Effect of structural modifications of ganglioside GM2 on intra-molecular carbohydrate-to-carbohydrate interaction and enzymatic susceptibility

The effect of inter-molecular carbohydrate-to-carbohydrate interaction on basic cell biological processes has been well documented and appreciated. In contrast, very little is known about the intra-molecular carbohydrate-to-carbohydrate interaction. The presence of an interaction between the GalNAc and the Neu5Ac in GM2 detected by NMR spectroscopy represents a well-defined intra-molecular carbohydrate-to-carbohydrate interaction. This intriguing interaction is responsible for the GM2-epitope, GalNAcbeta1-->4(Neu5Acalpha2-->3)Gal-, to exhibit a rigid and compact conformation. We hypothesized that this compact conformation may be the cause for both the GalNAc and the Neu5Ac in GM2 to be refractory to enzymatic hydrolysis and the GM2 activator protein is able to interact with the compact trisaccharide GM2-epitope, rendering the GalNAc and the Neu5Ac accessible to beta-hexosaminidase A and sialidase. We have used a series of structurally modified GM2 to study the effect of modifications of sugar chains on the conformation and enzymatic susceptibility of this ganglioside. Our hypothesis was borne out by the fact that when the GalNAcbeta1-->4Gal linkage in GM2 was converted to the GalNAcbeta1-->6Gal, both the GalNAc and the Neu5Ac became susceptible to beta-hexosaminidase A and sialidase, respectively, without GM2 activator protein. We hope our work will engender interest in identifying other intra-molecular carbohydrate-to-carbohydrate interactions in glycoconjugates.     

Specificity of the N1 and N2 sialidase subtypes of human influenza A virus for natural and synthetic gangliosides

Sialyl-linkage specificity of sialidases of the human influenza A virus strains, A/Aichi/2/68 (H3N2) and A/PR/8/34 (H1N1) were studied using natural and synthetic gangliosides. The sialidase of the A/Aichi/2/68 strain hydrolyzed the terminal Neu5Acalpha2-3Gal sequence but not the Neu5Acalpha2-3 linkage on the inner Gal of GM1a, which is a ganglioside that has the gangliotetraose chain (Galbeta1-3GalNAcbeta1-4- (Neu5Acalpha2-3)Galbeta1++ +-4Glcbeta1-Cer). The sialidase hydrolyzed the Neu5Ac on the inner Gal of GM2, which had a shorter gangliotriose chain. GM4, which had the shortest chain (Neu5Acalpha2-3Galbeta1-Cer) of the gangliosides, had a lower substrate specificity. The N1 and N2 sialidase subtypes of the human influenza A virus had no significant variation in their substrate specificity for the gangliosides. Analysis of 11 synthetic gangliosides, which contained various ceramide or sialic acid moieties, demonstrated that A/Aichi/2/68 (H3N2) sialidase recognized the ceramide and sialic acid moiety and the length and structure of the sialyl sugar chain.      

Interaction of the GM2-activator protein with phospholipid-ganglioside bilayer membranes and with monolayers at the air-water interface.

Differential scanning calorimetry (DSC) and film balance measurements were performed to study the interactions of the GalNAcbeta1-->4(NeuAcalpha2-->3)Galbeta1-->4Glc1 -->1'Cer (GM2)-activator protein with phospholipid/ganglioside vesicles and monolayers. The nonglycosylated form of the GM2-activator protein, added to unilamellar lipid vesicles of different composition, causes differential effects on the gel to liquid-crystalline phase transition peaks. The phase transition temperature (Tm) of pure dimyristoylglycerophosphocholine (DMPC) bilayer is slightly decreased. When lipids which specifically bind the GM2-activator protein are incorporated into the vesicles (e.g. a sulfatide or gangliosides) a shoulder in the thermograms at higher temperatures is observed, indicating an increase of the stability of the gel phase in relation to the liquid-crystalline phase. We also studied the surface activity of a glycosylated and a nonglycosylated GM2-activator protein at the air-water interface. The glycosylated form showed a slightly lower surface activity than the GM2-activator protein without oligosaccharide moiety. When the GM2-activator protein is added to the sub-phase of a surface covered with a lipid monolayer, it can only insert into the monolayer and reach the air-water interface below a monolayer pressure of 25 mN.m-1, depending on the lipid composition, and not when the monolayers are at the bilayer equivalence pressure of 30-35 mN.m-1. Particularly for Galbeta1-->3GalNAcbeta1-->4(NeuAcalpha2-->3)Galbeta 1-->4Glc1-->1'Cer (GM1) and GM2 containing films, the critical pressures (picrit) when no additional increase in surface pressure is observed after addition of the protein into the subphase, are much lower. This leads to the conclusion that binding of the GM2 activator protein to the ganglioside headgroups prevents the protein from reaching the air-water interface. The protein is then located preferentially at the lipid-water interface and cannot penetrate into the chain region.     

