Thomas A. Beyer

[41] Purification of mammalian glycosyltransferases

Publisher Summary It is reported that purified glycosyltransferases are powerful tools for the study of glycoconjugate structure and function, as well as the regulation of glycoprotein biosynthesis. Thus, this chapter presents the procedure for purification of seven mammalian glycosyltransferases. Affinity chromatographic methods are well suited to the isolation of glycosyltransferases. The technique tends to separate enzymically active species from inert related forms. Because there may be great specificity in both the adsorption and elution steps, the extraction of minor constituents from complex protein solutions is often feasible. The conditions for optimal adsorption are frequently similar to those for maximum enzymic activity, which for most enzymes is close to conditions for optimum stability. The addition of detergents, if necessary, does not generally interfere with the mechanism of affinity chromatography. Most methods of elution give a concentrated enzyme solution that may enhance the stability of labile proteins that tend to denature if diluted. Finally, knowledge of kinetic parameters permits a rational approach to the choice of an affinity ligand, as well as conditions for adsorption and elution.

The lactose synthase acceptor site: a structural map derived from acceptor studies.

SummaryA pictorial map of the lactose synthase (galactosyl transferase) acceptor binding site has been formulated from this and published studies on substrate analogs and inhibitors. The basic requirements are a pyranose, thiopyranose or inositol ring structure and equatorial substituents (if any) at C-2, C-3, C-4, and C-5. The aglycone (at C-1) may be either α or β-, but α- is somewhat preferred. In the absence of α-lactalbumin galactosyl transferase will accept long chain 2-N-acyl substituents on the glucosamine (GlcNH2) structure. An equatorial amino or N-acetyl substituent (e.g. mannosamine, N-acetylmannosamine) is also a suitable acceptor in the absence of α-lactalbumin since both N-acetylglucosamine and N-acetylmannosamine have complementary binding loci for the N-acyl moiety. The aglycone moiety must be equatorial (β-configuraation). However, upon α-lactalbumin binding the aglycone specificity allows for axial (α-configuration) as well as equatorial substituents. Furthermore, the 2-N-acyl substituent binding locus is blocked beyond a 2-N-hexanoyl group. It is suggested that α-lactalbumin binds to a hydrophobic site some distance from the C-2 group.

Characterization of a mutated IgA2 antibody of the m(1) allotype against the epidermal growth factor receptor for the recruitment of monocytes and macrophages.

Background: IgA constitutes a promising antibody isotype, which requires optimization before immunotherapeutic application. Results: P221R-mutated and wild type IgA2m(1) antibodies were similarly effective in killing tumor cells and in recruiting myeloid effector cells. Conclusion: Improved IgA antibodies constitute promising next generation antibodies for tumor therapy. Significance: These studies support the clinical development of therapeutic IgA antibodies. IgA antibodies constitute an important part of the mucosal immune system, but their immunotherapeutic potential remains rather unexplored, in part due to biotechnological issues. For example, the IgA2m(1) allotype carries an unusual heavy and light chain pairing, which may confer production and stability concerns. Here, we report the generation and the biochemical and functional characterization of a P221R-mutated IgA2m(1) antibody against the epidermal growth factor receptor (EGFR). Compared with wild type, the mutated antibody demonstrated heavy chains covalently linked to light chains in monomeric as well as in joining (J)-chain containing dimeric IgA. Functional studies with wild type and mutated IgA2m(1) revealed similar binding to EGFR and direct effector functions such as EGFR down-modulation and growth inhibition. Furthermore, both IgA molecules triggered similar levels of indirect tumor cell killing such as antibody-dependent cell-mediated cytotoxicity (ADCC) by isolated monocytes, activated polymorphonuclear cells, and human whole blood. Interestingly, the dimeric IgA antibodies demonstrated higher efficiency in direct as well as in indirect effector mechanisms compared with their respective monomeric forms. Both wild type and mutated antibody triggered effective FcαRI-mediated tumor cell killing by macrophages already at low effector to target cell ratios. Interestingly, also polarized macrophages mediated significant IgA2-mediated ADCC. M2 macrophages, which have been described as promoting tumor growth and progression, may convert to ADCC-mediating effector cells in the presence of EGFR-directed antibodies. In conclusion, these results provide further insight into the immunotherapeutic potential of recombinant IgA antibodies for tumor immunotherapy and suggest macrophages as an additional effector cell population.

