Non-reducing end labeling of heparan sulfate via click chemistry and a high throughput ELISA assay for heparanase.

Heparan sulfate (HS) is a linear polysaccharide found in the extracellular matrix (ECM) and on the cell membrane. It plays numerous roles in cellular events, including cell growth, migration and differentiation through binding to various growth factors, cytokines and other ECM proteins. Heparanase (HPSE) is an endoglycosidase that cleaves HS in the ECM and cell membrane. By degrading HS, HPSE not only alters the integrity of the ECM but also releases growth factors and angiogenic factors bound to HS chains, therefore, changes various cellular activities, including cell mobility that is critical for cancer metastasis. Accordingly, HPSE is an ideal drug target for cancer therapeutics. Here, we describe a method for non-reducing end labeling of HS via click chemistry (CC), and further use it in a novel HPSE assay. HS chains on a recombinant human syndecan-4 are first labeled at their non-reducing ends with GlcNAz using dimeric HS polymerase EXT1/EXT2. The labeled sample is then biotinylated through CC, immobilized on a multi-well plate and detected with ELISA. HPSE digestion of the biotinylated sample removes the label and, therefore, reduces the signal in ELISA assay. Non-reducing end labeling avoids the interference in an HPSE reaction caused by any internal labeling of HS. The assay is very sensitive with only 2.5 ng of labeled syndecan-4 needed in each reaction. The assay is also highly reproducible with a Z' > 0.6. Overall, this new method is suitable for high-throughput drug screening on HPSE.

Galectin CvGal2 from the Eastern Oyster (Crassostrea virginica) Displays Unique Specificity for ABH Blood Group Oligosaccharides and Differentially Recognizes Sympatric Perkinsus Species.

Galectins are highly conserved lectins that are key to multiple biological functions, including pathogen recognition and regulation of immune responses. We previously reported that CvGal1, a galectin expressed in phagocytic cells (hemocytes) of the eastern oyster (Crassostrea virginica), is hijacked by the parasite Perkinsus marinus to enter the host, where it causes systemic infection and death. Screening of an oyster hemocyte cDNA library revealed a novel galectin, which we designated CvGal2, with four tandemly arrayed carbohydrate recognition domains (CRDs). Phylogentic analysis of the CvGal2 CRDs suggests close relationships with homologous CRDs from CvGal1. Glycan array analysis, however, revealed that, unlike CvGal1 which preferentially binds to the blood group A tetrasaccharide, CvGal2 recognizes both blood group A and B tetrasaccharides and related structures, suggesting that CvGal2 has broader binding specificity. Furthermore, SPR analysis demonstrated significant differences in the binding kinetics of CvGal1 and CvGal2, and structural modeling revealed substantial differences in their interactions with the oligosaccharide ligands. CvGal2 is homogeneously distributed in the hemocyte cytoplasm, is released to the extracellular space, and binds to the hemocyte surface. CvGal2 binds to P. marinus trophozoites in a dose-dependent and ß-galactoside-specific manner. Strikingly, negligible binding of CvGal2 was observed for Perkinsus chesapeaki, a sympatric parasite species mostly prevalent in the clams Mya arenaria and Macoma balthica. The differential recognition of Perkinsus species by the oyster galectins is consistent with their relative prevalence in oyster and clam species and supports their role in facilitating parasite entry and infectivity in a host-preferential manner.

Galectins as self/non-self recognition receptors in innate and adaptive immunity: an unresolved paradox.

