Confirmation of covalently-linked structure and cell-death inducing activity in site-specific chemical conjugates of human Fas ligand extracellular domain.

OBJECTIVE: In this study, we aimed to identify the structural components and to clarify the biological activity in the site-specific conjugates of human Fas ligand extracellular domain (hFasLECD) with either fluorescein moiety (FL) or chicken egg-white avidin (Avi). The conjugates were characterized by molecular-weight measurement using MALDI-TOF mass-spectrometric analysis and by cell-death inducing activity measurement against a human colorectal cancer cell line, HT-29, using MTT cell-viability assay. Pretreatment effect with human interferon-γ (IFN-γ) on the cell-death inducing activity was evaluated. RESULTS: The mass-spectrometric analysis of the hFasLECD-Avi conjugate showed that it was possible to detect the signal peak of molecular-weight to electric charge (m/z) derived from the component involved in the covalent linking as the sum of the molecular-weight of unconjugated hFasLECD- and Avi-derivative subunits, in addition to the signals from each corresponding subunit component irrelevant to the covalent linking. The cell-viability assay revealed that both conjugates possessed a remarkable death-inducing activity against HT-29 cells in synergy with the pretreatment using human IFN-γ. Following 24 h pretreatment with 100 IU/ml of human IFN-γ, almost no viable cells existed after 72 h treatment with either 100 or 1000 ng/ml of FL-hFasLECD and hFasLECD-Avi conjugates.

Site-specific chemical conjugation of human Fas ligand extracellular domain using trans-cyclooctene - methyltetrazine reactions.

BACKGROUND: Fas ligand plays a key role in the human immune system as a major cell death inducing protein. The extracellular domain of human Fas ligand (hFasLECD) triggers apoptosis of malignant cells, and therefore is expected to have substantial potentials in medical biotechnology. However, the current application of this protein to clinical medicine is hampered by a shortage of the benefits relative to the drawbacks including the side-effects in systemic administration. Effective procedures for the engineering of the protein by attaching useful additional functions are required to overcome the problem. RESULTS: A procedure for the site-specific chemical conjugation of hFasLECD with a fluorochrome and functional proteins was devised using an inverse-electron-demand Diels-Alder reaction between trans-cyclooctene group and methyltetrazine group. The conjugations in the present study were attained by using much less molar excess amounts of the compounds to be attached as compared with the conventional chemical modification reactions using maleimide derivatives in the previous study. The isolated conjugates of hFasLECD with sulfo-Cy3, avidin and rabbit IgG Fab' domain presented the functional and the structural integrities of the attached molecules without impairing the specific binding activity toward human Fas receptor extracellular domain. CONCLUSIONS: The present study provided a new fundamental strategy for the production of the engineered hFasLECDs with additional beneficial functions, which will lead to the developments of the improved diagnostic systems and the effective treatment methods of serious diseases by using this protein as a component of novel molecular tools.

Preparation of a functional fluorescent human Fas ligand extracellular domain derivative using a three-dimensional structure guided site-specific fluorochrome conjugation.

BACKGROUND: Human Fas ligand extracellular domain has been investigated as an important target protein in the field of medical biotechnology. In a recent study, the author developed an effective method to produce biologically active human Fas ligand extracellular domain derivatives using site-specific chemical modifications. FINDINGS: A human Fas ligand extracellular domain derivative containing a reactive cysteine residue within its N-terminal tag sequence, which locates not proximal to the binding interface between the ligand and the receptor in terms of the three-dimensional structure, was modified by Fluorescein-5-Maleimide without impairing the specific binding activity toward human Fas receptor extracellular domain. The purified protein sample free of low molecular-weight contaminants showed a characteristic fluorescence spectrum derived from the attached Fluorescein moieties, and formed a stable binding complex with human Fas receptor extracellular domain-human IgG1 Fc domain fusion protein in solution. The conjugation number of the fluorochrome was estimated to be 2.5 per a single human Fas ligand extracellular domain trimer from the ratio of the absorbance value at 280 nm to that at 495 nm. CONCLUSIONS: A functional fluorescent human Fas ligand extracellular domain derivative was prepared via a site-specific conjugation of fluorochrome, which was guided by the three-dimensional structure information on the ligand-receptor complex. Fluorescent derivatives created by this method may contribute to the development of an improved diagnosis system for the diseases related to Fas receptor.

