Specificity of submaxillary gland sialyltransferases

A method has been developed to determine the activities of specific sialyltransferases by analysis of the products of the reaction. This method, which utilizes high performance liquid chromatography, distinguishes addition of sialic acid to the N-acetylgalactosamine vs. galactose residues of the mucin disaccharide Galβ(1→3)GalNac, and can be used to distinguish formation of the 3′- and 6′-isomers of sialyllactose. For the bovine, ovine, and porcine submaxillary extracts, more than 95% of the activity with asialo ovine submaxillary mucin is due to formation of NeuAc α(2→6)GalNAc. With lactose as the acceptor, more than 95% of the α(2→3) isomer is produced. Activity with asialofetuin is due solely to the O-linked chain, with relative activity toward the galactose vs. GalNAc residues of 0.32, 1.5, and 0.10 for bovine, ovine, and porcine, respectively. The rat submaxillary gland extract showed equal formation of 3′- and 6′-sialyllactose, and very low activity with asialo ovine submaxillary mucin. However, at least 40% of the activity toward the Galβ(1→3)GalNAc disaccharide of asialofetuin was directed toward the GalNAc residue. The relative preference of the N-acetylgalactosaminide α(2→6) sialyltransferase for a monosaccharide vs. a substituted GalNAc may play a role in regulation of chain length during mucin synthesis.     

Isolation and identification of a fucose-containing ganglioside from bovine thyroid gland

Bovine thyroid glands are known to contain a complex array of gangliosides. One of the predominant gangliosides was isolated and analyzed by gas-liquid chromatography and mass spectrometry. The carbohydrate composition was fucose, N-acetylneuraminic acid, galactose, N-acetylgalactosamine, and glucose in molar ratios of 1:1:2:1:1. The structure of the ganglioside was identified as:

     

The effect of linoleic acid and benzyl alcohol on the activity of glycosyltransferases of rat liver golgi membranes and some soluble glycosyltransferases

The effects of the membrane perturbing reagents linoleic acid and benzyl alcohol on the activities of four rat liver Golgi membrane enzymes, N-acetylglucosaminyl-, N-acetylgalactosaminyl-, galactosyl-, and sialytransferases and several soluble glycosyltransferases, bovine milk galactosyl- and N-acetylglucosaminyltransferases and porcine submaxillary N-acetylgalactosaminyltransferases have been studied. In rat liver Golgi membranes, linoleic acid inhibited the activities of N-acetylgalactosaminyl- and galactosyltransferases by 50% or greater, sialyltransferase by 10–15%, and N-acetylglucosaminyltransferase not at all. The isolated bovine milk N-acetylglucosaminyltransferase and porcine submaxillary N-acetylgalactosylaminyltranferase were not inhibited but bovine milk galactosyltransferase was inhibited by 95% or greater. The inhibition by linoleic acid on Golgi membrane galactosyltransferase appears to be a direct effect of the reagent on the enzyme. Incorporation of bovine milk galactosyltransferase into liposomes formed from saturated phospholipids, DMPC, DPPC, and DSPC (dimyristoyl-, dipalmitoyl-, and distearoylphosphatidylcholine) prevented inhibition of the enzyme activity suggesting that the lipid formed a barrier which did not allow linoleic acid access to the enzyme. The water soluble benzyl alcohol was more effective in inhibiting enzymes of the isolated rat liver Golgi complex. All four glycosyltransferases were inhibited, the N-acetylglucosaminyl- and N-acetylgalactosaminyltransferases by more than 95%. A higher concentration of benzyl alcohol was necessary to inhibit the galactosyltransferases than was required for the other Golgi enzymes. Benzyl alcohol also inhibited the isolated bovine milk N-acetylglucosaminyl- and galactosyltransferases 90% to 95%, respectively, but did not affect the isolated porcine submaxillary gland N-acetylgalactosaminyltransferase. Benzyl alcohol did not inhibit the milk galactosyltransferase incorporated into DMPC or DPPC liposomes but showed a complex effect on the activity of the enzyme incorporated into DSPC vesicles, a stimulation of activity at low concentrations followed by an inhibition. A lipid environment consisting of saturated lipids appears to present a barrier to inhibiting substances such as linoleic acid and benzyl alcohol, or lipid may stabilize the active conformation of the enzyme. The different effects of these reagents on four transferases of the Golgi complex suggest that the lipid environment around these enzymes may be different for each transferase.     

