In Vitro Glycosylation of Membrane Proteins Using N-Glycosyltransferase

Abstract

Glycoproteins are post-translationally modified proteins that take part in nearly every biological process and make up a large percent of the proteome. N-Linked glycosylation can be performed by N -glycosyltransferase (NGT), which recognizes the consensus amino acid sequence, -Asn-X-Ser/Thr- (NXT), within the protein. The enzyme catalyzes glycosidic bond formation between the oligosaccharide donor, containing nucleoside phosphatase, and the amide nitrogen of the asparagine residue. The attachment of the sugar moiety can influence physiological and biological properties of the protein by affecting their folding, modulating interactions with other biomolecules, and modifying their functions at the cellular level. We are specifically interested in the properties of membrane glycoproteins, which are key components in a number of different disease states. Therefore, the use of in vitro protein glycosylation can help further evaluate the effects of the properties for these important macromolecules. In vitro studies of N-linked glycosylation were done in a stepwise fashion in a membrane-mimetic environment to confirm that the methods for glycosylating soluble proteins could be applicable to membrane proteins. Detergent and lipid systems were used since hydrophobic peptides and membrane proteins are insoluble in aqueous solvents. The stepwise method consisted of the glycosylation of a soluble 7-residue peptide, a hydrophobic WALP-NVT peptide, and a γ-sarcoglycan membrane protein, all of which contained the glycosylation site Asn-Val-Thr (NVT). Glycosylation of the samples was performed using Escherichia coli -expressed NGT from the Actinobacillus pleuropneumoniae genome, and a single sugar moiety of glucose, provided from a nucleotide-linked donor, was added to the glycosylation site. Gel electrophoresis, mass spectrometry, and NMR studies were used for the detection of glycosyltransferase activity and to show the attachment of a single glucose molecule. Our experiments demonstrated that small or large membrane proteins that contain an N-glycosylation consensus sequence can be glycosylated by NGT in membrane-mimetic environments.

Lectin-Modified Magnetic Nano-PLGA for Photodynamic Therapy In Vivo

“…Since breast cancer cells overexpressed more mannose N-glycan units on the membrane proteins than that of normal cells, the binding of mPLGA/IR775-ConA occurred due to the interaction of ConA with mannose residues on several proteins types in cancer cells. N-glycosylation, the sequential addition of complex sugars to adhesion proteins, ion channels, and receptors, is one of the most frequent protein glycosylation processes, leading to the fact that ConA-based nanoparticles interact both with internalizing and non-internalizing proteins on the cell membrane, resulting in the nanoparticle accumulation in different cell compartments, resting on the cell membrane, localizing in endosomes and in the cytoplasm [ 43 , 44 , 45 ]. Maybe this feature is one of the most interesting aspects of lectin-modified nanoparticles’ interaction with cancer cells gathering the attention of researchers, since this fact allows for the affecting several types of cell compartments simultaneously, which is very important on the road to the development of multifunctional cancer-fighting strategies, especially those based on PDT.…”

In Vitro Glycosylation of the Membrane Protein γ-Sarcoglycan in Nanodiscs

“…The expression and purification of the glycosylation enzyme N -glycosyltransferase (NGT) can be found in the manuscript by Ahangama Liyanage et al Briefly, the enzyme NGT was recombinantly expressed in BL21 E. coli as a histidine-tagged protein.…”

“…Expression and Purification of N-Glycosyltransferase. The expression and purification of the glycosylation enzyme Nglycosyltransferase (NGT) can be found in the manuscript by Ahangama Liyanage et al 10 Briefly, the enzyme NGT was recombinantly expressed in BL21 E. coli as a histidine-tagged protein. Cells were harvested after 4 h of induction by Larabinose.…”

“…which are most commonly expressed through recombinant bacterial approaches or cell-free systems, neither of which produce protein that is glycosylated. Previously, several groups had shown that recombinantly expressed and purified soluble proteins could be glycosylated in vitro using recombinantly expressed N -glycosyltransferases. − Using similar methods, our group has previously shown that it is also possible to glycosylate membrane proteins in detergent and lipid micelle environments using an N-glycosyltransferase from Actinobacillus pleuropneumoniae (ApNGT) . The enzyme recognizes the consensus sequence N-(X)-T in which X is any amino acid besides proline and attaches the sugar to the side chain of the asparagine residue.…”

Investigation of the Catalytic Mechanism of a Soluble N-glycosyltransferase Allows Synthesis of N-glycans at Noncanonical Sequons

“…Additionally, a sequential glycosylation strategy was developed to install distinct pseudo-N-glycoforms at the specific sites within one protein by combination of ApNGT and endo -β- N -acetylglucosaminidase (ENGase) mutants . Notably, ApNGT is unable to transfer a GlcNAc moiety and therefore fails to create a natural GlcNAc–Asn linkage. ,, Despite that, the NGT-mediated N-glycosylation system has attracted growing attention for N-glycoprotein/glycopeptide production in vitro and in vivo . − …”