Pradman K. Qasba

Alkaline transfer of DNA to plastic membrane.

DNA forms a stable complex with Gene Screen Plus membrane at alkaline pH. Based on this, a method of alkaline transfer of DNA from agarose gel to Gene Screen Plus membrane was elaborated. The procedure entails the use of 0.4 M NaOH for both, the DNA denaturation and DNA transfer steps. The alkaline transfer offers a higher hybridization efficiency and simplifies the transfer procedure as compared with the standard method of DNA transfer at neutral pH. In addition, it can be used to remove RNA contamination from the transferred DNA.

Structure-based design of beta 1,4-galactosyltransferase I (beta 4Gal-T1) with equally efficient N-acetylgalactosaminyltransferase activity: point mutation broadens beta 4Gal-T1 donor specificity.

β1,4-Galactosyltransferase I (Gal-T1) normally transfers Gal from UDP-Gal to GlcNAc in the presence of Mn2+ ion. In the presence of α-lactalbumin (LA), the Gal acceptor specificity is altered from GlcNAc to Glc. Gal-T1 also transfers GalNAc from UDP-GalNAc to GlcNAc, but with only ∼0.1% of Gal-T activity. To understand this low GalNAc-transferase activity, we have carried out the crystal structure analysis of the Gal-T1·LA complex with UDP-GalNAc at 2.1-Å resolution. The crystal structure reveals that the UDP-GalNAc binding to Gal-T1 is similar to the binding of UDP-Gal to Gal-T1, except for an additional hydrogen bond formed between the N-acetyl group of GalNAc moiety with the Tyr-289 side chain hydroxyl group. Elimination of this additional hydrogen bond by mutating Tyr-289 residue to Leu, Ile, or Asn enhances the GalNAc-transferase activity. Although all three mutants exhibit enhanced GalNAc-transferase activity, the mutant Y289L exhibits GalNAc-transferase activity that is nearly 100% of its Gal-T activity, even while completely retaining its Gal-T activity. The steady state kinetic analyses on the Leu-289 mutant indicate that theK m for GlcNAc has increased compared to the wild type. On the other hand, the catalytic constant (k cat) in the Gal-T reaction is comparable with the wild type, whereas it is 3–5-fold higher in the GalNAc-T reaction. Interestingly, in the presence of LA, these mutants also transfer GalNAc to Glc instead of to GlcNAc. The present study demonstrates that, in the Gal-T family, the Tyr-289/Phe-289 residue largely determines the sugar donor specificity.

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.

Alpha-Lactalbumin (La) Stimulates Milk Beta-1,4-Galactosyltransferase I (Beta 4Gal-T1) to Transfer Glucose from Udp-Glucose to N-Acetylglucosamine. Crystal Structure of Beta 4Gal-T1 X La Complex with Udp-Glc.

β-1,4-Galactosyltransferase 1 (Gal-T1) transfers galactose (Gal) from UDP-Gal toN-acetylglucosamine (GlcNAc), which constitutes its normal galactosyltransferase (Gal-T) activity. In the presence of α-lactalbumin (LA), it transfers Gal to Glc, which is its lactose synthase (LS) activity. It also transfers glucose (Glc) from UDP-Glc to GlcNAc, constituting the glucosyltransferase (Glc-T) activity, albeit at an efficiency of only 0.3–0.4% of Gal-T activity. In the present study, we show that LA increases this activity almost 30-fold. It also enhances the Glc-T activity toward various N-acyl substituted glucosamine acceptors. Steady state kinetic studies of Glc-T reaction show that the K m for the donor and acceptor substrates are high in the absence of LA. In the presence of LA, the K m for the acceptor substrate is reduced 30-fold, whereas for UDP-Glc it is reduced only 5-fold. In order to understand this property, we have determined the crystal structures of the Gal-T1·LA complex with UDP-Glc·Mn2+ and withN-butanoyl-glucosamine (N-butanoyl-GlcN), a preferred sugar acceptor in the Glc-T activity. The crystal structures reveal that although the binding of UDP-Glc is quite similar to UDP-Gal, there are few significant differences observed in the hydrogen bonding interactions between UDP-Glc and Gal-T1. Based on the present kinetic and crystal structural studies, a possible explanation for the role of LA in the Glc-T activity has been proposed.

Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: Application for cell surface antigen detection

The Fc N-glycan chains of four therapeutic monoclonal antibodies (mAbs), namely, Avastin, Rituxan, Remicade, and Herceptin, released by PNGase F, show by MALDI analysis that these biantennary N-glycans are a mixture of G0, G1, and G2 glycoforms. The G0 glycoform has no galactose on the terminal GlcNAc residues, and the G1 and G2 glycoforms have one or two terminal galactose residues, respectively, while no N-glycan with terminal sialic acid residue is observed. We show here that under native conditions we can convert the N-glycans of these mAbs to a homogeneous population of G0 glycoform using beta1,4 galactosidase from Streptococcus pneumoniae. The G0 glycoforms of mAbs can be galactosylated with a modified galactose having a chemical handle at the C2 position, such as ketone or azide, using a mutant beta1,4-galactosyltransferase (beta1,4Gal-T1-Y289L). The addition of the modified galactose at a specific glycan residue of a mAb permits the coupling of a biomolecule that carries an orthogonal reactive group. The linking of a biotinylated or a fluorescent dye carrying derivatives selectively occurs with the modified galactose, C2-keto-Gal, at the heavy chain of these mAbs, without altering their antigen binding activities, as shown by indirect enzyme linked immunosorbent assay (ELISA) and fluorescence activated cell sorting (FACS) methods. Our results demonstrate that the linking of cargo molecules to mAbs via glycans could prove to be an invaluable tool for potential drug targeting by immunotherapeutic methods.