Improved production of recombinant human Fas ligand extracellular domain in Pichia pastoris: yield enhancement using disposable culture-bag and its application to site-specific chemical modifications.

BACKGROUND: A useful heterologous production system is required to obtain sufficient amounts of recombinant therapeutic proteins, which are often necessary for chemical characterization and engineering studies on the development of molecules with improved properties. Human Fas ligand extracellular domain (hFasLECD) is an agonistic death ligand protein that has potential applications for medical purposes. Site-specific chemical modifications can provide a powerful means for the development of engineered proteins with beneficial functions. This study aimed to enhance the yield of hFasLECD using a Pichia pastoris secretory expression system suitable for efficient production on a small laboratory scale, and further to provide procedures for its site-specific chemical modification without impairing the biological functions based on the developed production system. RESULTS: A convenient cultivation system using a disposable plastic bag provided a three-fold increase in purification yield of tag-free hFasLECD as compared with the conventional system using a baffled glass flask. The system was further applied to the production of a mutant, which contains an additional reactive cysteine residue in the N-terminal tag-sequence region. Site-specific conjugations and cross-linking without impairing biological functions were achieved by reaction of the mutant hFasLECD with single maleimide group containing compounds and a linear polyethylene glycol derivative containing two maleimide groups at either end, respectively. All purified tag-free and chemically modified hFasLECDs showed an evident receptor binding activity in co-immunoprecipitation experiments mediated by wild-type and N-glycosylation site deficient mutant human Fas receptor extracellular domain derivatives. An N-Ethylmaleimide conjugated hFasLECD derivative demonstrated a significant cytotoxic activity against human HT-29 colorectal cancer cells. CONCLUSIONS: A new, efficient cultivation system for enhanced secretory production of hFasLECD using P. pastoris and an effective strategy for site-specific chemical modifications of hFasLECD were devised. The results obtained constitute the basis for biomedical applications including developments of novel therapeutic proteins and diagnostic tools targeted to related diseases and their biomarkers.

Heterologous production of death ligands' and death receptors' extracellular domains: structural features and efficient systems.

The extracellular domains of death ligands and those of death receptors are closely related to many serious human diseases through the initiation of apoptosis. Recombinant production of the extracellular domains has been investigated due to demand for a large amount of purified samples, which are a prerequisite for their biochemical characterization and constitute the fundamentals of medical applications. This review focuses on the recombinant production of extracellular domains of the major members of death ligand and death receptor families using non-mammalian expression systems with an emphasis on Fas ligand and Fas receptor. In contrast to the efficient production of the functional extracellular domains of TRAIL, TNFα and LTα by intracellular expression systems using Escherichia coli or Pichia pastoris, that of Fas ligand requires the secretory expression systems using P. pastoris or Dictyostelium discoideum, and the productivity in P. pastoris was largely dependent on tag sequence, potential N-glycosylation site and expressed protein region. On the other hand, the exploitation of insect cell systems is generally useful for the preparation of functional extracellular domains of death receptors containing many disulfide bridges in the absence of extended secondary structure, and a Bombyx mori larvae secretion system presented a superior productivity for human Fas receptor extracellular domain. Based on the results obtained so far, further efforts should be devoted to the artificial control of death ligand - death receptor interactions in order to make a contribution to medicine, represented by the development of novel biopharmaceuticals.

Improved isolation and purification of functional human Fas receptor extracellular domain using baculovirus-silkworm expression system.

