Evidence for a malarial parasite interaction site on the major transmembrane protein of the human erythrocyte.

Soluble oligosaccharides derived from the surface of human erythrocytes were tested for their ability to competitively inhibit invasion of erythrocytes by Plasmodium falciparum, a malarial parasite. Invasion was most effectively inhibited by erythroglycan, a carbohydrate component of the band 3 transmembrane protein. The lactosamine chains of erythroglycan contributed much of the inhibitory activity. This indication of a primary parasite interaction site on band 3 supports a role for this protein in mediating the radical alterations of the erythrocyte cytoskeleton that accompany invasion.

Eicosapentaenoic and Arachidonic Acids from Phytophthora infestans Elicit Fungitoxic Sesquiterpenes in the Potato.

Mycelial extracts from Phytophthora infestans caused necrosis and elicited the accumulation of antimicrobial stress metabolites in potato tubers. A portion of the material with elicitor activity could be extracted from the mycelium by a mixture of chloroform and methanol. The most active elicitors of stress metabolites in these extracts were eicosapentaenoic and arachidonic acids. These fatty acids were found in either free or esterified form in all active fractions of the mycelial extracts.

On the relationship between glycerophosphoglycolipids and lipoteichoic acids of gram-positive bacteria. III. Di(glycerophospho)-acylkojibiosyldiacylglycerol and related compounds from Streptococcus lactis NCDO 712.

1. Streptococcus lactis NCDO 712 contains at lease three unusually polar glycerophosphoglycolipids. One of them was composed of D-glucose, glycerol, fatty acid ester, and phosphorus in the molar ratio of approx. 2 : 3 : 3 : 2. The structure was established as 1,2-di-O-acyl-3-O-[6-(sn-glycero-1-phospho-3-sn-glycero-1-phospho)-alpha-D-glucopyranosyl-(1 leads to 2)-(6-O-acyl-alpha-D-glucopyranosyl)]-glycerol. 2. The second glycerophosphoglycolipid was shown to have the same core structure but is lacking the carbohydrate-linked fatty acid. The third glycero-phosphoglycolipid is a glycosylated derivative of the first one bearing an alpha-galactosyl residue at position 2 of the inner glycolipid-linked glycerophosphate moiety. 3. These novel phosphoglycolipids are considered to be the so far missing link between simple glycerophosphoglycolipids and lipoteichoic acids of Gram-positive bacteria.

On the relationship between glycerophosphoglycolipids and lipoteichoic acids in Gram-positive bacteria. II. Structures of glycerophosphoglycolipids.

1. Eight glycerophosphoglycolipids were isolated from six Gram-positive bacteria. Besides sn-glycero-1-phospho-beta-gentiobiosyldiacylglycerol (i) and sn-glycero-1-phospho-alpha-kojibiosyldiacylglycerol (ii), three novel structures have been established: 1,2-di-O-acyl-3-O-[6-(sn-glycero-1-phospho)-alpha-D-glucopyranosyl-(1 leads to 2)-(6-O-acyl-alpha-D-glucopyranosyl)]glycerol (iii), 1,2-di-O-acyl-3-O-[6-(sn-glycero-1-phospho)-beta-D-glucopyranosyl-(1 leads to 6)-alpha-D-galactopyranosyl-(1 leads to 2)-alpha-D-glucopyranosyl]glycerol (iv), and 1,2-di-O-acyl-3-O-[6-(sn-glycero-1-phospho)-beta-D-glucopyranosyl-(1 leads to 6)-alpha-D-galactopyranosyl-(1 leads to 2)-(6-O-acyl-alpha-D-glucopyranosyl)]glycerol (v). 2. Compound i was isolated from Bacillus licheniformis, Bacillus subtilis and Staphylococcus aureus, compound ii from a group B Streptococcus, compounds ii and iii from Streptococcus lactis, compounds iv and v from Lactobacillus casei. Lactobacillus plantarum contained besides compounds iv and v a glycerophosphate derivative of 1,2-di-O-acyl-3-O-[alpha-D-galactopyranosyl (1 leads to 2)-alpha-D-glucopyranosyl]glycerol. 3. Identical structural features of the described glycerophosphoglycolipids and the corresponding lipoteichoic acids are discussed.

