Misao Hiroto

Biomedical and biotechnological applications of PEG- and PM-modified proteins

Chemical modification of proteins and other bioactive molecules with polyethylene glycol (PEG) or its derivatives (PM) can be used to tailor molecular properties to particular applications, eliminating disadvantageous properties or conferring new molecular functions. Complexes of therapeutic proteins and PEG or PM show reduced immunoreactivity, prolonged clearance times and improved biostability. Modification with PEG can also increase the solubility and activity of enzymes in organic solvents, thus extending their potential for application in organic syntheses and biotransformation processes.

Cell cycle arrest and apoptosis of leukemia cells induced by L -asparaginase

Apoptotic cell death of murine leukemia cells induced by E. coli L-asparaginase was studied. Deprivation of L-asparagine from the culture of L5178Y cells by L-asparaginase caused the fragmentation of chromosomal DNA of the leukemia cells within 24 h. Prior to the degradation of DNA, cell cycles of L5178Y cells were found to be arrested in G1 phase, and evidence of the DNA strand breaks was initially observed in G1 phase cells as early as 8 h after the asparaginase treatment. Therefore, apoptosis of leukemia cells induced by L-asparaginase is an event that is associated with the cell cycle arrest in G1 phase.

Bioconjugates of proteins and polyethylene glycol : potent tools in biotechnological processes

Abstract Chemical modification of enzymes and other bioactive molecules with polyethylene glycol derivatives, activated PEG and PM, can eliminate some of the drawbacks of the biomolecules and/or give them new functions in biotechnological processes. PEG- or PM-lipase becomes soluble and active in organic solvents so that the reverse reactions of hydrolysis proceed effectively, not only in organic media but also in straight substrates without any solvent. These include ester synthesis and ester exchange reactions including lactone synthesis and optical resolution Enzymes such as lipase and asparaginase modified with activated PMs gain stabilization towards heat and urea denaturation and, for asparaginase in vivo, prolongation of clearance time. Photostabilization of natural pigments, magnetization of enzymes and effective affinity partitioning are archieved by modification with PEG derivatives.

[7] Modification of proteins with polyethylene glycol derivatives

Publisher Summary This chapter discusses the chemical modification of proteins with synthetic macromolecules— polyethylene glycol (PEG) derivatives. The purposes of these modifications include alteration of immunoreactivity, immunogenicity, and suppression of immunoglobulin E production, or making enzymes soluble and active in organic solvents. Proteins can be modified with an activated PEG derivative. The methods of activation of polyethylene glycol were illustrated by Harris. The modifier is usually synthesized from monomethoxyPEG that has a hydroxy group at one end of the molecule amenable to manipulation. The chapter describes the syntheses of the modifiers. Most of the modifiers have a chain-shaped form, such as 2,4-bis( O -methoxyPEG)-6-chloro-s-triazine, abbreviated as “activated PEG 2 .” The chapter describes a new type of modifier with a comb-shaped form, which is a copolymer of maleic anhydride and a monomethoxyPEG derivative, abbreviated as “activated PM.” Each modifier reacts mainly with the ɛ-amino group of lysine residues and/or the N-terminal amino group.

Isolation of Biopterin-α-glucoside from Spirulina (Arthrospira) platensis and Its Physiologic Function

Abstract: A fluorescent substance was isolated from the cyanobacterium with a yield of 4.5 mg per 10 g of dried Spirulina (Arthrospira) platensis cells by gentle extraction and ethanol fractionation followed by column chromatography. The fluorescent substance, which has absorption maxima at 256 nm and 362 nm (pH 8.4), was identified as biopterin-α-glucoside by spectrophotometry and nuclear magnetic resonance spectroscopy. Biopterin-α-glucoside prevented decolorization of the photosynthetic pigments, chlorophyll a, phycocyanin, and carotenoids in photosynthetic vesicles of Spirulina platensis cells, by ultraviolet irradiation.

Chemical modification of lipase with a comb-shaped synthetic copolymer of polyoxyethylene allyl methyl diether and maleic anhydride

SummaryLipase fromPseudomonas fluorescens was coupled with a copolymer of polyoxyethylene allyl methyl diether and maleic anhydride, activated PM. The PM-lipase became soluble and active in organic solvents, and also heat stable. It catalyzed the ester synthesis in benzene and ester hydrolysis in an aqueous system with high enzymic activity.

