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Hyaluronic acid was obtained from filtrates of heat-killed cultures of Streptococcus pyogenes group A, strain K56, by simple ethanol precipitation and treatment with an adsorbent. The hyaluronic acid is pure as judged from chemical and sedimentation analyses. Particles of streptococcal bacteriophage 12/12 were isolated from phage-lysed group A streptococci by polyethylene glycol precipitation and isopyenic centrifugation. Electron micrographs of negatively stained preparations showed a typical Bradley group B virus with a long, flexible, cross-striated tail and a knob- or star-like structure at the distal tip of the tail. The hyaluronic acid is depolymerized upon incubation with the phage 12/12 virions. After extensive digestion, a mixture of at least four oligosaccharides is formed, the two smallest of which are a tetra- and octasaccharide terminating in reducing N-acetyl-D-glucosamine. The tetrasaccharide shows an absorption maximum at 231.5 nm with a molar extinction coefficient epsilon = 4820 litres X mole-1 X cm-1, and it is therefore concluded that the bacteriophage-borne hyaluronidase catalyses a beta-elimination. Accordingly it is classified as a hyaluronate lyase (EC 4.2.99.1).

Earlier studies are extended to the higher-numbered K-types of Klebsiella, several of which may now be fitted into the previously found groups. Additional correlations between chemical structure and serological specificity are given, not only among the higher K-types, but also for several which were previously uninterpretable because structures were not known at the time. K1, K5, K6, K7 and K56 are shown to form a related group, although the reasons for their cross-reactivities are not always the same.

An earlier study of more than 60 Klebsiella K-polysaccharides in anti-pneumococcal sera of many capsular types provided the impetus for the present work. With anti-Klebsiella sera, some of these cross-reactions are now shown to be reciprocal. In most instances in which several or many K-substances precipitated antipneumococcal sera of a given type, these same K-polysaccharides were cross-reactive with one or more anti-Klebsiella sera belonging to the same group. For example, the polysaccharides K1, 15, 24, 31, 39, 42, and 59 precipitated an equine antipneumococcal type X serum. Pneumococcal capsular polysaccharide type X and Klebsiella polysaccharides K15, 24, 31, 39, 42, and 59 precipitated one or both of the equine anti-Klebsiella K1 sera available. Similar results with anti-Klebsiella rabbit sera to other K-types show the validity of this approach in revealing hitherto unknown serological, and hence chemical, relationships between the K-types.

The thrombin-like serine protease ancrod from the Malayan pit viper Agkistrodon rhodostoma was expressed in mouse epithelial cells (C127). Oligosaccharide constituents were liberated from tryptic glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F. Neutral oligosaccharide alditols obtained after reduction and enzymic desialylation were separated by two-dimensional HPLC and characterized by methylation analysis, liquid secondary-ion mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sequential degradation with exoglycosidases. In contrast to natural ancrod, the recombinant glycoprotein carries exclusively diantennary, triantennary and tetraantennary N-glycans with Gal beta 4 GlcNAc beta (type-2) antennae which were, in part, further substituted by host-cell-specific structural elements such as Gal alpha 3 residues or N-acetyllactosamine repeats. As a characteristic feature, a substantial proportion of the oligosaccharides bears a GalNAc beta 4Glc-NAc antenna. Studies at the level of individual N-glycosylation sites demonstrated that glycans with N, N'-diacetyllactosediamine units are not specifically attached but occur at all sites in varying amounts. Hence, the putative recognition signal (Pro70-Lys-Lys) for glycoprotein hormone N-acetylgalactosaminyltransferase, present in this glycoprotein in close proximity to Asn79, does not convey site-specific transfer of GalNAc residues in these cells.

The disulfide-bonding pattern of glycoprotein 70 (gp70), the surface glycoprotein (SU) encoded by the envelope gene of polytropic Friend milk cell focus-inducing virus, was elucidated and compared with that of glycoprotein 71 (gp71), the corresponding glycoprotein of the ecotropic Friend murine leukemia virus, which had previously been determined (M. Linder, D. Linder, J. Hahnen, H.-H. Schott, and Stirm, Eur. J. Biochem. 203:65-73, 1992). In the carboxy-terminal constant domain, in which these glycoproteins have about 97% sequence homology, the location of the four disulfide bonds was found to be analogous. In the amino-terminal differential domain, with about 37% sequence homology, 8 of the 12 cysteine residues of the ecotropic SU are conserved in the polytropic SU. In this domain, a similar clustering of disulfide bonds was detected, which led to the identification of three distinct disulfide-bonded regions in both glycoproteins. However, because of deletions and sequence deviations, the glycoproteins must have significantly different three-dimensional structures in these regions. Since the receptor-binding functions of both glycoproteins have been attributed to their amino-terminal domains and since each binds to a different receptor, these disulfide-bonded structures are likely candidates for receptor-binding functions. Limited proteolysis of both glycoproteins with various endoproteinases led to the identification of preferential proteolytic sites between disulfide-bonded regions, at the beginning of the hypervariable proline-rich region, and between differential and constant domains, further confirming the structural organization of the folded glycoproteins.

