Stephan Stirm

Rat α1 macroglobulin inhibits hemagglutination by influenza C virus

Purified alpha 1-macroglobulin (RMG) isolated from rat plasma was found to be a potent inhibitor of hemagglutination by influenza C virus. Neuraminidase treatment of purified RMG reduced its inhibitory activity by more than 80% indicating that sialic acid is required for maximal HI-activity. The inhibitory activity of RMG was shown to be sensitive to the receptor-destroying activity (RDA) of influenza C virus. Methylation analysis of the glycopeptides of RMG indicated the presence of only one major type of oligosaccharide which is a complex N-linked oligosaccharide with a biantennary structure. Comparison of the glycopeptides before and after neuraminidase treatment revealed that the oligosaccharides are terminated by sialic acid residues attached to galactose residues at position C-6. Methylation analysis was also performed on RMG which had lost its inhibitory activity upon incubation with RDA of influenza C virus. No difference between the glycopeptides of native and inactive RMG could be detected. Galactose was found to be substituted at position C-6 in both samples, indicating that also the oligosaccharides of inactive RMG are terminated by sialic acid. The implications of these results are discussed.

The sheaths of Brugia microfilariae: isolation and composition.

Burgia malayi andB. pahangi microfilariae were isolated from the blood of infectedMastomys 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.

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.

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.

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.

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.

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.

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

Microfilarial sheaths ofLitomosoides 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>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 ofL. carinii-infectedMastomys 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.

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).

Inhibition with rat α1-macroglobulin of haemagglutination by influenza C virus

Abstract The nature of the cellular receptor for influenza C virus is investigated by studying the interaction of influenza C virus with rat α 1 -macroglobulin (RMG), a haemagglutination inhibitor. Inactivation of RMB by influenza C virus is not thought to be due to the loss of sialic acid or any other sugar.