimum blocking conditions was 1%BSA;the optimum buffer was 10 mM Tris-HCl,100 mM NaCl,pH 10.0;the optimum reaction time of aGCN-carboxyl graphene and GPC3 was 5 min,the reaction time of aGCN-carboxyl graphene-GPC3 complex and ScGFP was 15 min,and the reaction time of aGCN-carboxyl graphene-GPC3-ScGFP complex and aGCC-carboxyl graphene was 10 min.After the optimum reaction conditions were determined,GPC3 were quantitatively tested in buffer and serum.The results showed that the fluorescence quenching rate increased with the increasing concentration of GPC3,and there was a good linear between quenching rate and the log base 2 of the GPC3 concentration.So the amount of GPC3 in solution can be determined by the standard curve.Finally,the new method was successfully applied to detect GPC3 in the clinical HCC serum samples.The results showed that the average amount of GPC3 in HCC group was significantly higher than that in individuals with normal liver(NL)and patients with chronic hepatitis(CH).The GPC3 level in HCC was 12-fold higher than that in NL and 8-fold higher than that in CH.Setting the cut-off point at 3.71 ng/ml,the sensitivity was 59.5%,which was higher than the other methods.Compared with the traditional sandwich method,the new method had the high sensitivity and specificity,and shorten the time to less than 1h.So the new detection method had the important application value in the early diagnosis of HCC.In addition,this homogeneous detection method also established a common technology platform for the analysis and detection of other PGs.