Juvenile female Litomosoides sigmodontis produce an excretory/secretory antigen (Juv-p120) highly modified with dimethylaminoethanol.

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.

Glycosylation of recombinant ancrod from Agkistrodon rhodostoma after expression in mouse epithelial cells.

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.

Brugia spp. and Litomosoides carinii: identification of a covalently cross-linked microfilarial sheath matrix protein (shp2).

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.

Litomosoides carinii microfilarial sheaths: partial amino acid sequences of several major polypeptide constituents.

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.

Structural elements in glycoprotein 70 from polytropic Friend mink cell focus-inducing virus and glycoprotein 71 from ecotropic Friend murine leukemia virus, as defined by disulfide-bonding pattern and limited proteolysis.

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.

Trans-splicing of an early embryo mRNA in Litomosoides carinii, coding for the major microfilarial sheath protein gp22.

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.

Structure elucidation of sulphated oligosaccharides from recombinant human tissue plasminogen activator expressed in mouse epithelial cells.

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.

Carbohydrate structure of a thrombin-like serine protease from Agkistrodon rhodostoma. Structure elucidation of oligosaccharides by methylation analysis, liquid secondary-ion mass spectrometry and proton magnetic resonance.

The carbohydrate side chains of the thrombin-like serine protease ancrod from the venom of the Malayan pit viper Agkistrodon rhodostoma were liberated from tryptic glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and fractionated by high-performance liquid chromatography. Glycans obtained were characterized by digestion with exoglycosidases, methylation analysis and, in part, by liquid secondary-ion mass spectrometry and 1H-NMR spectroscopy. The results reveal that this snake venom glycoprotein contains partially truncated di-, tri- and tetraantennary complex type N-glycans carrying Fuc(alpha 1-6) residues at the innermost N-acetylglucosamine and solely (alpha 2-3)-linked sialic acid substituents. As a characteristic feature, ancrod oligosaccharides comprise mainly sialylated Gal beta 3GlcNAc beta lactosamine antennae. Furthermore, a small proportion of the sugar chains were found to carry a NeuAc alpha 3GalNAc beta 4GlcNAc beta antenna exclusively linked to C-2 of Man(alpha 1-3) residues of the pentasaccharide core. Thus, many of the glycans found represent novel glycoprotein-N-glycan structures.

Structure of the N-linked oligosaccharides of the human transferrin receptor.

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.

Chemical composition of Litomosoides carinii microfilarial sheaths.

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 degrees 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.

Litomosoides carinii: extraction of the microfilarial sheath components and antigenicity of the sheath fractions.

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.

Localization of the intrachain disulfide bonds of the envelope glycoprotein 71 from Friend murine leukemia virus.

Envelope glycoprotein 71 from Friend murine leukemia virus was purified to homogeneity by reversed-phase HPLC. It could be shown that all 20 cysteine residues of the molecule are linked by disulfide bonds. After complete tryptic digestion, peptides containing cystine were identified by comparison of the reversed-phase HPLC profile of the digest with that of a reduced aliquot which had been subjected to affinity chromatography on thiol-Sepharose. The locations of the 10 disulfide bonds were determined by isolation, further digestion and analysis of peptides containing cystine. The first cysteine residue of the sequence (Cys46) was shown to be coupled to the sixth (Cys98), leading to a large loop containing four additional cysteine residues. Computer model building and energy calculations led to the assignment of Cys72 to Cys87 and Cys73 to Cys83. The following four cysteine residues of the sequence also constitute a structural unit, with Cys121 bonded to Cys141 and Cys133 to Cys146, and the last two cysteine residues in the amino-terminal domain of glycoprotein 71 form a small loop (Cys178 to Cys184). The first two cysteine residues of the carboxy-terminal domain produce a very small hydrophobic loop (Cys312-Cys315). Cys361 is bound to Cys373, Cys342 to Cys396 and Cys403 to Cys416. A model for the folding pattern of the viral glycoprotein is proposed.

