Elizabeth Boeggeman - Academia.edu (original) (raw)

Papers by Elizabeth Boeggeman

Bioconjugate Chemistry, May 1, 2007

The mutant 1,4-galactosyltransferase ( 4Gal-T1), 4Gal-T1-Y289L, in contrast to wild-type 4Gal-T1,... more The mutant 1,4-galactosyltransferase ( 4Gal-T1), 4Gal-T1-Y289L, in contrast to wild-type 4Gal-T1, can transfer GalNAc from the sugar donor UDP-GalNAc to the acceptor, GlcNAc, with efficiency as good as that of galactose from UDP-Gal. Furthermore, the mutant can also transfer a modified sugar, C2 keto galactose, from its UDP derivative to O-GlcNAc modification on proteins that provided a functional handle for developing a highly sensitive chemoenzymatic method for detecting O-GlcNAc post-translational modification on proteins. We report herein that the modified sugar, C2 keto galactose, can be transferred to free GlcNAc residues on N-linked glycoproteins, such as ovalbumin or asialo-agalacto IgG1. The transfer is strictly dependent on the presence of both the mutant enzyme and the ketone derivative of the galactose. Moreover, the PNGase F treatment of the glycoproteins, which cleaves the N-linked oligosaccharide chain, shows that the modified sugar has been transferred to the N-glycan chains of the glycoproteins and not to the protein portion. The application of the mutant galactosyltransferase, 4Gal-T1-Y289L, to produce glycoconjugates carrying sugar moieties with reactive groups, is demonstrated. We envision a broad potential for this technology such as the possibilities to link cargo molecules to glycoproteins, such as monoclonal antibodies, via glycan chains, thereby assisting in the glycotargeting of drugs to the site of action or used as biological probes.

Bioconjugate Chemistry, Nov 1, 2007

Here, we describe a new method for the bioconjugation of a nonglycoprotein with biomolecules. Usi... more Here, we describe a new method for the bioconjugation of a nonglycoprotein with biomolecules. Using polypeptide-R-N-acetylgalactosaminyltransferase II (ppGalNAc-T2), we transfer a C2-modified galactose that has a chemical handle, such as ketone or azide, from its respective UDP-sugars to the Ser/Thr residue(s) of an acceptor polypeptide fused to the nonglycoprotein. The protein with the modified galactose is then coupled to a biomolecule that carries an orthogonal reactive group. As a model system for the nonglycoprotein, we engineered glutathione-Stransferase (GST) protein with a 17-amino-acid-long fusion peptide at the C-terminal end that was expressed as a soluble protein in E. coli. The ppGalNAc-T2 protein, the catalytic domain with the C-terminal lectin domain, was expressed as inclusion bodies in E. coli, and an in Vitro folding method was developed to produce milligram quantities of the active enzyme from a liter of bacterial culture. This ppGalNAc-T2 enzyme transfers from the UDP-sugars not only GalNAc but also C2-modified galactose with a chemical handle to the Ser/Thr residue(s) in the fusion peptide. The chemical handle at the C2 of galactose is used for conjugation and assembly of bionanoparticles and preparation of immuno-liposomes for a targeted drug delivery system. This novel method enables one to glycosylate, using ppGalNAc-T2, the important biological nonglycoproteins, such as single-chain antibodies, growth factors, or bacterial toxins, with an engineered 17-residue peptide sequence at the C-terminus of the molecule, for conjugation and coupling.

Bba Gene Struct Express, 1991

Biochemistry, Nov 1, 2004

Beta-1,4-galactosyltransferase (beta4Gal-T1) in the presence of manganese ion transfers galactose... more Beta-1,4-galactosyltransferase (beta4Gal-T1) in the presence of manganese ion transfers galactose from UDP-galactose (UDP-Gal) to N-acetylglucosamine (GlcNAc) that is either free or linked to an oligosaccharide. Crystallographic studies on bovine beta4Gal-T1 have shown that the primary metal binding site is located in the hinge region of a long flexible loop, which upon Mn(2+) and UDP-Gal binding changes from an open to a closed conformation. This conformational change creates an oligosaccharide binding site in the enzyme. Neither UDP nor UDP analogues efficiently induce these conformational changes in the wild-type enzyme, thereby restricting the structural analysis of the acceptor binding site. The binding of Mn(2+) involves an uncommon coordination to the Sdelta atom of Met344; when it is mutated to His, the mutant M344H, in the presence of Mn(2+) and UDP-hexanolamine, readily changes to a closed conformation, facilitating the structural analysis of the enzyme bound with an oligosaccharide acceptor. Although the mutant M344H loses 98% of its Mn(2+)-dependent activity, it exhibits 25% of its activity in the presence of Mg(2+). The crystal structures of M344H-Gal-T1 in complex with either UDP-Gal.Mn(2+) or UDP-Gal.Mg(2+), determined at 2.3 A resolution, show that the mutant enzyme in these complexes is in a closed conformation, and the coordination stereochemistry of Mg(2+) is quite similar to that of Mn(2+). Although either Mn(2+) or Mg(2+), together with UDP-Gal, binds and changes the conformation of the M344H mutant to the closed one, it is the Mg(2+) complex that engages efficiently in catalyses. Thus, this property enabled us to crystallize the M344H mutant for the first time with the acceptor substrate chitobiose in the presence of UDP-hexanolamine and Mn(2+). The crystal structure determined at 2.3 A resolution reveals that the GlcNAc residue at the nonreducing end of chitobiose makes extensive hydrophobic interactions with the highly conserved Tyr286 residue.