Studies on the relationship between glycerophosphoglycolipids and lipoteichoic acids. IV. Trigalactosylglycerophospho-acylkojibiosyldiacylglycerol and related compounds from Streptococcus lactis Kiel 42172.

Streptococcus lactis Kiel 42172 contains at least six unusually polar glycerophosphoglycolipids. The predominant one was composed of D-galactose, D-glucose, glycerol, acyl groups and phosphorus in a molar ratio of approx. 3 : 2 : 2 : 3 : 1. By analysis of the breakdown products of HF hydrolysis and Smith-degradation the structure was established to be [Galp (alpha 1 leads to 6)Galp(alpha 1 leads to 3)-sn-glycero(2 comes from 1 alpha Galp)-1-phospho] leads to 6Glcp(alpha 1 leads to 2), acyl leads to Glcp(alpha 1 leads to 3)-acyl2Gro. By HF hydrolysis the other compounds were shown to be in the main also derivatives of GroP leads to 6Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3)acyl2Gro but they released as water-soluble glycosides Gal(alpha 1 leads to 2)Gro, Gal(alpha 1 leads to 3)Gro, Gal(alpha 1 leads to 3)Gro(2 comes from 1 alpha Gal), Gal(alpha 1 leads to 6)Gal(alpha 1 leads to 3)Gro and Gal(alpha 1 leads to 6)Gal-(alpha 1 leads to 6)Gal(alpha 1 leads to 3)Gro(2 comes from 1 alpha Gal), respectively. In the lipid extract Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3)acyl2Gro and GroP leads to 6Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3) acyl2Gro were also observed. This set of compounds is proposed to constitute a biosynthetic series reflecting the individual steps in the synthesis of the lipoteichoic acid of Streptococcus lactis Kiel 42172 which is made up by the same lipid anchor and a non-classical poly(galabiosyl, galactosyl glycerophosphate)-chain (Koch, H.U. and Fischer, W. (1978) Biochemistry 17, 5275--5281).

On the relationship between glycerophosphoglycolipids and lipoteichoic acids in Gram-positive bacteria. I. The occurrence of phosphoglycolipids.

1. Gram-positive bacteria out of the families of Streptococcaceae, Lactobacillaceae, Micrococcaceae and Bacillaceae were investigated with respect to the occurrence and the concentration of phosphoglycolipids. 2. Phosphatidylglycolipids occur exclusively in group D Streptococci and in Streptococcus hemolyticus D-58. Phosphatidyl-alpha-kojibiosyldiacylglycerol, the prevalent species, accounts for up to 28% of the polar lipids. The related glycerophospho-phosphatidyl-alpha-kojibiosyldiacylglycerol is restricted to Streptococcus faecalis. 3. Glycerophosphoglycolipids, usually minor components, comprise thirteen compounds most of which have so far not been described. Except Micrococcus lysodeikticus all examined bacteria contained one or more glycerophosphoglycolipids. Their occurrence parallels, therefore, that of lipoteichoic acids, which supports the hypothesis of a metabolic relationship between these two membrane components.

Structures of the asparagine-linked sugar chains of laminin.

This investigation describes the isolation and characterization of oligosaccharides of the basement membrane glycoprotein, laminin. Pronase-released glycopeptides of isolated laminin, from a mouse Engelbreth-Holm-Swarm tumor, were fractionated using a combination of gel permeation chromatography and Con A-Sepharose affinity chromatography. The glycopeptides were analyzed for sugar linkage patterns by methylation analysis. Glycopeptides and hydrazine-released oligosaccharides were further analyzed using endo-beta-galactosidase, endo-beta-N-acetylglucosaminidase H and specific exoglycosidases in conjunction with calibrated gel permeation chromatography. Based on these experiments, murine tumor laminin was shown to contain asparagine-linked oligosaccharides with the following structures: bi-, tri- and tetraantennary complex-type oligosaccharides; polylactosaminyl side chains containing Gal(beta 1----4)GlcNAc(beta 1----3) repeating units attached to the trimannose core portion of the bi-, tri- and tetraantennary complex-type oligosaccharides; unusual complex-type oligosaccharides terminated at the nonreducing end with sialic acid, alpha-galactose, beta-galactose and beta-N-acetylglucosamine; alpha-galactosyl residues linked to N-acetyllactosamine sequences; high-mannose-type oligosaccharides. These results, in conjunction with analytical data, indicate that most of the carbohydrate of this laminin is N-linked to asparagine and that there are about 43 such N-linked oligosaccharides per laminin molecule.