Cell surface n-acetylneuraminic acid alpha2,3-galactoside-dependent intercellular adhesion of human colon cancer cells

Sialoglycans on the cell surface of human colon cancer (HCC) cells have been implicated in cellular adhesion and metastasis. To clarify the role of N-acetylneuraminic acid (NeuAc) linked alpha2,3 to galactose (Gal) on the surface of HCC cells, we studied the intercellular adhesion of HCC cell lines expressing increasing NeuAcalpha2,3Gal-R. Our model system consisted of the HCC SW48 cell line, which inherently possesses low levels of cell surface alpha2,3 and alpha2,6 sialoglycans. To generate SW48 clonal variants with elevated cell surface NeuAcalpha2,3Gal-R linkages, we transfected the expression vector, pcDNA3, containing either rat liver cDNA encoding Galbeta1,3(4)GlcNAc alpha2,3 sialyltransferase (ST3Gal III) or human placental cDNA encoding Galbeta1,3GalNAc/Galbeta1,4GlcNAc alpha2,3 sialyltransferase (ST3Gal IV) into SW48 cells. Selection of neomycin-resistant clones (600 microgram G418/ml) having a higher percentage of cells expressing NeuAcalpha2,3Gal-R (up to 85% positive Maackia amurenis agglutinin staining compared with 30% for wild type cells) was performed. These ST3Gal III and ST3Gal IV clonal variants demonstrated increased adherence to IL-1beta-activated human umbilical vein endothelial cells (HUVEC) (up to 90% adherent cells compared with 63% for wild type cells). Interestingly, ST3Gal III and ST3Gal IV clonal variants also bound non-activated HUVEC up to 4-fold more effectively than wild type cells. Cell surface NeuAcalpha2,3Gal-R expression within the various SW48 clonal variants correlated directly with increased adhesion to HUVEC (r=0.84). Using HCC HT-29 cells, which express high levels of surface NeuAcalpha2,3Gal-R, addition of synthetic sialyl, sulfo or GalNAc Lewis X structures were found to specifically inhibit intercellular adhesion. At 1.0mM, NeuAcalpha2,3Galbeta1,3(Fucalpha1, 4)GlcNAc-OH and Galbeta1,4(Fucalpha1,3)GlcNAcbeta1,6(SE-6Galbeta1++ +, 3)GalNAcalpha1-O-methyl inhibited HT-29 cell adhesion to IL-1beta-stimulated HUVEC by 100% and 68%, respectively. GalNAcbeta1, 4(Fucalpha1,3)GlcNAcbeta1-O-methyl and GalNAcbeta1,4(Fucalpha1, 3)GlcNAcbeta1,6Manalpha1,6Manbeta1-0-C30H61, however, did not possess inhibitory activity. In conclusion, these studies demonstrated that cell surface NeuAcalpha2,3Gal-R expression is involved in HCC cellular adhesion to HUVEC. These specific carbohydrate-mediated intercellular adhesive events may play an important role in tumor angiogenesis, metastasis and growth control.     

Molecular cloning and functional expression of two members of mouse NeuAcalpha2,3Galbeta1,3GalNAc GalNAcalpha2,6-sialyltransferase family, ST6GalNAc III and IV

Two cDNA clones encoding NeuAcalpha2,3Galbeta1,3GalNAc GalNAcalpha2, 6-sialyltransferase have been isolated from mouse brain cDNA libraries. One of the cDNA clones is a homologue of previously reported rat ST6GalNAc III according to the amino acid sequence identity (94.4%) and the substrate specificity of the expressed recombinant enzyme, while the other cDNA clone includes an open reading frame coding for 302 amino acids. The deduced amino acid sequence is not identical to those of other cloned mouse sialyltransferases, although it shows the highest sequence similarity with mouse ST6GalNAc III (43.0%). The expressed soluble recombinant enzyme exhibited activity toward NeuAcalpha2, 3Galbeta1, 3GalNAc, fetuin, and GM1b, while no significant activity was detected toward Galbeta1,3GalNAc or asialofetuin, or the other glycoprotein substrates tested. The sialidase sensitivity of the 14C-sialylated residue of fetuin, which was sialylated by this enzyme with CMP-[14C]NeuAc, was the same as that of ST6GalNAc III. These results indicate that the expressed enzyme is a new type of GalNAcalpha2,6-sialyltransferase, which requires sialic acid residues linked to Galbeta1,3GalNAc residues for its activity; therefore, we designated it mouse ST6GalNAc IV. Although the substrate specificity of this enzyme is similar to that of ST6GalNAc III, ST6GalNAc IV prefers O-glycans to glycolipids. Glycolipids, however, are better substrates for ST6GalNAc III.     