Galectins are characterized by their binding affinity for ß-galactosides, a unique binding site sequence motif, and wide taxonomic distribution and structural conservation in vertebrates, invertebrates, protista, and fungi. Since their initial description, galectins were considered to bind endogenous ("self") glycans and mediate developmental processes and cancer. In the past few years, however, numerous studies have described the diverse effects of galectins on cells involved in both innate and adaptive immune responses, and the mechanistic aspects of their regulatory roles in immune homeostasis. More recently, however, evidence has accumulated to suggest that galectins also bind exogenous ("non-self") glycans on the surface of potentially pathogenic microbes, parasites, and fungi, suggesting that galectins can function as pattern recognition receptors (PRRs) in innate immunity. Thus, a perplexing paradox arises by the fact that galectins also recognize lactosamine-containing glycans on the host cell surface during developmental processes and regulation of immune responses. According to the currently accepted model for non-self recognition, PRRs recognize pathogens via highly conserved microbial surface molecules of wide distribution such as LPS or peptidoglycan (pathogen-associated molecular patterns; PAMPs), which are absent in the host. Hence, this would not apply to galectins, which apparently bind similar self/non-self molecular patterns on host and microbial cells. This paradox underscores first, an oversimplification in the use of the PRR/PAMP terminology. Second, and most importantly, it reveals significant gaps in our knowledge about the diversity of the host galectin repertoire, and the subcellular targeting, localization, and secretion. Furthermore, our knowledge about the structural and biophysical aspects of their interactions with the host and microbial carbohydrate moieties is fragmentary, and warrants further investigation.

The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2.

In recent years, sugars with a unique chemical handle have been used to detect and elucidate the function of glycoconjugates. Such chemical handles have generally been part of an N-acetyl moiety of a sugar. We have previously developed several applications using the single mutant Y289L-ß1,4-galactosyltransferase I (Y289L-ß4Gal-T1) and the wild-type polypeptide-α-GalNAc-T enzymes with UDP-C2-keto-Gal. Here, we describe for the first time that the GlcNAc-transferring enzymes-R228K-Y289L-ß4Gal-T1 mutant enzyme, the wild-type human ß1,3-N-acetylglucosaminyltransferase-2 and human Maniac Fringe-can also transfer the GlcNAc analog C2-keto-Glc molecule from UDP-C2-keto-Glc to their respective acceptor substrates. Although the R228K-Y289L-ß4Gal-T1 mutant enzyme transfers the donor sugar substrate GlcNAc or its analog C2-keto-Glc only to its natural acceptor substrate, GlcNAc, it does not transfer to its analog C2-keto-Glc. Thus, these observations suggest that the GlcNAc-transferring glycosyltransferases can generally accommodate a chemical handle in the N-acetyl-binding cavity of the donor sugar substrate, but not in the N-acetyl-binding cavity of the acceptor sugar.

Bioconjugation using mutant glycosyltransferases for the site-specific labeling of biomolecules with sugars carrying chemical handles.

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.

Galectin-1 as a fusion partner for the production of soluble and folded human beta-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.

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.

Bioconjugation and detection of lactosamine moiety using alpha1,3-galactosyltransferase mutants that transfer C2-modified galactose with a chemical handle.

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 (Galbeta1-4GalNAcbeta)), we have mutated a bovine alpha1,3-galactosyltransferse (alpha3Gal-T)() enzyme which normally transfers Gal from UDP-Gal to the LacNAc acceptor, to transfer GalNAc or C2-modified galactose from their UDP derivatives. The alpha3Gal-T enzyme belongs to the alpha3Gal/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 Fucalpha1-2Galbeta1-4GlcNAc. On the basis of the sequence and structure comparison of these enzymes, we have carried out rational mutation studies on the sugar donor-binding residues in bovine alpha3Gal-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 alpha3Gal-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 2-keto-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.

Galactosylation of IgG from rheumatoid arthritis (RA) patients--changes during therapy.