Both genomic and cDNA clones have been isolated encoding the major sheath glycoprotein, gp22, of Litomosoides carinii microfilariae. The mature gp22 mRNA is shown to result from both trans-splicing of a 22-nucleotide 5'-leader sequence to an acceptor site at position 313 of the pre-mRNA, immediately upstream from the start codon, and from cis-splicing of a 117-nt intron located within the coding sequence. Cis-splicing precedes the trans-splicing reaction. The gp22 reading frame of 148 codons has the inferred structure of a prepro-protein and includes a leader peptide and a pro-segment ahead of the known N terminus of the mature, extracellular protein of 105 amino acids. The N-terminal part of that protein contains five repeats of an elastin-related pentapeptide sequence, which, together with a proline-threonine segment between two Cys clusters in the center and at its C terminus, may cause an elongated conformation with an apparent molecular size of 22 kDa in contrast to the calculated M(r) of 11,200.

Litomosoides carinii microfilariae were exsheathed by freezing and thawing, and the sheaths were separated by filtration. Samples of pure sheaths thus obtained were hydrolyzed, methanolyzed or oxidized with nitric acid under pressure at 300°C, respectively, and were analyzed for amino acids, sugars, fatty acids or for metal ions and phosphorus. Almost 75% of the sheath dry weight could thus be accounted for. Amino acids (55 weight %) were the major constituents, and amongst these glutamine and proline (approximately 11% each). The detection of 2% cysteine/cystine indicated the possible presence of disulfide crosslinks. Besides amino acids, approximately 8% of sugars — roughly equimolar amounts of ( N -acetyl)galactosamine and uronic acids — 1.5% of monovalent cations (Na + and K + ) and 9.5% of phosphate were detected. No appreciable amounts of fatty acids, neutral sugars, neuraminic acid, or ( N -acetyl)glucosamine (i.e. no chitin) were found.

Microfilarial sheaths of Litomosoides carinii were isolated and extracted with 2% sodium dodecyl sulfate (SDS) and 5% 2-mercaptoethanol (2ME). Extraction with SDS alone did not alter the ultrastructure of the sheaths and yielded five polypeptides (27-67 kDa) that were not recognized by antibodies of infected hosts but reacted with antibodies to host-serum proteins. 2ME treatment caused partial solubilization of the sheaths (45% as determined by amino acid analysis), which could be further improved by combining 2ME with SDS. The remainder showed filamentous/threadlike structures on electron microscopic examination. As compared with whole sheaths, the insoluble proportion was markedly enriched in alanine and cysteine but contained less galactosamine, serine, and threonine. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of 2ME/SDS-extractable components showed 12-16 bands of 14- greater than 120 kDa. A predominant component had an apparent molecular mass of 22 kDa. Two bands (42 and 120 kDa) could be stained with Coomassie blue but showed "negative" staining when gels were stained with silver. Several components (but not the 22-kDa polypeptide) bore phosphocholine epitopes. Apart from the negatively staining bands, most of the 2ME-soluble sheath components were recognized by antibodies of L. carinii-infected Mastomys coucha. Except for several polypeptides that had been unspecifically recognized by IgM, the antibody response to sheath components started at the end of the prepatent period.

Burgia malayi and B. pahangi microfilariae were isolated from the blood of infected Mastomys natalensis, and were exsheathed by freezing, thawing and agitation. Pure sheaths were obtained by a filtration procedure. The sheaths were found to contain about 95 mol% of amino acids, with proline, glutamic acid/glutamine, alanine, cysteine/cystine and glycine being the major components, and 5 mol% of carbohydrates, notably (N-acetyl)galactosamine, but no (N-acetyl)glucosamine.

In several filarial genera the first stage larvae (microfilariae) are enclosed by an eggshell-derived sheath that provides a major interface between the parasite and the host immune system. Analysis of the polypeptide constituents of the microfilarial sheath from the cotton rat filaria Litomosoides sigmodontis identified two abundant surface glycoproteins: Shp3a and Shp3. The corresponding genes and the orthologues of the human parasite Brugia malayi and the rodent filaria Brugia pahangi were cloned and sequenced. They encode secreted, mucin-like proteins with N-terminal Ser/Thr-rich repeats and a C-terminal anchor domain rich in aromatic amino acids. About 75% of the protein molecular masses result from post-translational modifications. The Ser/Thr-rich motifs are supposed to serve as targets for dimethylaminoethanol-phosphate substitutions. These modifications were detected only on the sheaths of the late developmental stage of stretched microfilariae, corresponding with the expression of the proteins in the epithelium of the distal part of the uterus and the specific transcription of shp3 and shp3a in the anterior female worm segment. Genomic analysis of all three species demonstrated a conserved linkage of the two genes. Their transcripts undergo cis- and trans-splicing. The transcription start sites of the primary transcripts were determined for the L. sigmodontis genes. The core promoter regions are remarkably conserved between the paralogue genes Ls-shp3a and Ls-shp3 and their orthologues in Brugia, implicating conserved regulatory elements.

A 120 kDa antigen produced by juvenile female Litomosoides sigmodontis (Juv-p120) was isolated and purified. The amino acid composition of the molecule was determined. Juv-p120 was shown to be highly modified with N,N-dimethyl-aminoethanol (28.4 mol%). Treatment of Juv-p120 with potassium hydroxide (beta-elimination) or with sodium m-periodate leads to the destruction of epitopes recognized by antibodies immune affinity-purified with isolated Juv-p120. Juvenile L. sigmodontis were shown to release Juv-p120 into the pleural cavity of infected Mastomys coucha before the onset of patency.