Structural analysis of murine zona pellucida glycans. Evidence for the expression of core 2-type O-glycans and the Sd(a) antigen

Murine sperm initiate fertilization by binding to specific oligosaccharides linked to the zona pellucida, the specialized matrix coating the egg. Biophysical analyses have revealed the presence of both high mannose and complex-type N-glycans in murine zona pellucida. The predominant high mannose-type glycan had the composition Man(5)GlcNAc(2), but larger oligosaccharides of this type were also detected. Biantennary, triantennary, and tetraantennary complex-type N-glycans were found to be terminated with the following antennae: Galbeta1-4GlcNAc, NeuAcalpha2-3Galbeta1-4GlcNAc, NeuGcalpha2-3Galbeta1-4GlcNAc, the Sd(a) antigen (NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc, NeuGcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc), and terminal GlcNAc. Polylactosamine-type sequence was also detected on a subset of the antennae. Analysis of the O-glycans indicated that the majority were core 2-type (Galbeta1-4GlcNAcbeta1-6[Galbeta1-3]GalNAc). The beta1-6-linked branches attached to these O-glycans were terminated with the same sequences as the N-glycans, except for terminal GlcNAc. Glycans bearing Galbeta1-4GlcNAcbeta1-6 branches have previously been suggested to mediate initial murine gamete binding. Oligosaccharides terminated with GalNAcbeta1-4Gal have been implicated in the secondary binding interaction that occurs following the acrosome reaction. The significant implications of these observations are discussed.     

Selection of a carbohydrate-binding domain with a helix-loop-helix structure

We obtained a novel carbohydrate-binding peptide having a helix-loop-helix scaffold from a random peptide library. The helix-loop-helix peptide library randomized at five amino acid residues was displayed on the major coat protein of a filamentous phage. Affinity selection with a ganglioside, Galbeta1-3GalNAcbeta1-4(Neu5Acalpha2-3)Galbeta1-4Glcbeta1-1'Cer (GM1), gave positive phage clones. Surface plasmon resonance spectroscopy showed that a corresponding 35-mer synthetic peptide had high affinity for GM1 with a dissociation constant of 0.24 microM. This peptide preferentially binds to GM1 rather than asialo GM1 and GM2, suggesting that a terminal galactose and sialic acid are required for the binding as for cholera toxin. Circular dichroism spectroscopic studies indicated that a helical structure is important for the affinity and specificity. Furthermore, alanine scanning at randomized positions showed that arginine and phenylalanine play an especially important role in the recognition of carbohydrates. Such a de novo helix-loop-helix peptide would be available for the design of carbohydrate-binding proteins.     

Carbohydrate recognition factors of a Talpha (Galbeta1-->3GalNAcalpha1-->Ser/Thr) and Tn (GalNAcalpha1-->Ser/Thr) specific lectin isolated from the seeds of Artocarpus lakoocha

Artocarpus lakoocha agglutinin (ALA), isolated from the seeds of A. lakoocha fruit, is a galactose-binding lectin and a potent mitogen of T and B cells. Knowledge obtained from previous studies on the affinity of ALA was limited to molecular and submolecular levels of Galbeta1-->3GalNAc (T) and its derivatives. In the present study, the carbohydrate specificity of ALA was characterized at the macromolecular level according to the mammalian Gal/GalNAc structural units and corresponding glycoconjugates by an enzyme-linked lectinosorbent (ELLSA) and inhibition assays. The results indicate that ALA binds specifically to tumor-associated carbohydrate antigens GalNAcalpha1-->Ser/Thr (Tn) and Galbeta1-->3 GalNAcalpha1-->Ser/Thr (Talpha). It barely cross-reacts with other common glycotopes on glycoproteins, including ABH blood group antigens, Galbeta1-->3/4GlcNAc (I/II) determinants, T/Tn covered by sialic acids, and N-linked plasma glycoproteins. Dense clustering structure of Tn/Talpha-containing glycoproteins tested resulted in 2.4 x 10(5)-6.7 x 10(5)-fold higher affinities to ALA than the respective GalNAc and Gal monomer. According to our results, the overall affinity of ALA for glycans can be ranked respectively: polyvalent Tn/Talpha glycotopes > monomeric Talpha and simple clustered Tn > monomeric Tn > GalNAc > Gal; while other glycotopes: Galalpha1-->3/4Gal (B/E), Galbeta1-->3/4GlcNAc (I/II), GalNAcalpha1-->3Gal/GalNAc (A/F), and GalNAcbeta1-->3/4Gal (P/S) were inactive. The strong specificity of ALA for Tn/Talpha cluster suggests the importance of glycotope polyvalency during carbohydrate-receptor interactions and emphasizes its value as an anti-Tn/T lectin for analysis of glycoconjugate mixtures or transformed carbohydrates.     