Plos One, 2011

Background: Alpha-lactalbumin (a-LA) is a calcium-bound mammary gland-specific protein that is fo... more Background: Alpha-lactalbumin (a-LA) is a calcium-bound mammary gland-specific protein that is found in milk. This protein is a modulator of b1,4-galactosyltransferase enzyme, changing its acceptor specificity from N-acetyl-glucosamine to glucose, to produce lactose, milk's main carbohydrate. When calcium is removed from a-LA, it adopts a molten globule form, and this form, interestingly, when complexed with oleic acid (OA) acquires tumoricidal activity. Such a complex made from human a-LA (hLA) is known as HAMLET (Human A-lactalbumin Made Lethal to Tumor cells), and its tumoricidal activity has been well established.

Biochem Biophys Res Commun, 2002

Bioconjugate Chemistry, 2009

Studies on wild-type and mutant glycosyltransferases have shown that they can transfer modified s... more Studies on wild-type and mutant glycosyltransferases have shown that they can transfer modified sugars with a versatile chemical handle, such as keto or azido group, that can be used for conjugation chemistry and detection of glycan residues on glycoconjugates. To detect the most prevalent glycan epitope, N-acetyllactosamine (LacNAc (Galβ1-4GalNAcβ)), we have mutated a bovine α1,3-galactosyltransferse (α3Gal-T) 1 enzyme which normally transfers Gal from UDP-Gal to the LacNAc acceptor, to transfer GalNAc or C2-modified galactose from their UDP derivatives. The α3Gal-T enzyme belongs to the α3Gal/GalNAc-T family that includes human blood group A and B glycosyltransferases, which transfer GalNAc and Gal, respectively, to the Gal moiety of the trisaccharide Fucα1-2Galβ1-4GlcNAc. Based on the sequence and structure comparison of these enzymes, we have carried out rational mutation studies on the sugar donor-binding residues in bovine α3Gal-T at positions 280 to 282. A mutation of His280 to Leu/Thr/Ser/Ala or Gly and Ala281 and Ala282 to Gly resulted in the GalNAc transferase activity by the mutant α3Gal-T enzymes to 5-19% of their original Gal-T activity. We show that the mutants 280 SGG 282 and 280 AGG 282 with the highest GalNAc-T activity can also transfer modified sugars such as 2keto-galactose or GalNAz from their respective UDP-sugar derivatives to LacNAc moiety present at the nonreducing end of glycans of asialofetuin, thus enabling the detection of LacNAc moiety of glycoproteins and glycolipids by a chemiluminescence method.

Protein Engineering, 1993

Biochemistry Usa, 2004

Methods in molecular biology (Clifton, N.J.), 2011

This chapter presents a technique that employs mutant glycosyltransferase enzymes for the site-sp... more This chapter presents a technique that employs mutant glycosyltransferase enzymes for the site-specific bioconjugation of biomolecules via a glycan moiety to facilitate the development of a targeted drug delivery system. The target specificity of this methodology is based on unique sugar residues that are present on glycoproteins or engineered glycopeptides. The glycosyltransferases used in this approach have been manipulated in a way that confers the ability to transfer a modified sugar residue with a chemical handle to a sugar moiety of the glycoprotein or to a polypeptide tag of an engineered nonglycoprotein. The availability of the modified sugar moiety thus makes it possible to link cargo molecules at specific sites. The cargo may be comprised of, for example, biotin or fluorescent tags for detection, imaging agents for magnetic resonance imaging (MRI), or cytotoxic drugs for cancer therapy.

Current drug targets, 2008

Beta-1,4-galactosylransferase (beta4Gal-T1) participates in the synthesis of Galbeta1-4-GlcNAc-di... more Beta-1,4-galactosylransferase (beta4Gal-T1) participates in the synthesis of Galbeta1-4-GlcNAc-disaccharide unit of glycoconjugates. It is a trans-Golgi glycosyltransferase (Glyco-T) with a type II membrane protein topology, a short N-terminal cytoplasmic domain, a membrane-spanning region, as well as a stem and a C-terminal catalytic domain facing the trans-Golgi-lumen. Its hydrophobic membrane-spanning region, like that of other Glyco-T, has a shorter length compared to plasma membrane proteins, an important feature for its retention in the trans-Golgi. The catalytic domain has two flexible loops, a long and a small one. The primary metal binding site is located at the N-terminal hinge region of the long flexible loop. Upon binding of metal ion and sugar-nucleotide, the flexible loops undergo a marked conformational change, from an open to a closed conformation. Conformational change simultaneously creates at the C-terminal region of the flexible loop an oligosaccharide acceptor b...

Protein expression and purification, 2003

Many recombinant proteins overexpressed in Escherichia coli are generally misfolded, which then a... more Many recombinant proteins overexpressed in Escherichia coli are generally misfolded, which then aggregate and accumulate as inclusion bodies. The catalytic domain (CD) of bovine and human beta1,4-galactosyltransferase (beta4Gal-T), expressed in E. coli, it also accumulates as inclusion bodies. We studied the effect of the fusion of the stem region (SR), as an N-terminal extension of the catalytic domain, on the in vitro folding efficiencies of the inclusion bodies. The stem region fused to the catalytic domain (SRCD) increases the folding efficiency of recombinant protein with native fold compared to the protein that contains only the CD. During in vitro folding, also promotes considerably the solubility of the misfolded proteins, which do not bind to UDP-agarose columns and exhibit no galactosyltransferase activity. In contrast, the misfolded proteins that consist of only the CD are insoluble and precipitate out of solution. It is concluded that a protein domain that is produced in...

Trends in Biochemical Sciences, 2005