A microfilarial sheath protein gene (shp2) coding for the major constituent of the insoluble, cross-linked sheath remnant (SR) from Brugia malayi, Brugia pahangi and Litomosoides carinii has been cloned and sequenced, based on peptide partial amino-acid sequences. All three closely related single-copy shp2 genes in the two genera carry a single intron in identical position; shp2 mRNAs are post-transcriptionally modified by both cis-splicing and trans-splicing. In accordance with their extracellular destinations the encoded proteins include signal peptide sequences; molecular masses of approx. 23 kDa are hence predicted for the mature secreted polypeptides. In their structures sheath matrix proteins shp2 may be regarded as extreme cases of a modular constitution, since these proteins largely consist of two different segments of multiple sequence repetitions, PAA and QYPQAP (or QYPQ), separated by elements of unique sequence. Extreme insolubility and cross-linking are likely to originate from these repetitive sequences within shp2, and to constitute the basic properties of a microfilarial matrix largely consisting of an shp2 network.

Isolated sheaths from Litomosoides carinii microfilariae were disintegrated by reduction with dithiothreitol and were 14C-carboxymethylated. Five major sheath proteins thus solubilized were purified by size exclusion chromatography and reversed-phase HPLC (rpHPLC). Proteolytic fragments of complete sheaths and of the single sheath proteins were isolated by rpHPLC and were N-terminally sequenced. A library of 27 partial sheath polypeptide sequences was thus established, 21 of which could be assigned to three L. carinii sheath structural genes (shp1,2, and 3/3a) isolated on the basis of this and of previous amino acid sequence information. The remaining peptides document the presence of at least one additional major sheath constituent.

Sulphated N-linked carbohydrate chains isolated from recombinant human tissue plasminogen activator expressed in mouse epithelial (C127) cells were analysed as oligosaccharide alditols by methylation analysis, liquid secondary ion mass spectrometry, and one- and two-dimensional 1H-NMR spectroscopy. The results demonstrate that the major component has the following novel structure: NeuAc-alpha 2-6Gal beta 1-4GlcNAc beta 1-2[NeuAc alpha 2-3Gal beta 1- 4GlcNAc beta 1-4]-Man alpha 1-3[NeuAc alpha 2-3(SO4-6)Gal beta 1- 4-GlcNAc beta 1-2Man alpha 1-6]-Man beta 1-4GlcNAc beta 1- 4[Fuc alpha 1-6]GlcNAc-o1.

Human transferrin receptor was isolated from placenta and from the hepatocarcinoma cell line Hep G2. Asparagine-linked oligosaccharides were released by treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Oligosaccharide alditols were fractionated by anion-exchange high-performance liquid chromatography and by high-pH anion-exchange chromatography. Glycans from placental transferrin receptor were further characterized, after desialylation, by methylation analysis and, in part, by liquid secondary-ion mass spectrometry. Sialylation of placental transferrin receptor was examined by lectin affinity blotting with Sambucus nigra agglutinin and Maackia amurensis agglutinin. In order to trace possible inter-individual differences in N-glycosylation of the receptor, two preparations of placental transferrin receptor purified from two donors were compared. The results demonstrate that human transferrin receptor from placenta predominantly carries diantennary and triantennary N-acetyllactosaminic glycans as well as hybrid-type species, the galactose residues of which being almost completely substituted with (alpha 2-3)-linked sialic acid residues. Distinct differences were noted in the glycosylation pattern of the receptor from different individuals. Transferrin receptor from donor A carried predominantly diantennary and triantennary complex-type glycans, in part fucosylated at the innermost N-acetylglucosamine residue, in addition to small amounts of bisected and of incomplete diantennary species. Placental transferrin receptor from donor B predominantly carried triantennary N-acetyllactosaminic glycans without fucose and hybrid-type oligosaccharides with four or five mannose residues. Distinct from placental transferrin receptor, the receptor from Hep G2 cells contained larger amounts of oligomannosidic glycans with six to nine mannose residues and tetrasialylated complex-type oligosaccharides apart from mono-, di- and trisialylated species.

Glycoprotein 71 from Friend murine leukemia virus was digested with proteases and the glycopeptides obtained were isolated and assigned, by amino acid sequencing, to the eight N-glycosylated asparagines in the molecule; only Asn334 and Asn341 could not be separated. The oligosaccharides liberated from each glycopeptide by endo-beta-N-acetylglucosaminidase H, or by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, were fractionated and subjected to structural analysis by one- and two-dimensional 1H NMR, as well as by methylation/gas-liquid-chromatography/mass-fragmentography. At each glycosylation site, the substituents were found to be heterogeneous including, at Asn334/341 and Asn410, substitution by different classes of N-glycans: oligomannosidic oligosaccharides, mainly Man alpha 1----6(Man alpha 1----3)Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc beta 1----, were detected at Asn168, Asn334/341 and Asn410. Hybrid species, partially sialylated, intersected and (proximally) funcosylated Man alpha 1----6(Man alpha 1----3)Man alpha 1----6 and Man alpha 1----3Man alpha 1----6 and Man alpha 1----3Man alpha 1----6(Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc beta 1----, were found at Asn12, as previously published [Schluter, M., Linder, D., Geyer, R., Hunsmann, H., Schneider, J. & Stirm, S. (1984) FEBS Lett. 169, 194-198] and at Asn334/341. N-Acetyllactosaminic glycans, mainly partially intersected and fucosylated NeuAc alpha 2----3 or Gal alpha 1----3Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(NeuAc alpha 2----6 or NeuAc alpha 2----3Gal-beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNac beta 1----4GlcNAc beta 1---- with some bifurcation at ----6Man alpha 1----6, were obtained from Asn266, Asn302, Asn334/341, Asn374 and Asn410. In addition, Thr268, Thr277, Thr279, Thr304/309, as well as Ser273 and Ser275, were found to be O-glycosidically substituted by Gal beta 1----3GalNAc alpha 1----, monosialylated or desialylated at position 3 of Gal or/and position 6 of GalNAc.