2-Keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN)- and N-acetylneuraminic acid-cleaving sialidase (KDN-sialidase) and KDN-cleaving hydrolase (KDNase) from the hepatopancreas of oyster, Crassostrea virginica.

KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid), a sialic acid analog, has been found to be widely distributed in nature. Despite the structural similarity between KDN and Neu5Ac, alpha-ketosides of KDN are refractory to conventional sialidases. We found that the hepatopancreas of the oyster, Crassostrea virginica, contains two KDN-cleaving sialidases but is devoid of conventional sialidase. The major sialidase, KDN-sialidase, effectively cleaves alpha-ketosidically linked KDN and also slowly cleaves the alpha-ketosides of Neu5Ac. The minor sialidase, KDNase, is specific for alpha-ketosides of KDN. We were able to separate these two KDN-cleaving enzymes using hydrophobic interaction and cation-exchange chromatographies. The rate of hydrolysis of 4-methylumbelliferyl-alpha-KDN (MU-KDN) by KDN-sialidase is 30 times faster than that of MU-Neu5Ac in the presence of 0.2 M NaCl, whereas in the absence of NaCl this ratio is only 8. KDNase hydrolyzes MU-KDN over 500 times faster than MU-Neu5Ac and is not affected by NaCl. KDN-sialidase purified to electrophoretically homogeneous form was found to have a molecular mass of 25 kDa and an isoelectric point of 8.4. One of the three tryptic peptides derived from KDN-sialidase contains the consensus motif, SXDXGXTW, that has been found in all conventional sialidases. Kinetic analysis of the inhibition of the hydrolysis of MU-KDN and MU-Neu5Ac by 2, 3-dehydro-2-deoxy-KDN (KDN2-en) and 2,3-dehydro-2-deoxy-(Neu5Ac2-en) suggests that KDN-sialidase contains two separate active sites for the hydrolysis of KDN and Neu5Ac. Both KDN-sialidase and KDNase effectively hydrolyze KDN-G(M3), KDNalpha2-->3Gal beta1-->4Glc, KDNalpha2-->6Galbeta1-->4Glc, KDNalpha2-->6-N-acetylgalactosaminitol, KDNalpha2-->6(KDNalpha2-->3)N-acetylgalactosaminitol, and KDNalpha2-->6(GlcNAcbeta1-->3)N-acetylgalactosaminitol. However, only KDN-sialidase also slowly hydrolyzes G(M3), Neu5Acalpha2-->3Galbeta1-->4Glc, and Neu5Acalpha2-->6Galbeta1-->4Glc. These two KDN-cleaving sialidases should be useful for studying the structure and function of KDN-containing glycoconjugates.     

Histochemical analysis of glycoconjugates in the domestic cat testis

The localization and characterization of oligosaccharide sequences in the cat testis was investigated using 12 lectins in combination with the beta-elimination reaction, N-Glycosidase F and sialidase digestion. Leydig cells expressed O-linked glycans with terminal alphaGalNAc (HPA reactivity) and N-glycans with terminal/internal alphaMan (Con A affinity). The basement membrane showed terminal Neu5Acalpha2,6Gal/GalNAc, Galbeta1,3GalNAc, alpha/betaGalNAc, and GlcNAc (SNA, PNA, HPA, SBA, GSA II reactivity) in O-linked oligosaccharides, terminal Galbeta1,4GlcNAc (RCA120 staining) and alphaMan in N-linked oligosaccharides; in addition, terminal Neu5acalpha2,3Galbeta1,4GlcNac, Forssman pentasaccharide, alphaGal, alphaL-Fuc and internal GlcNAc (MAL II, DBA, GSA I-B4, UEA I, KOH-sialidase-WGA affinity) formed both O- and N-linked oligosaccharides. The Sertoli cells cytoplasm contained terminal Neu5Ac-Galbeta1,4GlcNAc, Neu5Ac-betaGalNAc as well as internal GlcNAc in O-linked glycans, alphaMan in N-linked glycoproteins and terminal Neu5Acalpha2,6Gal/ GalNAc in both O- and N-linked oligosaccharides. Spermatogonia exhibited cytoplasmic N-linked glycoproteins with alphaMan residues. The spermatocytes cytoplasm expressed terminal Neu5Acalpha2,3Galbeta1,4 GlcNAc and Galbeta1,3GalNAc in O-linked oligosaccharides, terminal Galbeta1,4GlcNAc and alpha/betaGalNAc in N-linked glycoconjugates. The Golgi region showed terminal Neu5Acalpha2,3Galbeta1,4GlcNac, Galbeta1,4GlcNAc, Forssman pentasaccharide, and alphaGalNAc in O-linked oligosaccharides, alphaMan and terminal betaGal in N-linked oligosaccharides. The acrosomes of Golgi-phase spermatids expressed terminal Galbeta1,3GalNAc, Galbeta1,4GlcNAc, Forssmann pentasaccharide, alpha/betaGalNAc, alphaGal and internal GlcNAc in O-linked oligosaccharides, terminal alpha/betaGalNAc, alphaGal and terminal/internal alphaMan in N-linked glycoproteins. The acrosomes of cap-phase spermatids lacked internal Forssman pentasaccharide and alphaGal, while having increased alpha/betaGalNAc. The acrosomes of elongated spermatids did not show terminal Galbeta1,3GalNAc, displayed terminal Galbeta1,4GlcNAc and alpha/betaGalNAc in N-glycans and Neu5Ac-Galbeta1,3GalNAc in O-linked oligosaccharides.     