To achieve an efficient isolation of human Fas receptor extracellular domain (hFasRECD), a fusion protein of hFasRECD with human IgG1 heavy chain Fc domain containing thrombin cleavage sequence at the junction site was overexpressed using baculovirus-silkworm larvae expression system. The hFasRECD part was separated from the fusion protein by the effective cleavage of the recognition site with bovine thrombin. Protein G column treatment of the reaction mixture and the subsequent cation-exchange chromatography provided purified hFasRECD with a final yield of 13.5mg from 25.0 ml silkworm hemolymph. The functional activity of the product was examined by size-exclusion chromatography analysis. The isolated hFasRECD less strongly interacted with human Fas ligand extracellular domain (hFasLECD) than the Fc domain-bridged counterpart, showing the contribution of antibody-like avidity in the latter case. The purified glycosylated hFasRECD presented several discrete bands in the disulphide-bridge non-reducing SDS-PAGE analysis, and virtually all of the components were considered to participate in the binding to hFasLECD. The attached glycans were susceptible to PNGase F digestion, but mostly resistant to Endo Hf digestion under denaturing conditions. One of the components exhibited a higher susceptibility to PNGase F digestion under non-denaturing conditions.

Efficient production of human Fas receptor extracellular domain-human IgG1 heavy chain Fc domain fusion protein using baculovirus/silkworm expression system.

The fusion protein consisting of human Fas receptor extracellular domain and human IgG1 heavy chain Fc domain (hFasRECD-Fc) is a medically important protein that potentially has therapeutic uses. The fusion gene composed of a synthetic human Fas receptor extracellular domain gene and the cDNA encoding human IgG1 heavy chain Fc domain was investigated on the secretory expression using two baculovirus systems which employed either Spodoptera frugiperda 9 (Sf9) cell line or Bombyx mori (silkworm) larvae as the host organism. Both expression systems produced the functional hFasRECD-Fc as a dimer molecule linked by disulfide bridges. The secretion level per unit volume was much higher in the case of silkworm larvae as compared to Sf9 cell line, and was estimated to be more than 150 times. A substantially pure hFasRECD-Fc sample from silkworm larvae was obtained by single step Protein G-agarose affinity column chromatography. The affinity purified sample was further fractionated by anion-exchange chromatography with the final purification yield of 22.5 mg from 26 ml hemolymph. The hFasRECD-Fc from silkworm larvae and the tag-free human Fas ligand extracellular domain derivative from Pichia pastoris formed a stable complex in solution, which was verified by size-exclusion chromatography. This study demonstrated that the baculovirus/silkworm expression system provided the means for efficient production of highly pure hFasRECD-Fc with functional activity.

Improved secretion of human Fas ligand extracellular domain by N-terminal part truncation in Pichia pastoris and preparation of the N-linked carbohydrate chain trimmed derivative.

Human Fas ligand is a medically important transmembrane glycoprotein directing the induction of apoptosis. The influence of N-terminal part (Q103-P138) truncation of human Fas ligand extracellular domain (hFasLECD) on the expression of N-terminal FLAG-(Gly)(5)-tagged hFasLECD (NFG5-hFasLECD) with partial N-glycosylation-sites deletion in Pichia pastoris was investigated. The N-terminal part truncation significantly improved the secretion level of both singly (N184Q) and doubly (N184Q, N250Q) N-glycosylation-sites deleted NFG5-hFasLECD. The highly purified N-terminal truncated NFG5-hFasLECD with the double N-glycosylation-sites deletion mutation was obtained using single-step cation-exchange chromatography. The isolation yield was about 24mg from one liter culture supernatant, which amounted to approximately five times higher than that of the previously reported non-truncated NFG5-hFasLECD with N184Q mutation. The remaining N-linked carbohydrate chain in the purified product was digested with a high-mannose type glycochain specific endoglycosidase, Endo Hf, under non-denatured condition. The N-linked carbohydrate chain trimmed product was purified through Con A-agarose column fractionation and another cation-exchange chromatography from the reaction mixture. The final product showed the molecular weight exact to that of NFG5-hFasLECD-[Delta(103-138), N250Q] mutant with single N-acetylglucosamine residue in MALDI-TOF mass-spectrometric analysis, and existed as a trimer in solution. The N-terminal truncated product either with or without N-linked carbohydrate chain exhibited the specific binding activity toward soluble human Fas receptor extracellular domain-human IgG(1)-Fc domain fusion protein, which revealed that the presence of N-linked carbohydrate chain was not essential for the functional activity of hFasLECD. The sample preparation system developed here may be applicable to the structural analysis of hFasLECD.