There is a glycanase activity associated with the particles of Klebsiella bacteriophage No. 13 which catalyzes hydrolysis of O - β - d -glucopyranosyl-(1 → 4)- β - d -mannopyranose linkages in Klebsiella serotype 13 capsular polysaccharide. The initial glycanase reaction velocity (at 37° and the optimum pH of 7–8) exhibits hyperbolical dependence on low concentrations of substrate, with K m = 170 μ m (in repeating units), and V max = 140 nmol/min (glucose equivalents, for 10 10 plaque-forming units). The reaction is inhibited by reaction products (type-13 penta- and decasaccharides, one and two repeating-units), and also by the substrate at concentrations above ∼ 1m m . Of seventy-four heterologous Klebsiella , and two other bacterial capsular heteropolysaccharides, as well as two substrate derivatives tested, only the Klebsiella serotype-2, -22, and -37 glycans were also depolymerized by the phage-13 enzyme. The reacting polysaccharides consist of repeating units, characteristically containing (a) chain 3 eq ,1 eq → 4 eq ,1 eq dihexopyranosyl sequences around the susceptible linkages, with equatorial hydroxyl and hydroxymethyl groups at C-2 and C′-5 next to them, and ( b ) branch carboxyl groups that may have to be located within a certain distance from these linkages.

The NH2-terminal amino acid sequences (initial 23 residues) of Friend murine leukemia virus gp71 and gp69 were determined and found to be different but highly related. Friend murine leukemia virus gp71 differed from Rauscher murine leukemia virus gp70 in only one position. Friend murine leukemia virus gp69 showed approximately 41% homology to these glycoproteins but lacked the glycosylation site (sequon) occurring at position 12 in Rauscher murine leukemia virus gp70.

Mutants of Escherichia coli C were selected for resistance towards a set of cell wall LPS core specific bacteriophages, including oX174. Increasingly deficient LPS's from wt and mutant E. coli C were tested for inactivation of oX174, and the core oligosaccharides were subjected to structural analysis by methylation/g.l.c./m.s. Loss of the terminal galactose in the following basic structure of the E. coli C wt core was found to lead to adsorption resistance towards oX174

A version of the methylation analysis of complex carbohydrates by gas chromatography-mass spectrometry of the methylalditol acetates ( H. Bjorndal, C. G. Hellerquist, B. Lindberg, and S. Svensson (1970) Angew. Chem. 82 , 643–674) is described. With this version 100- to 500-pmol samples of N -glycosidically linked glycoprotein oligosaccharides may be analyzed. The method is based on the use of capillary columns which allow the separation of all partially methylated alditol acetates potentially obtained from this group of oligosaccharides and on their selective and sensitive detection by mass fragmentography after chemical ionization with ammonia.

Fibre X-ray diffraction patterns have been obtained from oriented, semi-crystalline films of the Klebsiella capsular polysaccharide secrotype K57. K57 is a polytetrasaccharide that contains galactosyluronic acid, mannosyl, and galactosyl residues in the backbone, and an additional mannosyl group as a side-appendage. The simplest interpretation of the diffraction pattern is that the molecule crystallizes as a three-fold helix with an axially projected repeat of 1.143 nm which correlates directly with the chemical repeat. The chain is highly extended, even though it incorporates a 1,2-diaxial linkage in the main backbone. Molecular models have been built using least-squares techniques to minimise interatomic compression and simultaneously meet the observed helical parameters. These models have been compared with the experimental data by using cylindrically averaged, Fouriertransform calculations.

Different interactions between particles of Escherichia coli capsule bacteriophage 29 and its receptor, the E. coli serotype 29 capsular polysaccharide have been studied. The inactivation of phage 29 (8 x 10(3) PFU/ml) by isolated host capsular glycan was found to be physiologically insignificant (50% inactivation dose equals 100 mug after 1 h at 37 C). No adsorption (less than 2 x 10(4) PFU/mug) of the viruses to K29 polysaccharide-coated erythroyctes (at 0 or 37 C) was observed either. The phage particles were, however, found to catalyze the hydrolysis of beta-D-glucosido-(1leads to 3)-D-glucuronic acid bonds (arrow) in the receptor polymer, leading, ultimately, to the formation of a mixture of K29 hexasaccharide (one repeating unit), dodecasaccharide, and octadecasaccharide: (see article). Testing derivatives of K29 polysaccharide, as well as 82 heterologous bacterial (mainly Enteriobactericeae) capsular glycans, the viral glycanase was found to be highly specific; in accordance with the host range of phage 29, only one enzymatic cross-reaction (with the Klebsiella K31 polysaccharide) was observed. These and previous results, as well as the electron optical findings of M. E. Bayer and H. Thurow (submitted for publication), are discussed in terms of a unifying mechanism of phage 29-host capsule interaction. We propose that the viruses penetrate the capsules by means of their spike-associated glycanase activity, which leads them along capsular polysaccharide strands to membrane-cell wall adhesions where ejection of the viral genomes occurs.