Lectinochemical characterization of a GalNAc and multi-Galbeta1-->4GlcNAc reactive lectin from Wistaria sinensis seeds.

An agglutinin that has high affinity for GalNAcbeta1-->, was isolated from seeds of Wistaria sinensis by adsorption to immobilized mild acid-treated hog gastric mucin on Sepharose 4B matrix and elution with aqueous 0.2 M lactose. The binding property of this lectin was characterized by quantitative precipitin assay (QPA) and by inhibition of biotinylated lectin-glycan interaction. Of the 37 glycoforms tested by QPA, this agglutinin reacted best with a GalNAcbeta1-->4 containing glycoprotein (GP) [Tamm-Horsfall Sd(a+) GP]; a Galbeta1-->4GlcNAc containing GP (human blood group precursor glycoprotein from ovarian cyst fluid and asialo human alpha1-acid GP) and a GalNAcalpha1-->3GalNAc containing GP (asialo bird nest GP), but poorly or not at all with most sialic acid containing glycoproteins. Among the oligosaccharides tested, GalNAcalpha1-->3GalNAcbeta1-->3Galalpha1-->4Galbeta 1-->4Glc (Fp) was the most active ligand. It was as active as GalNAc and two to 11 times more active than Tn cluster mixtures, Galbeta1--> 3/4GlcNAc (I/II), GalNAcalpha1-->3(L-Fucalpha1-->2)Gal (Ah), Galbeta1-->4Glc (L), Galbeta1-->3GalNAc (T) and Galalpha1--> 3Galalpha-->methyl (B). Of the monosaccharides and their glycosides tested, p-nitrophenyl betaGalNAc was the best inhibitor; it was approximately 1.7 and 2.5 times more potent than its corresponding alpha anomer and GalNAc (or Fp), respectively. GalNAc was 53.3 times more active than Gal. From the present observations, it can be concluded that the Wistaria agglutinin (WSA) binds to the C-3, C-4 and C-6 positions of GalNAc and Gal residues; the N-acetyl group at C-2 enhances its binding dramatically. The combining site of WSA for GalNAc related ligands is most likely of a shallow type, able to recognize both alpha and beta anomers of GalNAc. Gal ligands must be Galbeta1-->3/4GlcNAc related, in which subterminal beta1-->3/4 GlcNAc contributes significantly to binding; hydrophobicity is important for binding of the beta anomer of Gal. The decreasing order of the affinity of WSA for mammalian structural carbohydrate units is Fp >/= multi-II > monomeric II >/= Tn, I and Ah >/= E and L > T > Gal.     

alpha 2,3-Sialylation of terminal GalNAc beta 1-3Gal determinants by ST3Gal II reveals the multifunctionality of the enzyme. The resulting Neu5Ac alpha 2-3GalNAc linkage is resistant to sialidases from Newcastle disease virus and Streptococcus pneumoniae

