Novel Method for in Vitro O-Glycosylation of Proteins: Application for Bioconjugation (original) (raw)
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Construction and analysis of a novel peptide tag containing an unnatural N-glycosylation site
FEBS Letters, 2011
The addition of N-glycans to clinically used proteins enhances their therapeutic features. Here we report the design of a novel peptide tag with an unnatural N-glycosylation site, which may increase the N-glycan content of generally any protein. The designed GlycoTags were attached to A1AT, EPO and AGP and constructs were expressed in HEK293 or CHO cells. Hereby we could prove that the attached unnatural N-glycosylation site is decorated with complex-type N-glycans and that the spacer as well as the C-terminal ''tail'' sequence are critical for the usage of the novel N-glycosylation site. This demonstrates that the novel GlycoTag is a convenient tool to provide proteins with extra Nglycan moieties by simply adding a peptide tag sequence as small as 22 amino acids.
Journal of Molecular Catalysis B: Enzymatic, 2008
The question whether proteins fused to 1,4galactosyltransferase (4GalT-1) influence the biocatalytic properties of the glycosyltransferase has not been addressed so far. In the present study we have chosen a novel approach to express the gene encoding for human 4GalT-1 from placenta in an N-terminal fusion with the pre-propeptide of the lipase from Staphylococcus hyicus. The pre-propeptide provides a secretion signal in Escherichia coli and was reported to protect fused proteins against proteolytic degradation. Expression of the fusion protein was challenged with an almost full-length version of human 4GalT-1 including parts of the signal anchor and the stem region (propeptide-nat4GalT-1) and the full catalytic domain (His 6 propeptide-cat4GalT-1), respectively. We demonstrate that the fusion protein in propeptide-nat4GalT-1 is cleaved off during purification using immobilized metal ion chromatography IMAC, most probably catalyzed by the immobilized Zn 2+ ions. Cleavage can be avoided by deletion of five C-terminal amino acids of the propeptide and the stem region yielding His 6 propeptide-cat4GalT-1. Kinetic data reveal that both enzyme constructs possess specific biocatalytic characteristics when compared to a recombinant luminal 4GalT-1 construct. The catalytic efficiency towards more hydrophobic acceptor glycosides, e.g. benzyl 2-acetamido-2-deoxy--d-glucopyranoside and p-nitro phenyl 2-acetamido-2-deoxy--d-glucopyranoside, and the N-glycans of IgG from rat is significantly increased. In summary, the His 6 propeptide-cat4GalT-1 is very useful for biocatalytic applications involving hydrophobic acceptor glycosides and the propeptide-nat4GalT-1 is able to glycosylate glycoproteins in an efficient way.
Synthetic glycosylation of peptides using unprotected saccharide ?-glycosylamines
Glycoconjugate Journal, 1993
Glycopeptides can be valuable tools in determining the influence of carbohydrate moieties on the intrinsic properties of glycoproteins. However, glycopeptides of sufficient quantity and purity are as yet not readily available from biological sources. The chemical coupling of a/~-glycosylamino group of an unprotected carbohydrate with an activated aspartic acid residue of an unprotected peptide is a simple method for synthesizing asparagine-linked glycopeptides. In this report we demonstrate that the use of this method is not restricted to/~-glycosylamines of simple monosaccharides or short aspartic acid-containing pentapeptides. This is illustrated by the syntheses of several glycopentapeptides containing N,N'-diacetylchitobiose, a glutamine-linked glycopentapeptide containing a biantennary complex oligosaccharide, and glycosylated variants of two analogs of a polypeptide hormone, atriopeptin, containing N,N'-diacetylchitobiose.
Journal of the American Chemical Society, 2007
Synthesis of well-defined neoglycopolymer-protein biohybrid materials and a preliminary study focused on their ability of binding mammalian lectins and inducing immunological function is reported. Crucial intermediates for their preparation are well-defined maleimide-terminated neoglycopolymers (M n = 8-30 kDa; M w /M n = 1.20-1.28) presenting multiple copies of mannose epitope units, obtained by combination of transition-metal-mediated living radical polymerization (TMM LRP) and Huisgen [2+3] cycloaddition. Bovine serum albumin (BSA) was employed as single thiol-containing model protein, and the resulting bioconjugates were purified following two independent protocols and characterized by circular dichroism (CD) spectroscopy, SDS PAGE, and SEC HPLC. The versatility of the synthetic strategy presented in this work was demonstrated by preparing a small library of conjugating glycopolymers that only differ from each other for their relative epitope density were prepared by coclicking of appropriate mixtures of mannopyranoside and galactopyranoside azides to the same polyalkyne scaffold intermediate. Surface plasmon resonance binding studies carried out using recombinant rat mannose-binding lectin (MBL) showed clear and dose-dependent MBL binding to glycopolymer-conjugated BSA. In addition, enzyme-linked immunosorbent assay (ELISA) revealed that the neoglycopolymerprotein materials described in this work possess significantly enhanced capacity to activate complement via the lectin pathway when compared with native unmodified BSA.
Toward fully synthetic, homogeneous glycoproteins: Advances in chemical ligation
Biopolymers, 2010
Traditionally, in the pharma sciences, there has been an unstated but operative bifurcation into small molecules and biologics. Small molecules were seen to be, at the discovery level, in the province of chemistry, based on targets provided through biology. By contrast, "biologics" were seen to arise solely from the province of biology exploiting its accessible replicative mechanisms. Our laboratory has been dedicated to the proposition that explosive advances in chemical synthesis have been such as to render so called "biologics" as being accessible to chemical synthesis. In this paper, we focus particularly on the area of glycopeptides. Chemical synthesis, in principle, offers an advantage, in that it can lead to homogeneous glycopeptides characterized by a single glycoform of the glycosidic domain mounted at a particular amino acid in the polypeptide domain. In support of this defining goal, a variety of new methods have been developed. The key problem addressed is that of ligation. In this paper, we review how insights available from mechanistic organic chemistry have been used to create an imposing framework for the synthesis of structures which would, in an earlier day, have been seen to be strictly in the realm of chemically inaccessible "biologics". We dedicate this paper to the remarkable accomplishments of Professor Steven Kent-particularly, his discovery of NCL, which changed the course of peptide ligations.
Journal of Chromatography A, 2013
In this paper, we report on a novel oriented peptide-N-glycosidase F (PNGase F) immobilization approach onto methacrylate based monolithic support for rapid, reproducible and efficient release of the N-linked carbohydrate moieties from glycoproteins. The glutathione-S-transferase-fusion PNGase F (PNGase F-GST) was expressed in Escherichia coli using regular vector technology. The monolithic pore surface was functionalized with glutathione via a succinimidyl-6-(iodoacetyl-amino)-hexanoate linker and the specific affinity of GST toward glutathione was utilized for the oriented coupling. This novel immobilization procedure was compared with reductive amination technique commonly used for non-oriented enzyme immobilization via primary amine functionalities. Both coupling approaches were compared using enzymatic treatment of several glycoproteins, such as ribonuclease B, fetuin and immunoglobulin G followed by MALDI/MS and CE-LIF analysis of the released glycans. Orientedly immobilized PNGase F via GST-glutathione coupling showed significantly higher activity, remained stable for several months, and allowed rapid release of various types of glycans (high-mannose, core fucosylated, sialylated, etc.) from glycoproteins. Complete protein deglycosylation was obtained as fast as in several seconds when using flow-through immobilized microreactors.