The sugar composition of the surface glycoprotein from Friend murine leukemia virus was determined by gas-liquid chromatography of the alditol acetates and by the thiobarbituric acid method, respectively. N-Acetylglucosamine, mannose, galactose, sialic acid and fucose were found in a molar ratio around 15.2:11.6:7.4:3.3:1.0. Ten oligosaccharide fractions were obtained from glycoprotein preparations by a suitable sequence of degradation (with pronase, endo-beta-N-acetylglucosaminidase H, neuraminidase, and by hydrazinolysis) and separation procedures (concanavalin A-affinity chromatography and gel filtration). The qualitative sugar composition of these fractions was analyzed by in vivo labelling with D-[6-(3)H]glucosamine, D-[2-(3)H]mannose, D-[6-(3)H]galactose, or L-[6-(3)H]fucose, and their molecular weights were estimated from the gel elution volumina. Four fractions of N-glycosidically linked oligosaccharides of the oligomannosidic ('high mannose') type oligomannosidic7-oligomannosidic10, about seven to ten sugar residues), two of the mixed (M11 and M12), and four of the N-acetyllactosaminic ('complex') type (N-acetyllactosaminic9, probably nine sugar residues; N-acetyllactosaminica-N-acetyllactosaminic c, size unknown) were thus identified.

Virus-associated glycanases acting on bacterial capsular polysaccharides or on the 0-specific side chains of cell wall lipopolysaccharides, carboxylic ester and amide hydrolases removing 0— and N—acetyl residues from these glycans, as well as lyases for bacterial 2xopolysaccharides are known to occur on bacteriophages for different bacterial species, notably for many belonging to the family of Enterobacteriaceae. A review of the current knowledge on this group of enzymes is presented, covering the following aspects: (i) Isolation and selection of appropriate bacteriophages, (ii) purification of bacteriophage particles and of the virus organelles ('spikes" or thick "fibers") carrying the active center(s) , (iii) electron microscopy of the viruses, (iv) molecular properties of the virus organelles, (v) enzymology (pH dependence, kinetics, substrate specificity etc.) of the reactions as catalyzed by complete bacteriophage particles, (vi) applications of the viral glycanases, which allow the preparative isolation of oligosaccharides consisting of one and more repeating units of the host surface heteropolysaccharides.

Summary Using pairs of smooth and rough forms of Enterobacteriaceae, six smooth-specific bacteriophages were isolated from sewage and another was obtained from Dr Hedda Milch, Budapest. Upon incubation of the individual extracted (smooth) host cell wall lipopolysaccharides with the homologous purified viruses, liberation of reducing groups (i.e. of about di- to nonasaccharides) was observed in four cases, indicating the action of glycanases — but no liberation of acetic acid, indicating the absence of esterase activity. Under the electron microscope, all phages were seen to exhibit Bradley group B or C morphology and to carry tail spikes.

From Escherichia coli R4, and from some deeper rough mutants of it, the cell wall lipopolysaccharides were isolated and subjected to mild acid hydrolysis. By methylation/g.l.c./m.s. and other analyses of the core oligosaccharides thus obtained, the primary structure of the E. coli R4 core (hexose and heptose region) was elucidated

Two bacteriophages degrading 3-deoxy-D-manno-2-octulosonic acid-(KDO)-containing capsules of Escherichia coli strains were identified. Using modifications of the thiobarbituric acid assay, it was shown that each phage contains a glycan hydrolase activity cleaving one type of ketosidic linkage of KDO. Thus, the enzyme from phage phi 95 catalyzes the hydrolysis of beta-octulofuranosidonic linkages of the K95 glycan; and phi 1092, the alpha-octulopyranosidonic linkages of the K? antigen of E. coli LP1092. No cross-reactivity of the phage enzymes with other KDO-containing capsular polysaccharides was observed.

The primary structure of the Klebsiella serotype 16 capsular polysaccharide consists of tetrasaccharide repeating-units comprising a/ar3)-α- D -Glc p -(1/ar4)β- D -GlcA p -(1/ar4)-α- L -Fuc p -(1/ar chain with a β- D -Gal p -(1→ branch at position 4 of the D -glucosyl residue.

The lipopolysaccharides of ampicillin-resistant cell-wall-defective mutants of Escherichia coli K-12 were analyzed. From their lipopolysaccharides the respective core oligosaccharides were obtained. Following dephosphorylation, the core oligosaccharides were methylated and analyzed by gas chromatography/mass spectrometry. From core-defective mutants substructures of the K-12 core were obtained. Analysis of the lipopolysaccharide preparations from wild-type K-12 indicated the presence of several core structures with different degrees of completion. The lipopolysaccharide preparation was degraded and the oligosaccharide mixture was partially resolved by gel filtration chromatography. Methylation, gas chromatography and mass spectrometry of the oligosaccharides permitted the tentative formulation of the K-12 core structure. Alternative interpretations for this heterogeneity are discussed.

Purified particles of two small bacteriophages, active on Klebsiella K20 or K24 strains, were found to exhibit galactosidase or glucosidase activities, respectively, that cause an extensive depolymerization of isolated host capsular polysaccharides.

The primary structure of the Klebsiella serotype 6 capsular polysaccharide has been shown to consist of →3)-α- l -Fuc p -(1→3)-β- d -GlC p -(1→3)-β- d -Man p -(1→4)-α- d -GlcA p -(1 → repeating-units. substituted by pyruvate acetal on positions 4 and 6 of the mannosyl residue.