A major Litomosoides carinii microfilarial sheath glycoprotein (gp22): amino terminal sequence and immunological studies with corresponding synthetic peptides.

The major glycoprotein of the sheath of Litomosoides carinii microfilariae (gp22) was analysed for its amino acid and amino sugar composition. It is rich in proline, glutamine/glutamic acid and glycine and contains (N-acetyl)galactosamine. The N-terminal amino acid sequence was determined up to position 37. It consists of a group of 6 repeats of the pentapeptide sequence methionine-glycine-proline-glutamine-proline with two minor modifications in repeats 3-6, while the first two repeats follow the general pattern more loosely. Identical N-terminal amino acid sequences were found in at least two other sheath polypeptides (33 kDa, 39 kDa). Antisera prepared against 3 overlapping synthetic peptides corresponding to the amino terminus of gp22 recognized different epitopes. They all reacted with identical patterns of sheath polypeptides. The antisera failed to recognize antigens of 4th-stage larvae of L. carinii. In contrast, cross-reacting epitopes were detected in other parasite stages. Antisera reacted with material surrounding embryos and microfilariae in the uterus of females, and caused patchy fluorescence on the sheath of blood-derived and in vitro-released microfilariae.

The sheaths of Brugia microfilariae: isolation and composition.

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.

Oligosaccharides at individual glycosylation sites in glycoprotein 71 of Friend murine leukemia virus.

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 [Schlüter, 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.

Primary structure of Klebsiella serotype K22 capsular polysaccharide: another glycan containing 4-O-[(S)-1-carboxyethyl]-D-glucuronic acid.

The primary structure of the acidic capsular polysaccharide isolated from Klebsiella serotype K22 has been investigated using methylation analysis, hydrolysis, bacteriophage-borne enzyme degradation, and n.m.r. spectroscopy. The repeating unit comprises the chain disaccharide----3)-beta-D-Galp-(1----4)-beta-D-Glcp-(1---- substituted by 4-O-[(S)-1-carboxyethyl]-beta-D-GlcpA-(1----6)-alpha-D-Glcp-(1---- at O-4 of the galactose. The galactose carries an O-acetyl group on position 6.

Carbohydrate analysis of chicken heart glycolipids.

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).

Glycosphingolipid composition of human semen.

Glycosphingolipids were extracted from human semen and purified. Based on the fluorometric assay of sphingosine, in spermatozoa a content of 4.4 +/- 0.9 nmol/10(8) cells of gangliosides and 22.1 +/- 1.7 nmol/10(8) cells of neutral glycosphingolipids was determined. Seminal plasma contained 4.1 +/- 0.6 nmol gangliosides and 29.3 +/- 1.5 nmol neutral glycosphingolipids per milliliter. The glycosphingolipid component patterns of human spermatozoa and seminal plasma were determined by thin-layer chromatography. Four neutral glycolipids were isolated and their carbohydrate moieties were characterized. All of these glycolipid components belonged to the globo-series. Gas chromatography, combined gas chromatography/mass fragmentography, and exoglycosidase treatments revealed the following structures for the glycosphingolipids of human semen: Glc1-Cer, Gal beta 1-4Glc1-Cer, Gal alpha 1-4Gal beta 1-4Glc1-Cer, and Gal-NAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc1-Cer. In addition, the occurrence of trace amounts of lactoneotetraosyl- and lactoneohexaosylceramide was detected by immunostaining after thin-layer chromatographic separation. Human spermatozoa, as well as seminal plasma, contained the gangliosides Glac1,Glac2, a sialolactoneotetraosylceramide, and a sialolactoneohexaosylceramide. The gangliosides were identified on the basis of their running characteristics by high-performance thin-layer chromatography, exoglycosidase treatment, and immunostaining after thin-layer chromatography. The ceramide composition of the glycolipids in human spermatozoa, as well as in seminal plasma, was dominated by C22:0-behenic acid and the saturated sphingoid d18:0, sphinganine.