One-pot enzymatic production of 2-acetamido-2-deoxy-d-galactose (GalNAc) from 2-acetamido-2-deoxy-d-glucose (GlcNAc)

2-Acetamido-2-deoxy-d-galactose (GalNAc) is a common monosaccharide found in biologically functional sugar chains, but its availability is often limited due to the lack of abundant natural sources. In order to produce GalNAc from abundantly available sugars, 2-acetamido-2-deoxy-d-glucose (GlcNAc) was converted to GalNAc by a one-pot reaction using three enzymes involved in the galacto-N-biose/lacto-N-biose I pathway of bifidobacteria. Starting the reaction with 600 mM GlcNAc, 170 mM GalNAc was produced at equilibrium in the presence of catalytic amounts of ATP and UDP-Glc under optimized conditions. GalNAc was separated from GlcNAc using water-eluting cation-exchange chromatography with a commonly available cation-exchange resin.     

Specificities of Ricinus communis agglutinin 120 interaction with sulfated galactose

Lectins are used extensively as research tools to detect and target specific oligosaccharide sequences. Ricinus communis agglutinin I (RCA₁₂₀) recognizes non-reducing terminal β-d-galactose (Galβ) and its specificities of interactions with neutral and sialylated oligosaccharides have been well documented. Here we use carbohydrate arrays of sulfated Galβ-containing oligosaccharide probes, prepared from marine-derived galactans, to investigate their interactions with RCA₁₂₀. Our results showed that RCA₁₂₀ binding to Galβ1–4 was enhanced by 2-O- or 6-O-sulfation but abolished by 4-O-sulfation. The results were corroborated with competition experiments. Erythrina cristagalli lectin is also a Galβ-binding protein but it cannot accommodate any sulfation on Galβ.     

The expanding roles of the Sd/Cad carbohydrate antigen and its cognate glycosyltransferase B4GALNT2

Background

The histo-blood group antigens are carbohydrate structures present in tissues and body fluids, which contribute to the definition of the individual immunophenotype. One of these, the Sd^a antigen, is expressed on the surface of erythrocytes and in secretions of the vast majority of the Caucasians and other ethnic groups.

Scope of review

We describe the multiple and unsuspected aspects of the biology of the Sd^a antigen and its biosynthetic enzyme β1,4-N-acetylgalactosaminyltransferase 2 (B4GALNT2) in various physiological and pathological settings.

Major conclusions

The immunodominant sugar of the Sd^a antigen is a β1,4-linked N-acetylgalactosamine (GalNAc). Its cognate glycosyltransferase B4GALNT2 displays a restricted pattern of tissue expression, is regulated by unknown mechanisms - including promoter methylation, and encodes at least two different proteins, one of which with an unconventionally long cytoplasmic portion. In different settings, the Sd^a antigen plays multiple and unsuspected roles. 1) In colon cancer, its dramatic down-regulation plays a potential role in the overexpression of sialyl Lewis antigens, increasing metastasis formation. 2) It is involved in the lytic function of murine cytotoxic T lymphocytes. 3) It prevents the development of muscular dystrophy in various dystrophic murine models, when overexpressed in muscular fibers. 4) It regulates the circulating half-life of the von Willebrand factor (vWf), determining the onset of a bleeding disorder in a murine model.

General significance

The expression of the Sd^a antigen has a wide impact on the physiology and the pathology of different biological systems.