Neutral and acidic glycolipids were extracted from chicken hearts. The neutral and acidic compounds were separated by preparative thin-layer chromatography into eight and two fractions, respectively. Total hydrolysis by mineral acid, permethylation analysis, and sequential cleavage with exoglycosidases showed the presence of glycolipids that belong to the globo- and gala-oligosaccharide series, i.e., the monohexosylceramides Glc-Cer and Gal-Cer, the dihexosylceramides Gal beta 1-4Glc-Cer and Gal alpha 1-4Gal-Cer, the tetrahexosylceramides GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer and GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal-Cer (III3GalNAc alpha-Ga3Cer) and four subfractions of the Forssman glycolipid GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer. With the notable exception of III3GalNAc alpha 1-Ga3Cer, all glycolipids with terminal GalNAc alpha 1-3GalNAc1 reacted on thin-layer chromatograms with a monoclonal anti-Forssman antibody. The major components of the acidic fraction glycolipids were characterized as the lactose-based gangliosides Glac1 (GM3) and Glac2 (GD3).

Fibre type X-ray diffraction patterns have been obtained from oriented, semi-crystalline films prepared from the sodium salt of the capsular polysaccharide of Klebsiella serotype K25. This molecule has a tetrasaccharide repeating structure consisting of a disaccharide backbone and a disaccharide side chain. The backbone contains a di-equatorially 1,4 linked β- d -glucose residue followed by a di-equatorially 1,3 linked β- d -galactose residue. The side chain is attached to the axial O(4) position of the galactose residue and consists of a di-equaltorially 1,2 linked β- d -glucoronic acid with a β- d -glucose residue attached terminally. An interesting feature of the backbone linkage geometry of this polysaccharide is its similarity with those of the animal connective tissue polydisaccharides. Analysis of diffraction patterns gives rise to an extended three fold helical conformation with an axially projected advance per chemical repeat of 0.97 nm. Molecular models have been computer generated using least squares techniques to optimize interatomic contacts and simultaneously meet the observed helical parameters. A left handed helix with inter-residue stabilizing hydrogen bonds was found to be most favourable and comparison of this model with other relevant polysaccharide structures is male.

Using periodate oxidation, methylation analysis, the characterization of oligosaccharides obtained by partial acid hydrolysis, p.m.r. spectroscopy, and analytical ultracentrifugation, the structure of the (mildly alkali-treated) Klebsiella serotype 11 capsular polysaccharide has been elucidated. The tetrasaccharide repeating-unit comprises the sequence ₒ3)-β- D -Glc p -(1ₒ3)-β- D -GlcUA p -(1ₒ3)-α- D -Gal p -(1→ with a 4,6- O -(1-car☐yethylidene)-α- D -galactosyl residue linked to O-4 of the glucuronic acid residue. The structural basis for some serological cross-reactions of the Klebsiella K11 antigen is discussed, and it is shown that rabbit antisera against the Klebsiella K11 test-strain predominantly contain K agglutinins specific for branch-terminal 4,6- O -(1-car☐yethylidene)- D -galactose.

Two size classes of O-glycosidically linked oligosaccharides were liberated from glycoprotein E1 of mouse hepatitis virus (MHV) A59 by reductive beta-elimination and separated by h.p.l.c. The structures of the reduced oligosaccharides were determined by successive exoglycosidase digestions and by methylation analyses involving combined capillary gas chromatography-mass spectrometry and mass fragmentography after chemical ionization with ammonia. Oligosaccharide A (Neu5Ac alpha 2----3 Gal beta 1----3 GalNAc) comprised 35% of the total carbohydrate side chains, while the remaining 65% of the oligosaccharides of E1 had the branched structure B: Neu5Ac alpha 2----3 Gal beta 1----3 (Neu5Ac alpha 2----6) GalNAc. Both oligosaccharides were linked to the E1 polypeptide via N-acetylgalactosamine, and 20% of the sialic acids present in E1 glycopeptides were found to consist of N-acetyl-9-mono-O-acetylneuraminic acid. The reported structures of the O-linked glycans are discussed in the context of the amino acid sequence of E1, which exhibits a cluster of four hydroxyamino acids (Ser-Ser-Thr-Thr) as potential O-glycosylation sites at the amino terminus. Oligosaccharides with identical structures and an identical O-glycosylated tetrapeptide sequence are present in the blood group M-active glycophorin A of the human erythrocyte membrane.

As previously shown, there is a host capsule deploymerase activity associated with the spikes of Klebsiella bacteriophage No. 11 (Bessler et al. , 1973). Purified particles of phage No. 11 were now subjected to a variety of denaturing conditions, and it was found that mild acid treatment (3 to 10 min at pH 3.5 and 20°) causes a pronounced decay of the viruses into their structural elements, but a comparatively moderate loss of depolymerase activity. From batches of phage fragments obtained in this manner, (I) detached pairs of spikes, (II) spike subunits still exhibiting host capsule depolymerase activity, and (III) head capsomeres were isolated. Electron optical visualization and SDS PAA gel electrophoresis of the preparations showed that the enzymatically active spike subunits are small (possibly hollow) particles of about 50–60 A diameter consisting of one 62,500-dalton polypeptide only, and that the head capsomeres are doughnut-shaped structures of approximately 80 A diameter, containing polypeptides of about 41,000 daltons. Previous results concerning the morphology and polypeptide composition of the whole phage spikes were verified.