Sequence of the V. parahaemolyticus gene for cytoplasmic N, N'-diacetylchitobiase and homology with related enzymes.

The nucleotide sequence of the gene encoding the cytoplasmic N, N'-diacetylchitobiase [EC 3.2.1.14] from Vibrio parahaemolyticus (ATCC #27969) has been determined. The deduced peptide sequence of this unusual beta-hexosaminidase surprisingly shows minimum evolutionary relationship to two other reported N, N'-diacetylchitobiases from vibrios, except in highly conserved regions which are also homologous to lysosomal beta-hexosaminidases from eukaryotes including humans. In contrast, the two other beta-hexosaminidases from vibrios with reported sequences are much more closely related to each other. This novel 85 kDa cytoplasmic glycosyl hydrolase with restricted specificity participates in the high level utilization of chitin-derived 2-deoxy-2-acetamido-D-glucose (GlcNAc) by vibrios as one of two parallel pathways for the metabolism of N,N'-diacetylchitobiose [Bassler, B.L., Yu, C., Lee, Y.C., and Roseman, S. (1991) J. Biol. Chem. 266, 24276-24286]. These pathways use chitin-binding proteins for the adherence of the bacterial chitinase to the substrate, and an extracellular chitinase and a periplasmic chitodextrinase to produce N,N'-diacetylchitobiose. The V. parahaemolyticus cytoplasmic N,N'-diacetyl-chitobiase reported herein appears to be a unique protein, lacking a signal sequence, and genetically distant from other known chitinoclastic beta-N,N'-diacetyl-hexosaminidases. This is consistent with its limited substrate specificity to small GlcNAc terminated oligosaccharides and its cytoplasmic rather than periplasmic localization.

Thermostable, salt tolerant, wide pH range novel chitobiase from Vibrio parahemolyticus: isolation, characterization, molecular cloning, and expression.

A chitobiase gene from Vibrio parahemolyticus was cloned into plasmid pUC18 in Escherichia coli strain DH5 alpha. The plasmid construct, pC120, contained a 6.4 kb Vibrio DNA insert. The recombinant gene expressed chitobiase [EC 3.2.1.30] activity similar to that found in the native Vibrio. The enzyme was purified by ion exchange, hydroxylapatite and gel permeation chromatographies, and exhibited an apparent molecular weight of 80 kDa on SDS-polyacrylamide gel electrophoresis. Chitobiose and 6 more substrates, including beta-N-acetyl galactosamine glycosides, were hydrolyzed by the recombinant chitobiase, indicating its putative classification as an hexosaminidase [EC 3.2.1.52]. The enzyme was resistant to denaturation by 2 M NaCl, thermostable at 45 degrees C and active over a very unusual (for glycosyl hydrolases) pH range, from 4 to 10. The purified cloned chitobiase gave 4 closely focussed bands on an isoelectric focusing gel, at pH 4 to 6.5. The N-terminal 43 amino acid sequence shows no homology with other proteins in commercial databanks or in the literature, and from its N-terminal sequence, appears to be a novel protein, unrelated in sequence to chitobiases from other Vibrios reported and unrelated to hexosaminidases from other organisms.

Occurrence of an unusual amount of an odd-numbered fatty acid in glycosphingolipids from human cataracts.

Human cataractous glycosphingolipids were isolated and purified according to previously established procedures. Fatty acid analyses of the purified glycosphingolipid revealed the presence of a significant amount of an odd-chain fatty acid. This was confirmed by methane (CH4) chemical ionization mass spectrometry, which showed characteristic ions at m/z 397, 425, 437, and 365. These ions facilitated the determination of molecular weight and the assignment of C25:0 (n-pentacosanoic acid) to this fatty acid in question. This may be the first time that such an unusual distribution of a single odd-chain fatty acid has been shown to occur in glycosphingolipid from any tissue.

Chemistry of human erythrocyte polylactosamine glycopeptides (erythroglycans) as related to ABH blood group antigenic determinants.