Secretory expression of synthetic human Fas ligand extracellular domain gene in Pichia pastoris: influences of tag addition and N-glycosylation site deletion, and development of a purification method.

Human Fas ligand is a medically important membrane glycoprotein that induces the apoptosis of harmful cells. A new secretory expression and purification method was devised for the production of a large amount of recombinant human Fas ligand extracellular domain (hFasLECD) by Pichia pastoris. The expression plasmid containing a synthetic hFasLECD gene designed using yeast optimal codons was constructed for the secretion of hFasLECD. The secreted product exhibited the specific binding activity toward soluble human Fas receptor extracellular domain-human IgG(1)-Fc domain fusion protein, and the receptor-ligand complex was immunoprecipitated by Protein A conjugated agarose-gel beads. The influences of the N- and C- terminal addition of FLAG/(His)(6) tag spaced by pentaglycine sequence and the sequentially accumulative deletions of N-glycosylation sites within hFasLECD were investigated. The secretion of functional hFasLECD was retained after the N-terminal tagging and the deletion of either single or double N-glycosylation sites. As judged from SDS-PAGE analysis of the culture supernatant, the N-terminal addition of FLAG-(Gly)(5) tag and the deletion of single N-glycosylation site via N184Q mutation increased the secretion level of the product. In contrast, the C-terminal tagged genes and all N-glycosylation sites deleted gene failed to direct the secretion of functional hFasLECD. The secreted products in the culture medium were purified using a cation-exchange chromatography and a gel-filtration chromatography. The purified hFasLECDs existed as trimers composed of a mixture of monomer species in different glycosylation states. Approximately five milligram of functional N-terminal FLAG-(Gly)(5) tagged hFasLECD N184Q mutant was obtained from one liter culture supernatant.

On the importance of carbohydrate-aromatic interactions for the molecular recognition of oligosaccharides by proteins: NMR studies of the structure and binding affinity of AcAMP2-like peptides with non-natural naphthyl and fluoroaromatic residues.

The specific interaction of a variety of modified hevein domains to chitooligosaccharides has been studied by NMR spectroscopy in order to assess the importance of aromatic-carbohydrate interactions for the molecular recognition of neutral sugars. These mutant AcAMP2-like peptides, which have 4-fluoro-phenylalanine, tryptophan, or 2-naphthylalanine at the key interacting positions, have been prepared by solid-phase synthesis. Their three-dimensional structures, when bound to the chitin-derived trisaccharide, have been deduced by NMR spectroscopy. By using DYANA and restrained molecular dynamics simulations with the AMBER 5.0 force field, the three-dimensional structures of the protein-sugar complexes have been obtained. The thermodynamic analysis of the interactions that occur upon complex formation have also been carried out. Regarding binding affinity, the obtained data have permitted the deduction that the larger the aromatic group, the higher the association constant and the binding enthalpy. In all cases, entropy opposes binding. In contrast, deactivation of the aromatic rings by attaching fluorine atoms decreases the binding affinity, with a concomitant decrease in enthalpy. The role of the chemical nature of the aromatic ring for establishing sugar contacts has been thus evaluated.

X-ray structural analysis of the ligand-recognition mechanism in the dual-affinity labeling of c-type lysozyme with 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine.

