Boopathy Ramakrishnan

Crystal Structure of β1,4-Galactosyltransferase Complex with UDP-Gal Reveals an Oligosaccharide Acceptor Binding Site

The crystal structure of the catalytic domain of bovine beta1,4-galactosyltransferase (Gal-T1) co-crystallized with UDP-Gal and MnCl(2) has been solved at 2.8 A resolution. The structure not only identifies galactose, the donor sugar binding site in Gal-T1, but also reveals an oligosaccharide acceptor binding site. The galactose moiety of UDP-Gal is found deep inside the catalytic pocket, interacting with Asp252, Gly292, Gly315, Glu317 and Asp318 residues. Compared to the native crystal structure reported earlier, the present UDP-Gal bound structure exhibits a large conformational change in residues 345-365 and a change in the side-chain orientation of Trp314. Thus, the binding of UDP-Gal induces a conformational change in Gal-T1, which not only creates the acceptor binding pocket for N-acetylglucosamine (GlcNAc) but also establishes the binding site for an extended sugar acceptor. The presence of a binding site that accommodates an extended sugar offers an explanation for the observation that an oligosaccharide with GlcNAc at the non-reducing end serves as a better acceptor than the monosaccharide, GlcNAc. Modeling studies using oligosaccharide acceptors indicate that a pentasaccharide, such as N-glycans with GlcNAc at their non-reducing ends, fits the site best. A sequence comparison of the human Gal-T family members indicates that although the binding site for the GlcNAc residue is highly conserved, the site that binds the extended sugar exhibits large variations. This is an indication that different Gal-T family members prefer different types of glycan acceptors with GlcNAc at their non-reducing ends.

Iterative cluster-NMA: A tool for generating conformational transitions in proteins

Computational models provide insight into the structure–function relationship in proteins. These approaches, especially those based on normal mode analysis, can identify the accessible motion space around a given equilibrium structure. The large magnitude, collective motions identified by these methods are often well aligned with the general direction of the expected conformational transitions. However, these motions cannot realistically be extrapolated beyond the local neighborhood of the starting conformation. In this article, the iterative cluster‐NMA (icNMA) method is presented for traversing the energy landscape from a starting conformation to a desired goal conformation. This is accomplished by allowing the evolving geometry of the intermediate structures to define the local accessible motion space, and thus produce an appropriate displacement. Following the derivation of the icNMA method, a set of sample simulations are performed to probe the robustness of the model. A detailed analysis of β1,4‐galactosyltransferase‐T1 is also given, to highlight many of the capabilities of icNMA. Remarkably, during the transition, a helix is seen to be extended by an additional turn, emphasizing a new unknown role for secondary structures to absorb slack during transitions. The transition pathway for adenylate kinase, which has been frequently studied in the literature, is also discussed. Proteins 2009.

The N-terminal stem region of bovine and human β1,4-galactosyltransferase I increases the in vitro folding efficiency of their catalytic domain from inclusion bodies

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 a soluble form does not guarantee the presence of the protein molecules in a properly folded and active form. The stem domain has a positive effect on the in vitro folding efficiency of the catalytic domain of both human and bovine beta4Gal-T1, suggesting that the stem region acts as a chaperone during protein folding. Furthermore, investigation of the folding conditions of the sulphonated inclusion bodies resulted in identifying a condition in which the presence of PEG-4000 and L-arginine, compared to their absence, increased the yields of native CD and SRCD 7- and 3-fold, respectively.

Site‐Specific Linking of Biomolecules via Glycan Residues Using Glycosyltransferases