The highly microheterogeneous, N-glycosidically linked oligosaccharides in the glycoproteins of Friend murine leukemia virus (as produced by Eveline cells) were liberated with endo-beta-N-acetylglucosaminidase H and by alkaline hydrolysis. They were fractionated (as desialylated oligosaccharitols) by gel filtration and by concanavalin A affinity chromatography, and the major fractions were analyzed by methylation-gas chromatography-mass spectrometry, by digestion with exoglycosidases, and, especially, by one- and two-dimensional proton nuclear magnetic resonance spectroscopy. Guidelines for qualitative and quantitative analysis of complex oligosaccharide mixtures by NMR were worked out and the results compared with those obtained by methylation analysis. It was found that these major fractions consist of bi-, tri-, and tetraantennary oligosaccharitols of the "complex" type (comprising a minority of species with N-acetyllactosamine repeating units), which are, in part, substituted by nonreducing terminal Gal alpha (1----3) and/or bisecting GlcNAc beta (1----4) residues.

Using 74 serological test strains for different Klebsiella K antigens as propective hosts, 50 new bacteriophages were isolated, the particles of which catalyze host capsular glycan depolymerization. As seen under the electron microscope, these viruses most often belong to Bradley's morphology group C (36 cases), occasionally to group B (11), and rarely to group A (3); they all carry tail spikes (or thick fibers) with which, presumably, the glycanase activity is associated. Employing the same set of Klebsiella strains, the host range of a total of 55 such bacteriophages was tested and 28 cross-reactions (host capsule depolymerization and plaque formation at a relative efficiency of plating ⪕ 10 5 ) were detected; in addition, one abortive infection (host capsule depolymerization without plaque formation) was found. The viral depolymerases thus proved to be very specific (33 cross-reacting with no, 18 with one, 2 with two, and 1 each with three or four heterologous polysaccharides). For further characterization of the over 80 phage-catalyzed depolymerization reactions thus identified, the purified virus particles were incubated with the isolated bacterial capsular polysaccharides, and the oligosaccharides produced were analyzed for reducing end sugar (glucose, galactose, mannose, fucose, or rhamnose), and for size (mainly one to three repeating unit oligosaccharides in about 85% of the cases). In those 49 phage-host systems, where the primary structures of the substrate polysaccharides were known, the reducing end sugar analyses led to unequivocal (36 reactions) or tentative (13) identifications of the phage enzyme susceptible glycosidic linkages, and hence to the following general conclusions: (I) in most cases cleavage occurs on either side of the (a) sugar unit carrying the negative charge(s), but reducing glycuronic acids are not produced; (II) most often, the reducing end sugar formed is substituted at position 3; (III) in the majority of cases, β-glycosidic linkages are hydrolyzed; (IV) in most polysaccharides which are acted upon by several phage enzymes, the same glycosidic bonds are split by the different agents.

The preparation and use of particles of Klebsiella bacteriophage No. 11 are described. A glycanase activity associated with the viruses catalyses the depolymerization of (alkali-treated)Klebsiella serotype 11 capsular polysaccharide, ultimately to a mixture of oligosaccharides consisting of one or two repeating units. Mainly glucosidic bonds are hydrolysed. The substrate specificity of the viral enzyme has been characterized by using derivatives of serotype-11 polysaccharide, as well as 81 heterologous, bacterial, capsular glycans. It is concluded that the glycanase will (at least) also depolymerize all polysaccharides containing the unsubstituted chain-trisaccharide repeating-unit of its natural substrate.

Using periodate oxidation, methylation analysis, characterization of oligosaccharides by Smith degradation or partial acid hydrolysis, as well as proton magnetic resonance, the primary structure of the Escherichia coli serotype 29 capsular polysaccharide (the receptor of E. coli K phage 29) was reinvestigated. The polymer was found to consist of hexasaccharide repeating units of the following structure: (see article).

The surface glycoprotein (mixture of isoglycoproteins with Mr 69 000 and 71 000) was isolated from the particles of Friend murine leukemia virus, and was successively digested with protease and with endo-beta-N-acetylglucosaminidase from Streptomyces griseus. Roughly 20% (w/w) of the carbohydrates in this glycoprotein were thus released, and they were fractionated by high-performance liquid chromatography after reduction with KB3H4/NaBH4. The radioactive oligosaccharitol fractions obtained were analyzed by exoglycosidase digestion, by acetolysis, and, after permethylation, by fast atom-bombardment mass spectrometry, as well as by capillary gas-liquid chromatography/mass spectrometry following hydrolysis, reduction and peracetylation. Around 85% (mol/mol) of the endo-H-sensitive viral glycans were thus found to be oligomannosidic oligosaccharitols of size classes Man5GlcNAcOH, Man6GlcNAcOH, Man8GlcNAcOH, Man7GlcNAcOH, and Man9GlcNAcOH (in order of prevalence), and the major structural isomers of each size class were identified. About another 15% (mol/mol) of the oligosaccharitols were shown to be of the 'mixed type', comprising mainly four species in which the Man(alpha 1----6)-branch of the Man alpha 1----6 (Man alpha 1----3) Man beta 1----4GlcNAcOH core is substituted by one or two additional alpha-mannoses, while the Man(alpha 1----3)-branch carries an N-acetyllactosamine unit substituted by sialic acid, or by Gal(alpha 1----3).