It is well documented that serum IgG from rheumatoid arthritis (RA) patients exhibits decreased galactosylation of its conservative N-glycans (Asn-297) in CH2 domains of the heavy chains; it has been shown that this agalactosylation is proportional to disease severity. In the present investigation we analyzed galactosylation of IgG derived from the patients using a modified ELISA-plate test, biosensor BIAcore and total sugar analysis (GC-MS). For ELISA and BIAcore the binding of IgG preparations, purified from the patients' sera, to two lectins: Ricinus communis (RCA-I) and Griffonia simplicifolia (GSL-II) was applied. Based on ELISA-plate test an agalactosylation factor (AF, a relative ratio of GSL-II/RCA-I binding) was calculated, which was proportional to actual disease severity. Repeated testing of several patients before and after treatment with methotrexate (MTX) alone or in combination with Remicade (a chimeric antibody anti-TNF-alpha) supplied results indicating an increase of IgG galactosylation during the treatment. This introductory observation suggests that IgG galactosylation may be an additional indicator of the RA patients' improvement.

Interaction between muscle aldolase and muscle fructose 1,6-bisphosphatase results in the substrate channeling.

Fructose 1,6-bisphosphatase (FBPase) is known to form a supramolecular complex with alpha-actinin and aldolase on both sides of the Z-line in skeletal muscle cells. It has been proposed that association of aldolase with FBPase not only desensitizes muscle FBPase toward AMP inhibition but it also might enable the channeling of intermediates between the enzymes [Rakus et al. (2003) FEBS Lett. 547, 11-14]. In the present paper, we tested the possibility of fructose 1,6-bisphosphate (F1,6-P(2)) channeling between aldolase and FBPase using the approach in which an inactive form of FBPase competed with active FBPase for binding to aldolase and thus decreased the rate of aldolase-FBPase reaction. The results showed that F1,6-P(2) is transferred directly from aldolase to FBPase without mixing with the bulk phase. Further evidence that F1,6-P(2) is channeled from aldolase to FBPase comes from the experiments investigating the inhibitory effect of a high concentration of magnesium ions on aldolase-FBPase activity. FBPase in a complex with aldolase, contrary to free muscle FBPase, was not inhibited by high Mg(2+) concentrations, which suggests that free F1,6-P(2) was not present in the assay mixture during the reaction. A real-time interaction analysis between aldolase and FBPase revealed a dual role of Mg(2+) in the regulation of the aldolase-FBPase complex stability. A physiological concentration of Mg(2+) increased the affinity of muscle FBPase to muscle aldolase, whereas higher concentrations of the cation decreased the concentration of the complex. We hypothesized that the presence of Mg(2+) stabilizes a positively charged cavity within FBPase and that it might enable an interaction with aldolase. Because magnesium decreased the binding constant (K(a)) between aldolase and FBPase in a manner similar to the decrease of K(a) caused by monovalent cations, it is postulated that electrostatic attraction might be a driving force for the complex formation. It is presumed that the biological relevance of F1,6-P(2) channeling between aldolase and FBPase is protection of this glyconeogenic, as well as glycolytic, intermediate against degradation by cytosolic aldolase, which is one of the most abundant enzyme of glycolysis.

Interaction of glycophorin A with lectins as measured by surface plasmon resonance (SPR).

Glycophorin A (GPA), the major sialoglycoprotein of the human erythrocyte membrane, was isolated from erythrocytes of healthy individuals of blood groups A, B and O using phenol-water extraction of erythrocyte membranes. Interaction of individual GPA samples with three lectins (Psathyrella velutina lectin, PVL; Triticum vulgaris lectin, WGA and Sambucus nigra I agglutinin SNA-I) was analyzed using a BIAcore biosensor equipped with a surface plasmon resonance (SPR) detector. The experiments showed no substantial differences in the interaction between native and desialylated GPA samples originating from erythrocytes of either blood group and each of the lectins. Desialylated samples reacted weaker than the native ones with all three lectins. PVL reacted about 50-fold more strongly than WGA which, similar to PVL, recognizes GlcNAc and Neu5Ac residues. SNA-I lectin, recognizing alpha2-6 linked Neu5Ac residues, showed relatively weak reaction with native and only residual reaction with desialylated GPA samples. The data obtained show that SPR is a valuable method to determine interaction of glycoproteins with lectins, which potentially can be used to detect differences in the carbohydrate moiety of individual glycoprotein samples.