The structural information on glycosyltransferases has revealed that the sugar‐donor specificity of these enzymes can be broadened to include modified sugars with a chemical handle that can be utilized for conjugation chemistry. Substitution of Tyr289 to Leu in the catalytic pocket of bovine β‐1,4‐galactosyltransferase generates a novel glycosyltransferase that can transfer not only Gal but also GalNAc or a C2‐modified galactose that has a chemical handle, from the corresponding UDP‐derivatives, to the non‐reducing end GlcNAc residue of a glycoconjugate. Similarly, the wild‐type polypeptide‐ N‐acetyl‐galactosaminyltransferase, which naturally transfers GalNAc from UDP‐GalNAc, can also transfer C2‐modified galactose with a chemical handle from its UDP‐derivative to the Ser/Thr residue of a polypeptide acceptor substrate that is tagged as a fusion peptide to a non‐glycoprotein. The potential of wild‐type and mutant glycosyltransferases to produce glycoconjugates carrying sugar moieties with chemical handle makes it possible to conjugate biomolecules with orthogonal reacting groups at specific sites. This methodology assists in the assembly of bio‐nanoparticles that are useful for developing targeted drug‐delivery systems and contrast agents for magnetic resonance imaging.

The role of tryptophan 314 in the conformational changes of β1,4-galactosyltransferase-I

beta1,4-Galactosyltransferase-I (beta4Gal-T1) undergoes critical conformational changes upon substrate binding from an open conformation (conf-I) to the closed conformation (conf-II). This change involves two flexible loops: the small (residues 313-316) and the long loop (residues 345-365). Upon substrate binding, Trp314 in the small flexible loop moves towards the catalytic pocket and interacts with the donor and the acceptor substrates. For a better understanding of the role played by Trp314 in the conformational changes of beta4Gal-T1, we mutated it to Ala and carried out substrate-binding, proteolytic and crystallographic studies. The W314A mutation reduces the enzymatic activity, binding to substrates and to the modifier protein, alpha-lactalbumin (LA), by over 99%. The limited proteolysis with Glu-C or Lys-C proteases shows differences in the rate of cleavage of the long loop of the wild-type and mutant W314A, indicating conformational differences in the region between the two proteins. Without substrate, the mutant crystallizes in a conformation (conf-I) (1.9A resolution crystal structure), that is not identical with, but close to an open conformation (conf-I), whereas its complex with the substrates and alpha-lactalbumin, crystallizes in a conformation (2.3A resolution crystal structure) that is identical with the closed conformation (conf-II). This study shows the crucial role Trp314 plays in the conformational state of the long loop, in the binding of substrates and in the catalytic mechanism of the enzyme.

Applications of site-specific labeling to study HAMLET, a tumoricidal complex of α-lactalbumin and oleic acid.

Background Alpha-lactalbumin (α-LA) is a calcium-bound mammary gland-specific protein that is found in milk. This protein is a modulator of β1,4-galactosyltransferase enzyme, changing its acceptor specificity from N-acetyl-glucosamine to glucose, to produce lactose, milks main carbohydrate. When calcium is removed from α-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 α-LA (hLA) is known as HAMLET (Human A-lactalbumin Made Lethal to Tumor cells), and its tumoricidal activity has been well established. Methodology/Principal Findings In the present work, we have used site-specific labeling, a technique previously developed in our laboratory, to label HAMLET with biotin, or a fluoroprobe for confocal microscopy studies. In addition to full length hLA, the α-domain of hLA (αD-hLA) alone is also included in the present study. We have engineered these proteins with a 17–amino acid C-terminal extension (hLA-ext and αD-hLA-ext). A single Thr residue in this extension is glycosylated with 2-acetonyl-galactose (C2-keto-galactose) using polypeptide-α-N-acetylgalactosaminyltransferase II (ppGalNAc-T2) and further conjugated with aminooxy-derivatives of fluoroprobe or biotin molecules. Conclusions/Significance We found that the molten globule form of hLA and αD-hLA proteins, with or without C-terminal extension, and with and without the conjugated fluoroprobe or biotin molecule, readily form a complex with OA and exhibits tumoricidal activity similar to HAMLET made with full-length hLA protein. The confocal microscopy studies with fluoroprobe-labeled samples show that these proteins are internalized into the cells and found even in the nucleus only when they are complexed with OA. The HAMLET conjugated with a single biotin molecule will be a useful tool to identify the cellular components that are involved with it in the tumoricidal activity.