Enzymatic alpha 2,3-sialylation of GalNAc has not been described previously, although some glycoconjugates containing alpha 2,3-sialylated GalNAc residues have been reported. In the present experiments, recombinant soluble alpha 2,3-sialyltransferase ST3Gal II efficiently sialylated the X(2) pentasaccharide GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, globo-N-tetraose GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and the disaccharide GalNAc beta 1-3Gal in vitro. The purified products were identified as Neu5Ac alpha 2-3GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, Neu5Ac alpha 2-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and Neu5Ac alpha 2-3GalNAc beta 1-3Gal, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, enzymatic degradations, and one- and two-dimensional NMR-spectroscopy. In particular, the presence of the Neu5Ac alpha 2-3GalNAc linkage was firmly established in all three products by a long range correlation between Neu5Ac C2 and GalNAc H3 in heteronuclear multiple bond correlation spectra. Collectively, the data describe the first successful sialyltransfer reactions to the 3-position of GalNAc in any acceptor. Previously, ST3Gal II has been shown to transfer to the Gal beta 1-3GalNAc determinant. Consequently, the present data show that the enzyme is multifunctional, and could be renamed ST3Gal(NAc) II. In contrast to ST3Gal II, ST3Gal III did not transfer to the X(2) pentasaccharide. The Neu5Ac alpha 2-3GalNAc linkage of sialyl X(2) was cleaved by sialidases from Arthrobacter ureafaciens and Clostridium perfringens, but resisted the action of sialidases from Newcastle disease virus and Streptococcus pneumoniae. Therefore, the latter two enzymes cannot be used to differentiate between Neu5Ac alpha 2-3GalNAc and Neu5Ac alpha 2-6GalNAc linkages, as has been assumed previously.     

Fucosyl-GM1a, an endoglycoceramidase-resistant ganglioside of porcine brain

The use of bovine brain has been prohibited in many countries because of the world-wide prevalence of mad cow disease, and thus porcine brain is expected to be a new source for the preparation of gangliosides. Here, we report the presence of a ganglioside in porcine brain which is strongly resistant to hydrolysis by endoglycoceramidase, an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. Five major gangliosides (designated PBG-1, 2, 3, 4, 5) were extracted from porcine brain by Folch's partition, followed by mild alkaline hydrolysis and PBA column chromatography. We found that PBG-2, but not the others, was strongly resistant to hydrolysis by the enzyme. After the purification of PBG-2 with Q-Sepharose, Silica gel 60 and Prosep-PB chromatographies, the structure of PBG-2 was determined by GC, GC-MS, FAB-MS and NMR spectroscopy as Fucalpha1-2Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-4Glcbeta1-1'Cer (fucosyl-GM1a). The ceramide was mainly composed of C18:0 and C20:0 fatty acids and d18:1 and d20:1 sphingoid bases. The apparent kcat/Km for fucosyl-GM1a was found to be 30 times lower than that for GM1a, indicating that terminal fucosylation makes GM1a resistant to hydrolysis by the enzyme. This report indicates the usefulness of endoglycoceramidase to prepare fucosyl-GM1a from porcine brain.     

Selection of ganglioside GM1-binding peptides by using a phage library.

Ganglioside Gal beta1 --> 3GalNAc beta1 --> 4(NeuAc alpha2 --> 3) Gal beta1 --> 4Glc beta1 -->1'Cer (GM1)-binding peptides were obtained from a phage-displayed pentadecapeptide library by an affinity selection. The selection processes were in situ-monitored by a quartz-crystal microbalance method, on which a ganglioside GM1 monolayer was transferred. After five rounds of biopanning, the DNA sequencing of 18 selected phages showed that only three individual clones were selected. The peptide sequences of the random region were found to be DFRRLPGAFWQLRQP, GWWYKGRARPVSAVA and VWRLLAPPFSNRLLP. Binding constants of these phage clones to the GM1 monolayer were 10(10) M(-1). Three synthetic pentadecapeptides inhibited the binding of cholera toxin B subunit to the GM1 monolayer with an IC50 of 24, 13 and 1.0 microM, respectively. These peptides will be useful for searching functional roles of ganglioside GMI.     

The major acidic glycolipids from the kidney of the Pacific salmon (Oncorhynchus keta): characterization of a novel ganglioside, fucosyl-N-acetylgalactosaminyl-GM1.

Two fractions of a major ganglioside from the kidney of the pacific salmon, Oncorhynchus keta, were eluted from a DEAE-Sephadex column in the monosialosyl fraction. The faster moving ganglioside (X1) on TLC was separated from the slower moving one (X2) by HPLC using a silica beads column. By methylation analysis, chemical and enzymatic degradation, reaction with monoclonal antibodies, LSIMS, and (1)H-NMR spectroscopy, X1 was determined to be a monosialosyl ganglioside belonging to the ganglio-series with a unique Fucalpha1-3GalNAc linkage at the nonreducing terminal: Fucalpha1-3GalNAcbeta1-3Galbeta1-3GalNAcbeta1-4[ NeuAcalpha2-3]Galbeta 1-4Glcbeta1-1Cer. Analysis of the lipophilic moiety indicated predominance of 24:1 fatty acid in combination with sphingenine. X2 was found to have a glycon structure identical to X1. The ceramide of X2 consisted predominantly of saturated fatty acids (18:0 and 16:0). The tissue concentrations of X1 and X2 in kidney were 3.7 and 2.8 nmol/g, respectively.     

