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cDNA clones of the mouse GM2/GD2 synthase (EC 2.4.1.92) gene were isolated, and their analyses revealed that the protein has a type II transmembrane structure with 533 amino acids, which was very similar to the human homolog except for the mRNA size. The mRNA level in thymocytes dramatically increased after treatment with anti-CD3 monoclonal antibody, whereas it was not elevated when treated with prostaglandin E2. In situ hybridization showed an elevation of mRNA levels in medullar thymocytes, suggesting that T cell receptor-mediated signaling induces up-regulation of the GM2/GD2 synthase gene in mature thymocytes.

Bombyx mori (Shunrei × Shogetsu) is sensitive to Cry1Aa and resistant to Cry1Ac, both insecticidal proteins of Bacillus thuringiensis . Cry1Aa passed through the peritrophic membrane (PM) much faster (0.37 μg/mm 2 PM/h) than Cry1Ac (0.05 μg/mm 2 PM/h) during the initial observation period. Both Cry1Aa and Cry1Ac bound to the PM but only the binding of Cry1Ac was specifically inhibited by N -acetylgalactosamine (GalNAc). When Cry1Ac was pretreated with GalNAc, Cry1Ac permeated the PM much faster. These results suggested that Cry1Ac bound a PM protein via GalNAc on a sugar side chain. The role of the PM on Cry1Ac resistance of B. mori was briefly discussed.

A novel human nucleotide sugar transporter (NST) which transports both UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylgalactosamine (UDP-GalNAc) has been identified, cloned and characterized. The strategy for the identification of the novel NST involved a search of the expressed sequence tags database for genes related to the human UDP-galactose transporter-related isozyme 1, followed by heterologous expression of a candidate gene (hUGTrel7) in Saccharomyces cerevisiae and biochemical analyses. Significantly more UDP-GlcA and UDP-GalNAc were translocated from the reaction medium into the lumen of microsomes prepared from the hUGTrel7-expressing yeast cells than into the control microsomes from cells not expressing hUGTrel7. The possibility that this transporter participates in glucuronidation and/or chondroitin sulfate biosynthesis is discussed.

The two δ-endotoxins comprising the Bacillus thuringiensis var. kurstaki HD1 insecticidal protein crystal were separated. The lepidopteran-specific protoxin was activated in vitro and its mechanism of action investigated. Toxicity towards Choristoneura fumiferana CF1 cells was specifically inhibited by preincubation of the toxin with N -acetylgalactosamine and N -acetylneuraminic acid. The lectins soybean agglutinin and wheat germ agglutinin, which bind N -acetylgalactosamine, also inhibited toxicity. Since N -acetylneuraminic acid is not known to occur in insects, these results suggest that the toxin may recognise a specific plasma membrane glycoconjugate receptor with a terminal N -acetylgalactosamine residue.

Detergent extracts of the albumen gland of the snail Lymnaea stagnalis contain an enzyme activity that transfers Gal from UDP-Gal to acceptor substrates with terminal non-reducing β-galactose residues. The products formed with lactose (Galβl→4Glc) as the acceptor were characterized by HPLC, and subjected to 400-MHz 1H-NMR and methylation analysis. The main product was shown to have the structure Gal-βl→3Galβl→4Glc. Therefore, the galactosyltransferase can be identified as a UDP-Gal:β-galactoside βl→3-galactosyltransferase. In view of its linkage and acceptor specificity, the enzyme may be essential to the biosynthesis of galactogen, the main polysaccharide produced by albumen glands of L. stagnalis.

Examination of the three-dimensional structure of Erythrina corallodendron lectin (ECorL) in complex with a ligand (lactose), the first of its kind for a Gal/GalNAc-specific lectin [(1991) Science 254, 862-866], revealed the presence of a hydrophobic cavity, surrounded by Tyr108 and Pro134-Trp135, which can accommodate bulky substituents such as acetamido or dansylamido (NDns) at C-2 of the lectin-bound galactose. Comparison of the primary sequence of ECorL with that of soybean agglutinin, specific for galactose and its C-2 substituted derivatives, and of peanut agglutinin, specific for galactose only, showed that in soybean agglutinin, Tyr108 is retained, and Pro134-Trp135 is replaced by Ser-Trp, whereas in peanut agglutinin, the former residue is replaced by Thr and the dipeptide by Ser-Glu- Tyr-Asn. Three mutants of ECorL were therefore constructed: L2, in which Pro134-Trp135 was replaced by Ser-Glu-Tyr-Asn; Y108T, in which Tyr108 was replaced by Thr and the double mutant L2; Y108T. They were expressed in Escherichia coli, as done for recombinant ECorL [(1992) Eur. J. Biochem. 205, 575-581]. The mutants had the same hemagglutinating activity as native or rECorL. Their specificity for galactose, GalNAc and Me beta GalNDns was examined by inhibition of hemagglutination and of the binding of the lectin to immobilized asialofetuin; in addition, their association constants with Me alpha GalNDns and Me beta GalNDns were measured by spectrofluorimetric titration. The results showed that Y108T had essentially similar specificity as the native and recombinant lectins. The affinity of L2 and L2;Y108T for galactose was also the same as ECorL, but they had a lower affinity for GalNAc and markedly diminished affinity for the dansyl sugars (up to 43 times, or 2 kcal, less). This appears to be largely due to steric hindrance by the two additional amino acids present in the cavity region in these mutants. Our findings also provide an explanation for the inability of PNA to accommodate C-2-substituted galactose derivatives at its primary subsite.

Bacillus thuringiensis Cry1Ac δ-endotoxin specifically binds a 115-kDa aminopeptidase-N purified from Manduca sexta midgut. Cry1Ac domain III mutations were constructed around a putative sugar-binding pocket and binding to purified aminopeptidase-N and brush border membrane vesicles (BBMV) was compared to toxicity. Q509A, R511A, Y513A, and 509–511 (QNR-AAA) eliminated aminopeptidase-N binding and reduced binding to BBMV. However, toxicity decreased no more than two-fold, indicating activity is not directly correlated with aminopeptidase-N binding. Analysis of toxin binding to aminopeptidase-N in M. sexta is therefore insufficient for predicting toxicity. Mutants retained binding, however, to another BBMV site, suggesting alternative receptors may compensate in vivo.

A lectin was isolated from Rhizoctonia solani mycelium by affinity chromatography on gum arabic-Sepharose. It is a dimeric protein composed of 2 identical subunits of 13 kDa with high contents of asparagine/ aspartic acid, valine, glycine, glutamine/glutamic acid and lysine. The R. solani agglutinin (RSA) exhibits specificity towards N-acetylgalactosamine, and preferentially agglutinates human type A over type B and type O erythrocytes.

GalNAc transferase activities of 6 human intestinal cancerous tissues were examined using bovine submaxillary gland mucin and its desialylated derivative, asialomucin, as acceptors. A Triton X-100 extract of these tissues was used as an enzyme source. All the tissues examined had GalNAc transferase that catalyzes the transfer of GalNAc from UDP-GalNAc to serine or threonine residues of the polypeptide chain. One of 6 specimens showed in addition UDP-GalNAc:GalNAc-mucin α-GalNAc transferase activity, synthesizing a disaccharide unit, GalNAcα→ GalNAc, when asialomucin was used as an acceptor. This carbohydrate structure was deduced on the basis of results of gel filtration, exoglycosidase digestion, and high-voltage paper electrophoresis.GalNAc transferaseHuman intestinal cancerous tissueBovine submaxillary gland mucin O-Glycosidically linked sugar chain