Galectin-1 as a fusion partner for the production of soluble and folded human β-1,4-galactosyltransferase-T7 in E. coli

The expression of recombinant proteins in Escherichia coli often leads to inactive aggregated proteins known as the inclusion bodies. To date, the best available tool has been the use of fusion tags, including the carbohydrate-binding protein; e.g., the maltose-binding protein (MBP) that enhances the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. We hypothesized that galectins, which are also carbohydrate-binding proteins, may help as fusion partners in folding the mammalian proteins in E. coli. Here we show for the first time that a small soluble lectin, human galectin-1, one member of a large galectin family, can function as a fusion partner to produce soluble folded recombinant human glycosyltransferase, beta-1,4-galactosyltransferase-7 (beta4Gal-T7), in E. coli. The enzyme beta4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, beta4Gal-T7 is expressed in E. coli as inclusion bodies. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for human galectin-1, followed by the Tev protease cleavage site, a 6x His-coding sequence, and a multi-cloning site where a cloned gene is inserted. After lactose affinity column purification of galectin-1-beta4Gal-T7 fusion protein, the unique protease cleavage site allows the protein beta4Gal-T7 to be cleaved from galectin-1 that binds and elutes from UDP-agarose column. The eluted protein is enzymatically active, and shows CD spectra comparable to the folded beta4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.

Mutant glycosyltransferases assist in the development of a targeted drug delivery system and contrast agents for MRI

The availability of structural information on glycosyltransferases is beginning to make structure-based reengineering of these enzymes possible. Mutant glycosyltransferases have been generated that can transfer a sugar residue with a chemically reactive unique functional group to a sugar moiety of glycoproteins, glycolipids, and proteoglycans (glyco-conjugates). The presence of modified sugar moiety on a glycoprotein makes it possible to link bioactive molecules via modified glycan chains, thereby assisting in the assembly of bionanoparticles that are useful for developing the targeted drug delivery system and contrast agents for magnetic resonance imaging. The reengineered recombinant glycosyltransferases also make it possible to (1) remodel the oligosaccharide chains of glycoprotein drugs, and (2) synthesize oligosaccharides for vaccine development.

Structure-based evolutionary relationship of glycosyltransferases: a case study of vertebrate β1,4-galactosyltransferase, invertebrate β1,4-N-acetylgalactosaminyltransferase and α-polypeptidyl-N-acetylgalactosaminyltransferase.

Cell surface glycans play important cellular functions and are synthesized by glycosyltransferases. Structure and function studies show that the donor sugar specificity of the invertebrate β1,4-N-acetyl-glactosaminyltransferase (β4GalNAc-T) and the vertebrate β1,4-galactosyltransferase I (β4Gal-T1) are related by a single amino acid residue change. Comparison of the catalytic domain crystal structures of the β4Gal-T1 and the α-polypeptidyl-GalNAc-T (αppGalNAc-T) shows that their protein structure and sequences are similar. Therefore, it seems that the invertebrate β4GalNAc-T and the catalytic domain of αppGalNAc-T might have emerged from a common primordial gene. When vertebrates emerged from invertebrates, the amino acid that determines the donor sugar specificity of the invertebrate β4GalNAc-T might have mutated, thus converting the enzyme to a β4Gal-T1 in vertebrates.

Applications of glycosyltransferases in the site-specific conjugation of biomolecules and the development of a targeted drug delivery system and contrast agents for MRI.

Background: The delivery of drugs to the proposed site of action is a challenging task. Tissue and cell-specific guiding molecules are being used to carry a cargo of therapeutic molecules. The cargo molecules need to be conjugated in a site-specific manner to the therapeutic molecules such that the bioefficacy of these molecules is not compromised. Methods: Using wild-type and mutant glycosyltransferases, the sugar moiety with a unique chemical handle is incorporated at a specific site in the cargo or therapeutic molecules, making it possible to conjugate these molecules through the chemical handle present on the modified glycan. Results/conclusions: The modified glycan residues introduced at specific sites on the cargo molecule make it possible to conjugate fluorophores for ELISA-based assays, radionuclides for imaging and immunotherapy applications, lipids for the assembly of immunoliposomes, cytotoxic drugs, cytokines, or toxins for antibody-based cancer therapy and the development of a targeted drug delivery system.