In spite of the belonging to the same c-type lysozyme family, hen egg-white lysozyme (HEWL) was much less susceptible to the dual-affinity labeling with 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine (Galbeta1,4GlcNAc-Epo) than human lysozyme (HL). The three-dimensional structures of the HEWL labeled with single Galbeta1,4GlcNAc-Epo and the Glu102-mutant HL labeled with double Galbeta1,4GlcNAc-Epo were determined by X-ray crystallography at resolutions of 1.85 and 2.0 A, respectively. The overall conformation and the interaction mode of the carbohydrate ligand part in the singly labeled HEWL and the doubly labeled Glu102-mutant HL were basically identical to those of the correspondingly labeled wild-type HL with minor alterations in some stereochemical parameters. A detailed comparison of the structures revealed the key protein-carbohydrate and carbohydrate-carbohydrate interactions essential for the dual labeling. It was suggested that the difference in the efficiency of the dual labeling was caused by the structural difference between Gln104 in HL and Asn103 in HEWL. The relevance to our previous study and the carbohydrate-carbohydrate interaction on cell-surface membranes were discussed.

Cytoagglutination and cytotoxicity of Wheat Germ Agglutinin isolectins against normal lymphocytes and cultured leukemic cell lines--relationship between structure and biological activity.

The relationships between degree of lectin-cell binding, cytotoxicity and cytoagglutinating activity of three Wheat Germ Agglutinin isolectins (WGA-1, WGA-2, WGA-3) against normal lymphocytes and cultured leukemic cell lines (Jurkat, MOLT-4, Raji, Daudi, K-562) were studied. All WGA-isolectins interacted in a similar degree with normal lymphocytes, while in the case of leukemic cells, the degree of isolectin-cell binding increased in the order: WGA-1< or =WGA-3

The importance of CH/pi interactions to the function of carbohydrate binding proteins.

It is suggested that the interactions between the hydrophobic C-H groups of carbohydrate residues and the pi-electron systems of aromatic amino-acid residues play an important role in the ligand-recognition function of carbohydrate-binding proteins. This review focuses on our recent structural and functional studies of human lysozyme and hevein-domain type lectins (wheat-germ agglutinin and Ac-AMP2) aimed at understanding how CH/pi interactions are involved in the actual binding events.

Interactions of wheat-germ agglutinin with GlcNAc beta 1,6Gal sequence.

The interactions of wheat-germ agglutinin (WGA) with the GlcNAc beta 1,6Gal sequence, a characteristic component of branched poly-N-acetyllactosaminoglycans, were investigated using isothermal titration calorimetry and X-ray crystallography. GlcNAc beta 1,6Gal exhibited an affinity greater than GlcNAc beta 1,4GlcNAc to all WGA isolectins, whereas Gal beta 1,6GlcNAc showed much less affinity than GlcNAc beta 1,4GlcNAc. X-ray structural analyses of the glutaraldehyde-crosslinked WGA isolectin 3 crystals in complex with GlcNAc beta 1,6Gal, GlcNAc beta 1,4GlcNAc and GlcNAc beta 1,6Gal beta 1,4Glc were performed at 2.4, 2.2 and 2.2 A resolution, respectively. In spite of different glycosidic linkages, GlcNAc beta 1,6Gal and GlcNAc beta 1,4GlcNAc exhibited basically similar binding modes to each other, in contact with side chains of two aromatic residues, Tyr64 and His66. However, the conformations of the ligands in the two primary binding sites were not always identical. GlcNAc beta 1,6Gal showed more extensive variation in the parameters defining the glycosidic linkage structure compared to GlcNAc beta 1,4GlcNAc, demonstrating large conformational flexibility of the former ligand in the interaction with WGA. The difference in the ligand binding conformation was accompanied by alterations of the side chain conformation of the amino acid residues involved in the interactions. The hydrogen bond between Ser62 and the non-reducing end GlcNAc was always observed regardless of the ligand type, indicating the key role of this interaction. In addition to the hydrogen bonding and van der Waals interactions, CH--pi interactions involving Tyr64, His66 and Tyr73 are suggested to play an essential role in determining the ligand binding conformation in all complexes. One of the GlcNAc beta 1,6Gal ligands had no crystal packing contact with another WGA molecule, therefore the conformation might be more relevant to the interaction mode in solution.