Stabilization of trypsin by modification with comb-shaped copolymers of poly(ethylene glycol) derivative and maleic anhydride

Trypsin from bovine pancreas was coupled with copolymers of poly(ethylene glycol) derivative and maleic anhydride with the molecular weights of 13 kDa and 100 kDa (activated PM13 and PM100). The modified trypsins were more stable towards autolysis and heat- or urea-treatment than nonmodified trypsin. Stabilization of trypsin caused by the chemical modification with activated PMs is discussed in relation to the protein conformation.

Tolerogenic capacity of poly(ethylene glycol) (PEG)--modified ovalbumins in relation to their immunoreactivity towards anti-ovalbumin antibody.

Ovalbumin (OVA) was chemically modified with poly(ethylene glycol) (PEG) derivatives: activated PEG2, 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chloro-s-triazine and activated PM13, copolymer of poly(oxyethylene) allyl methyl diether and maleic anhydride. Pretreatment of BALB/c mice with PEG2- or PM13-OVA suppressed the production of both IgG- and IgE-class anti-OVA antibodies induced by subsequent immunizations with unmodified OVA. PEG2-OVA had higher potency to suppress OVA-specific immune response than PM13-OVA. Although extensive modification (62%) of amino groups in the OVA molecule with activated PEG2 was required to diminish most of its immunoreactivity towards anti-OVA antibodies, only a low degree of modification (27%) was sufficient to induce tolerogenicity to OVA. Thus, the loss of immunoreactivity of PEG2-OVA was not prerequisite for its tolerogenic capacity. Moreover, the use of completely denatured PEG2-OVA immunogen lead to the loss of its ability to induce immune tolerance to native OVA. These observations are discussed in relation to the regulatory mechanism of immune response.

Regioselective deacetylation of peracetylated monosaccharide derivatives by polyethylene glycol-modified lipase for the oligosaccharide synthesis

Lipase modified with polyethylene glycol became soluble and active in organic solvents, and catalyzed regioselective deacetylation of peracetylated monosaccharide derivatives in 1,1,1-trichloroethane. The deacetylation occurred only at the positions of C-4 and C-6 of the glycopyranoside ring. Especially, peracetylated methyl β-D-xylopyranoside and peracetylated L-serine-β-D-xylopyranoside were hydrolyzed only at the position of C-4. Subsequently, one of the resulting products, that is L-serine-2,3-di-O-acetyl-β-D-xylopyranoside, was coupled with galactose residue to obtain L-serine-4-O-(β-D-galactopyranosyl)-β-D-xylopyranoside, a model compound of the carbohydrate-protein linkage region of proteoglycans.

Immune tolerance induced by polyethylene glycol-conjugate of protein antigen: clonal deletion of antigen-specific Th-cells in the thymus.

Polyethylene glycol (PEG) conjugates of protein antigens induce antigen-specific immune tolerance of helper T (Th)-cells. However, the mechanism of this Th-cell tolerance has remained unelucidated. Using transgenic mice with ovalbumin (OVA)-specific T-cell receptor (TCR) genes, we examined the response of OVA-specific Th-cells towards tolerogenic PEG-conjugate of OVA in vitro and in vivo. When stimulated with PEG-OVA in vitro, transgenic OVA-specific Th-cells proliferated and produced interleukin 2, the levels of which were comparable to those induced by unmodified OVA. In contrast, PEG-OVAadministered into the circulation of transgenic mice induced unresponsiveness in peripheral OVA-specific Th-cells. Moreover, in the thymus of these transgenic mice, the frequency of immature CD4+CD8+ (double positive) thymocytes was reduced. A similar phenomenon was not observed in transgenic mice treated with unmodified OVA. As autoreactive T-cells are known to be clonally deleted at the immature double positive stage in the thymus, Th-cell tolerance induced by PEG-protein antigens is at least in part mediated by central tolerance in the thymus, and is likely caused by the markedly enhanced stability of PEG-protein conjugates in the circulatory system.