Structure and Function of β-1,4-Galactosyltransferase

Beta-1,4-galactosylransferase (β4Gal-T1) participates in the synthesis of Galβ1-4-GlcNAc-disaccharide unit of glycoconjugates. It is a trans-Golgi glycosyltransferase (Glyco-T) with a type II membrane protein topology, a short Nterminal 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 binding site that did not exist before. The loop acts as a lid covering the bound donor substrate. After completion of the transfer of the glycosyl unit to the acceptor, the saccharide product is ejected; the loop reverts to its native conformation to release the remaining nucleotide moiety. The conformational change in β4Gal-T1 also creates the binding site for a mammary gland-specific protein, α-lactalbumin (LA), which changes the acceptor specificity of the enzyme toward glucose to synthesize lactose during lactation. The specificity of the sugar donor is generally determined by a few residues in the sugar-nucleotide binding pocket of Glyco-T, conserved among the family members from different species. Mutation of these residues has allowed us to design new and novel glycosyltransferases, with broader or requisite donor and acceptor specificities, and to synthesize specific complex carbohydrates as well as specific inhibitors for these enzymes.

Transcriptional and post-transcriptional roles of glucocorticoid in the expression of the rat 25,000 molecular weight casein gene.

In the presence of hydrocortisone, plus insulin and prolactin, rat mammary explants transcribe the 25K casein gene, and the half-life of the transcript is about 20 h. The first detectable effect of the withdrawal of glucocorticoid is reduction of the half-life of the casein mRNA to about 1 h, with full retention of the rate of transcription of the casein gene. A later effect of the withdrawal of glucocorticoid is loss of the ability to transcribe the casein gene. Both aberrations can be rectified by hydrocortisone in vitro. The instability of the 25K casein mRNA in the absence of glucocorticoid appears to be related to degradation in the nucleus. Accumulation of actin mRNA in the nuclear fraction is not dependent on glucocorticoid. The results indicate that glucocorticoid exerts a profound, selective effect on the stabilization of rat 25K casein mRNA, and is essential for the transcription of the casein gene. These effects can be dissociated temporally from one another.

Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar

Conjugation of small molecule drugs to specific sites on the antibody molecule has been increasingly used for the generation of relatively homogenous preparations of antibody-drug conjugates (ADCs) with physicochemical properties similar or identical to those of the naked antibody. Previously a method for conjugation of small molecules to glycoproteins through existing glycans by using an engineered glycotransferase and a chemically reactive sugar as a handle was developed. Here, for the first time, we report the use of this method with some modifications to generate an ADC from a monoclonal antibody, m860, which we identified from a human naïve phage display Fab library by panning against the extracellular domain of human HER2. M860 bound to cell surface-associated HER2 with affinity comparable to that of Trastuzumab (Herceptin®), but to a different epitope. The m860ADC was generated by enzymatically adding a reactive keto-galactose to m860 using an engineered glycotransferase and conjugating the reactive m860 to aminooxy auristatin F. It exhibited potent and specific cell-killing activity against HER2 positive cancer cells, including trastuzumab-resistant breast cancer cells. This unique ADC may have utility as a potential therapeutic for HER2 positive cancers alone or in combination with other drugs. Our results also validate the keto-galactose/engineered glycotransferase method for generation of functional ADCs, which could potentially also be used for preparation of ADCs targeting other disease markers.

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.

Deoxygenated Disaccharide Analogs as Specific Inhibitors of β1–4-Galactosyltransferase 1 and Selectin-mediated Tumor Metastasis

The disaccharide peracetylated GlcNAcβ1–3Galβ-O-naphthalenemethanol (disaccharide 1) diminishes the formation of the glycan sialyl Lewis X (Neu5Acα2–3Galβ1–4(Fucα1–3) GlcNAc; sLeX) in tumor cells. Previous studies showed that the mechanism of action of disaccharide 1 involves three steps: (i) deacetylation by carboxyesterases, (ii) action as a biosynthetic intermediate for downstream enzymes involved in sLeX assembly, and (iii) generation of several glycans related to sLeX. In this report, we show that GlcNAcβ1–3Galβ-O-naphthalenemethanol binds to the acceptor site of human β1–4-galactosyltransferase much like the acceptor trisaccharide, GlcNAcβ1–2Manβ1–6Man, which is present on N-linked glycans. The 4′-deoxy analog, in which the acceptor hydroxyl group was replaced by -H, did not act as a substrate but instead acted as a competitive inhibitor of the enzyme. The acetylated form of this compound inhibited sLeX formation in U937 monocytic leukemia cells, suggesting that it had inhibitory activity in vivo as well. A series of synthetic acetylated analogs of 1 containing -H, -F, -N3, -NH2, or -OCH3 instead of the hydroxyl groups at C-3′- and C-4′-positions of the terminal N-acetylglucosamine residue also blocked sLeX formation in cells. The reduction of sLeX by the 4′-deoxy analog also diminished experimental tumor metastasis by Lewis lung carcinoma in vivo. These data suggest that nonsubstrate disaccharides have therapeutic potential through their ability to bind to glycosyltransferases in vivo and to alter glycan-dependent pathologic processes.