Receptor specificities of human respiroviruses

Through their hemagglutinin-neuraminidase glycoprotein, parainfluenza viruses bind to sialic acid-containing glycoconjugates to initiate infection. Although the virus-receptor interaction is a key factor of infection, the exact nature of the receptors that human parainfluenza viruses recognize has not been determined. We evaluated the abilities of human parainfluenza virus types 1 (hPIV-1) and 3 (hPIV-3) to bind to different types of gangliosides. Both hPIV-1 and hPIV-3 preferentially bound to neolacto-series gangliosides containing a terminal N-acetylneuraminic acid (NeuAc) linked to N-acetyllactosamine (Galbeta1-4GlcNAc) by the alpha2-3 linkage (NeuAcalpha2-3Galbeta1-4GlcNAc). Unlike hPIV-1, hPIV-3 bound to gangliosides with a terminal NeuAc linked to Galbeta1-4GlcNAc through an alpha2-6 linkage (NeuAcalpha2-6Galbeta1-4GlcNAc) or to gangliosides with a different sialic acid, N-glycolylneuraminic acid (NeuGc), linked to Galbeta1-4GlcNAc (NeuGcalpha2-3Galbeta1-4GlcNAc). These results indicate that the molecular species of glycoconjugate that hPIV-1 recognizes are more limited than those recognized by hPIV-3. Further analysis using purified gangliosides revealed that the oligosaccharide core structure is also an important element for binding. Gangliosides that contain branched N-acetyllactosaminoglycans in their core structure showed higher avidity than those without them. Agglutination of human, cow, and guinea pig erythrocytes but not equine erythrocytes by hPIV-1 and hPIV-3 correlated well with the presence or the absence of sialic acid-linked branched N-acetyllactosaminoglycans on the cell surface. Finally, NeuAcalpha2-3I, which bound to both viruses, inhibited virus infection of Lewis lung carcinoma-monkey kidney cells in a dose-dependent manner. We conclude that hPIV-1 and hPIV-3 preferentially recognize oligosaccharides containing branched N-acetyllactosaminoglycans with terminal NeuAcalpha2-3Gal as receptors and that hPIV-3 also recognizes NeuAcalpha2-6Gal- or NeuGcalpha2-3Gal-containing receptors. These findings provide important information that can be used to develop inhibitors that prevent human parainfluenza virus infection.     

Carbohydrate specificity of an agglutinin isolated from the root of Trichosanthes kirilowii

The root of Trichosanthes kirilowii, which has been used as Chinese folk medicine for more than two thousand years, contains a Gal specific lectin (TKA). In order to elucidate its binding roles, the carbohydrate specificities of TKA were studied by enzyme linked lectinosorbent assay (ELLSA) and by inhibition of lectin-glycoform binding. Among glycoproteins (gp) tested, TKA reacted strongly with complex carbohydrates with Galbeta1-->4GlcNAc clusters as internal or core structures (human blood group ABH active glycoproteins from human ovarian cyst fluids, hog gastric mucin, and fetuin), porcine salivary glycoprotein and its asialo product, but it was inactive with heparin and mannan (negative control). Of the sugar inhibitors tested for inhibition of binding, Neu5Ac alpha2-->3/6Galbeta1-->4Glc was the best and about 4, 14.6 and 27.7 times more active than Galbeta1-->4GlcNAc(II), Galbeta1-->3GalNAc(T) and Gal, respectively. From these results, it is suggested that this agglutinin is specific for terminal or internal polyvalent Galbeta1-->4GlcNAcbeta1-->, terminal Neu5Ac alpha2-->3/6Galbeta1-->4Glc and cluster forms of Galbeta1-->3GalNAc alpha residues. The unusual affinity of TKA for terminal and internal Galbeta1-->glycotopes may be used to explain the possible attachment roles of this agglutinin in this folk medicine to target cells.     

Enzymatic characterization of beta D-galactoside alpha2 leads to 3 sialyltransferase from porcine submaxillary gland.