Human erythrocytes bear carbohydrates linked to both proteins and lipids. The majority of the carbohydrates is carried on two proteins: 1) Band 3 (which carries a high molecular weight polylactosamine, variously termed "Erythroglycan", "poly(glycosyl)peptide" or "lactosaminoglycan" and 2) Glycophorin A (which carries 15 O-linked tetrasaccharides and 1 triantennary N-linked structure). The remainder of carbohydrates are carried mainly by a few other glycoproteins (glycophorins B,C, the glucose transporter and others) with a minor amount carried by glycosphingolipids. This report concerns the Band 3 carbohydrate and its content of potential ABH-active sites. We have determined that an average number of two [Fuc1----2Ga11----4GlcNAc] sequences are carried by each "erythroglycan", polylactosamine N-linked oligosaccharide. One such large oligosaccharide occurs on each molecule of Band 3 polypeptide of which there are 1,000,000 copies per erythrocyte. Therefore, about 2,000,000 possible ABH sites are borne by Band 3 on each erythrocyte. This approximates the number of immunologically estimated ABH sites on human erythrocytes. Thus, Band 3 carbohydrate probably carries the majority of ABH substance on human red cells, while other glycoproteins and glycosphingolipids carry a minor fraction.

Efficacy of vetiver oil and nootkatone as soil barriers against Formosan subterranean termite (Isoptera: Rhinotermitidae).

Vetiver oil and its components nootkatone and cedrene were assessed as sand treatments for their efficacy to disrupt food recruitment by Coptotermes formosanus Shiraki. Termites were required to tunnel through sand treated with vetiver oil, nootkatone, cedrene, or untreated sand to reach a food source. Results showed that sand treated with vetiver oil or nootkatone disrupted termite tunneling behavior. As a consequence, after 21 d, wood consumption and termite survival were significantly lower compared with cedrene-treated or untreated sand treatments. Sand treated with vetiver oil or nootkatone at 100 microg/g substrate were effective barriers to termites.

A calculation of all possible oligosaccharide isomers both branched and linear yields 1.05 x 10(12) structures for a reducing hexasaccharide: the Isomer Barrier to development of single-method saccharide sequencing or synthesis systems.

The number of all possible linear and branched isomers of a hexasaccharide was calculated and found to be > 1.05 x 10(12). This large number defines the Isomer Barrier, a persistent technological barrier to the development of a single analytical method for the absolute characterization of carbohydrates, regardless of sample quantity. Because of this isomer barrier, no single method can be employed to determine complete oligosaccharide structure in 100 nmol amounts with the same assurance that can be achieved for 100 pmol amounts with single-procedure Edman peptide or Sanger DNA sequencing methods. Difficulties in the development of facile synthetic schemes for oligosaccharides are also explained by this large number. No current method of chemical or physical analysis has the resolution necessary to distinguish among 10(12) structures having the same mass. Therefore the 'characterization' of a middle-weight oligosaccharide solely by NMR or mass spectrometry necessarily contains a very large margin of error. Greater uncertainty accompanies results performed solely by sequential enzyme degradation followed by gel-permeation chromatography or electrophoresis, as touted by some commercial advertisements. Much of the literature which uses these single methods to 'characterize' complex carbohydrates is, therefore, in question, and journals should beware of publishing structural characterizations unless the authors reveal all alternate possible structures which could result from their analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental study of human fetal placental fibronectin: alterations in carbohydrates of tissue fibronectin during gestation.

Human term placental tissue fibronectin contains about twice the carbohydrate content of human adult plasma fibronectin or fetal plasma fibronectin. The chief difference is the presence of substantial amounts of large N-linked polylactosamine chains on the placental fibronectin. The large carbohydrate on placental tissue fibronectin weakens the binding of fibronectin to denatured collagen. To examine whether a developmental change takes place in the placental fibronectin during gestation, fibronectin was isolated from placentas of different developmental stages beginning with the first trimester and ending with term. Polylactosamine carbohydrate, as well as total quantity of carbohydrate, increased steadily during gestation, reaching a maximum at term of more than 9% carbohydrate. Weakened binding of fibronectin to collagen occurred near the end of gestation concomitant with an increase in the quantity of larger polylactosamine glycopeptides. Relationships among these developmental changes, the impending birth, and the end of the function of the placenta remain to be investigated.