Fowl plague virus, strain Dutch, was metabolically labeled withd-[2-3H]mannose, or withd-[6-3H]glucosamine, and the small subunit (HA2; 0.8 mg in total) of the viral hemagglutinin was isolated by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis. After proteolytic digestion, the radioactive oligosaccharides were sequentially liberated from the glycopeptides by treatment with different endo-β-N-acetylglucosaminidases and with peptide:N-glycosidase or, finally, by hydrazinolysis. In this manner, four groups of glycans could be obtained by consecutive gel filtrations and were subfractionated by HPLC. The structures of the individual oligosaccharides were analyzed by micromethylation, by acetolysis or by digestion with exoglycosidases. The major species amongst the high mannose glycans at Ans-406 of the viral glycopolypeptide were found to be Manα1-2Manα1-3(Manα1-2Manα1-6)Manα1-6(Manα1-2Manα1-2Manα1-3)Manβ1-4GlcNacβ1-4GlcNAc and Manα1-3(Manα1-2Manα1-6)Manα1-6(Manα1-2Manα1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc, while the complex glycans at Asn-478 are predominantly GlcNAcβ1-2Manα1-3(GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc (lacking, in part, one of the outerN-acetylglucosamine residues) and GlcNAcβ1-2Manα1-3(Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc.

The proteins p40 (Mr = 40 000), p32 (Mr = 32 000), p28 (Mr = 28 000), p20 (Mr = 20 000) and p10 (Mr = 10 000) are described which occur in noncovalent association with the polymorphic alpha, beta heterodimer of class II antigens. They were investigated with respect to their molecular characteristics and their mutual structural relationship. p32, the predominant species of this group corresponds to the invariant chain gamma (Ii). In contrast to the polymorphic subunits alpha and beta, proteins p40, p28, p20 and p10 migrated like gamma in electrophoretically constant positions, when class II molecules of different subregions and different alleles were assessed by two-dimensional gel electrophoresis [1st dimension, isoelectric focusing; 2nd dimension, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis]. Analogous to gamma, they are therefore designated invariant chains. The low Mr species of this group do not arise from higher Mr forms as preparation artefacts. Short-term pulse-chase analysis and cell-free translation of sucrose gradient-fractionated mRNA in conjunction with specific immunoprecipitation rendered the possibility unlikely that individual components of this set of proteins existed in a precursor-product relationship within the cell. Comparative enzymatic fragmentation on SDS polyacrylamide gels as well as tryptic peptide map comparisons by high performance liquid chromatography revealed a high structural relatedness among all members of this group of invariant proteins.

The carbohydrate moieties in the four isotypes of a variant surface glycoprotein from Trypanosoma congolense were analyzed. All variant surface glycoprotein isotypes were found to contain up to 15% by weight of d-galactose, d-mannose, and N-acetyl-d-glucosamine in molar ratios approaching 1:3.2:3.9 (isotypes I–III) or 1:2,4:2.4 (isotype IV); in addition, the presence of sialic acid could be demonstrated. After metabolic labelling with d-[6-3H]glucosamine, the four isoglycoproteins were successively digested with pronase and with endo-β-N-acetylglucosaminidase H. Up to two thirds of the oligosaccharides were thus liberated and were separated by gel filtration, and by high performance liquid chromatography. Using methylation, gas chromatography, mass spectrometry and digestion with α-mannosidase, they were shown to be mainly typical oligomannosidic oligosaccharides of size classes Man5GlcNAc to Man9GlcNAc. The residual glycans were liberated by hydrazinolysis, and were fractionated by serotonin affinity chromatography. After separation by gel filtration, the neutral oligosaccharides from isotype I were subjected to methylation analysis and successive exoglycosidase digestions. They were found to be biantennary oligosaccharides of the N-acetyllactosaminic type: (GalGlcNAc)2Man3GlcNAc1–2. Only about 30% of the sialylated glycans were susceptible to neuraminidases. The T. congolense variant surface glycoprotein studied here contains mainly high mannose and biatennary ‘complex’ oligosaccharides as found in many other eukaryotic glycoproteins, except that they seem to carry unusually substituted/linked sialic acid residues.

Depolymerization of bacterial, capsular polysaccharides by phage enzymes is a convenient method of preparing oligosaccharides that correspond to one, or several, repeating unit(s). Thus, the capsular polysaccharide from Klebsiella K21 yields a linear pentasaccharide, and that from Klebsiella K32, a linear tetrasaccharide. Both oligosaccharides contain acetal substituents, but, whereas the 4,6- O -(1-carboxyethylidene)- D -galactosyl residue in the K21 structure is relatively acid-stable, the corresponding 3,4- O -(1-carboxyethylidene)- l -rhamnosyl residue in K32 is extremely acid-labile. Phage degradation may, therefore, be the only way by which an oligosaccharide corresponding to an intact repeating-unit may be obtained in such circumstances.

The Escherichia coli capsule bacteriophages (K phages) described herein are specific for certain capsular strains of E. coli , all of them test strains for different E. coli K antigens. The phages are not adsorbed to the acapsular mutants of their host organisms nor to similar strains with serologically and chemically different capsular polysaccharides. Thirteen E. coli (and one Klebsiella ) K phages were visualized in the electron microscope. Most viruses are similar to P22 and thus belong to Bradley group C; however, one each of group A (long, contractile tail) and group B (long, noncontractile tail) was also found. All K phages were seen to carry spikes but no tail fibers were detected. These results suggest that the structures responsible for the recognition of the thick (about 400 nm or more) capsular polysaccharide gels are located in these spikes.