Affinity chromatography of glycosyltransferases.

This review summarizes the use of biospecific chromatography techniques in the purification of mammalian glycosyltransferases. Ligands that are analogues of donor or acceptor substrates have been linked to cyanogen bromide-activated agarose for use as affinity adsorbents. Immobilized lectins have been employed to recognize the carbohydrate moieties of glycosyltransferase and remove them from complex mixtures. The application of these methods has permitted extensive purification of many membrane-bound glycosyltransferases, some to homogeneity.

Characterization of Two Highly Purified Fucosyltransferases

Publisher Summary This chapter describes the characters of the two highly purified fucosyltransferases. Fucosyltransferases catalyzes the transfer of L-fucose from GDP–Fuc to carbohydrate acceptors by linking α-(1→2) to Gal, α-(1→4) to GlcNAc, α-(1→3) to GlcNAc and Glc, and α-(1→6) to GlcNAc.Two of these enzymes, a GDP-Fuc:β-D-Gal 2-α-L-fucosayltransferase [α(l→2)FucT] from porcine submaxillary glands, and a GDP–Fuc : (β-D-Gal 3-α-L-fucosyltransferase [α(l→3)FucT] from human milk, are purified and partially characterized. This represents a124,000-fold purification in an overall yield of 6%. Comparison of the apparent Km values for acceptors indicates a preference for structures having the β-D-Gal-(1→3)-HexNAc linkage. The cells were not agglutinated by η-specific lectins after treatment with the α(l→2) FucT alone but were agglutinated if pretreated with neuraminidase. However, it was also found that fucose were incorporated at approximately106 residues per cell when treated with the α(l→2) FucT alone and that these cells could be agglutinated with the Hspecific lectins. This suggests that sialic acid does not block the incorporation of fucose but rather interferes with the binding of the lectin to the cells.

GLYCOSYL TRANSFERASES IN OLIGOSACCHARIDE BIOSYNTHESIS AND THEIR USE IN STRUCTURE - FUNCTION ANALYSIS OF GLYCOPROTEINS

Abstract Eleven different glycosyl transferases have been highly purified, or purified to homogeneity, and each has been found to have a very strict acceptor substrate specificity, in accord with the hypothesis that one enzyme is required for the synthesis of each type of disaccharide sequence found in the oligosaccharides of glycoconjugates. Several of the transferases act reciprocally, that is, each have the same acceptor substrate, but the product of one transferase may be a poor acceptor for another transferase, or not an acceptor at all. The combined actions of the transferases not only indicate the types of oligosaccharide structures permitted in animal oligosaccharides, but also give some insight into the basis of the structural heterogeneity observed in many oligosaccharides. Moreover, it is also possible to predict the preferred pathways of biosynthesis of certain oligosaccharides. The pure glycosyl transferases have been shown to be very useful reagents for the structural analysis of oligosaccharides and for the assessment of the structure-function relationships of glycoconjugates. Examples of their use for this purpose include studies on the rabbit hepatic galactose binding protein, α1-acid glycoprotein, human blood coagulation factor VIII and a heretofore unrecognized oligosaccharide binding activity of hepatocytes that binds specifically certain fucose containing ligands. The structure-function relationships of cell surface oligosaccharides can also be assessed with the aid of the transferases, as illustrated by studies on the role of sialic acid in expression of erythrocyte MN antigens and in viral adherence to erythrocytes.