The substrate requirements, linkage specificity, and kinetic mechanism of a pure sialyltransferase from porcine submaxillary glands have been examined. The enzyme transfers sialic acid from the donor nucleotide, CMP-NeuAc, into the sequence NeuAcalpha2 leads to 3Galbeta1 leads to 3GalNAc, which is found in both glycoproteins and gangliosides. It forms only the alpha2 leads to 3 linkage with the disaccharide Gal/beta1 leads to 3GalNAc or antifreeze glycoprotein, which, along with asialoglycoproteins containing the sequence Gal/beta1 leads to 3GalNAcalpha1 leads to O-Thr/Ser, are the best acceptor substrates. Low molecular weight galactosides linked beta1 leads to 3 to glycose residues other than N-acetylgalactosamine are poor acceptors with relatively high Km values, while those in beta1 leads to 4 or beta1 leads to 6 linkages have both high Km and low Vmax. With glycoprotein and ganglioside acceptors this substrate specificity appears to be even more strict, with the sequence Gal/beta1 leads to 3GalNAc serving as the exclusive acceptor. Thus the present enzyme is not responsible either for the sequence, NeuAcalpha2 leads to 3Galbeta1 leads to 4GlcNAc, found in the asparagine-linked chains of certain glycoproteins, or for the synthesis of hematoside, NeuAcalpha2 leads to 3Galbeta1 leads to 4Glcbeta1 leads to 1Cer. Initial rate kinetic studies, with and without inhibitors, suggest that the transferase has an equilibrium random order mechanism.     

Binding profile of Artocarpus integrifolia agglutinin (Jacalin)

Artocarpus integrifolia agglutinin (Jacalin) from the seeds of jack fruits has attracted considerable attention for its diverse biological activities and has been recognized as a Galbeta1-->3GalNAc (T) specific lectin. In previous studies, the information of its binding was limited to the inhibition results of monosaccharides and several T related disaccharides, but its interaction with other carbohydrate structural units occurring in natural glycans has not been characterized. For this reason, the binding profile of this lectin was studied by enzyme linked lectinosorbent assay (ELLSA) with our glycan/ligand collection. Among glycoproteins (gps) tested for binding, high density of multi-Galbeta1-->3GalNAcalpha1--> (mT(alpha)) and GalNAcalpha1-->Ser/Thr (mTn) containing gps reacted most avidly with Jacalin. As inhibitors expressed as nanograms yielding 50% inhibition, these mT(alpha) and mTn containing glycans were about 7.1 x 10(3), 4.0 x 10(5), and 7.8 x 10(5) times more potent than monomeric T(alpha), GalNAc, and Gal. Of the sugars tested and expressed as nanomoles for 50% inhibition, Tn containing peptides, T(alpha), and the human P blood group active disaccharide (P(alpha), GalNAcbeta1-->3Galalpha1-->) were the best and about 283 times more active than Gal. We conclude that the most potent ligands for this lectin are mTn, mT, and possibly P(alpha) glycotopes, while GalNAcbeta1-->4Galbeta1-->, GalNAcalpha1-->3Gal, GalNAcalpha1-->3GalNAc, and Galalpha1-->3Gal determinants were poor inhibitors. Thus, the overall binding profile of Jacalin can be defined in decreasing order as high density of mTn, and mT(alpha) > simple Tn cluster > monomeric T(alpha) > monomeric P(alpha) > monomeric Tn > monomeric T > GalNAc > Gal > Methylalpha1-->Man z.Gt; Man and Glc (inactive). Our finding should aid in the selection of this lectin for biological applications.     

Synthesis of Neu5Ac(alpha2-->6)[Gal(alpha1-->3)]GalNAcalpha and Neu5Ac(alpha2-->6)[Gal(beta1-->3)]GalNAcalpha trisaccharides as protected trifluoroacetamidopropyl glycosides

Protected sialo-containing trisaccharides, fragments of oligosaccharide chains of mucin glycoproteins, were synthesized. Regioselective sialylation of the primary hydroxyl group of (3-fluoroacetamidopropyl)-2-azido-2-deoxy-3-O-(2,3,4,6-tetra-O-ben zyl)-alpha -D-galactopyranosyl)-alpha-D-galactopyranoside with methyl ester of peracetyl-beta-ethylthioglycoside of N-acetylneuraminic acid in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid (or its trimethylsilyl ester) yielded 39 and 25% of alpha- and beta-sialyl-(2-->6)biosides, respectively. Catalytic hydrogenolysis of the azide and benzyl groups of the alpha-anomer followed by N- and O-acetylation gave target trifluoroacetamidopropyl glycoside, Neu5Ac(alpha 2-->6)[Gal(alpha 1-->3)]GalNAc alpha-OSp, as a peracetate. An analogous coupling of the sialyl donor with (3-fluoroacetamidopropyl)-2-acetamido-2-deoxy-3-O- (2,3,4,6-tetra-O-acetyl)-beta-D-galactopyranosyl)-alpha-D-galactopyranos ide affords acetylated trifluoroacetamidopropyl glycoside Neu5Ac(alpha 2-->6)[Gal(beta 1-->3)]GalNAc alpha-OSp in a yield of 15% and the corresponding Neu5Ac(beta 2-->6)-anomer in a yield of 12%. After O-deacetylation and N-detrifluoroacetylation, these sialylbiosides can be used as ligands in preparing neoglycoconjugates.