The isolation, characterization and cloning of a globin-like, host-protective antigen from the excretory-secretory products of Trichostrongylus colubriformis.

An 18-kDa component from the excretory-secretory (ES) products of adults of Trichostrongylus colubriformis was isolated and characterized, and was shown to induce 60-84% protection of guinea pigs from challenge infection following a single intraperitoneal injection. Amino-terminal sequence analysis of gel-purified protein enabled oligonucleotides to be synthesized and used to screen a lambda gt10 cDNA library made from young adult worm mRNA, and to synthesize full-length clones from cDNA using the polymerase chain reaction (PCR). The full-length clones coded for a 20-kDa precursor protein of 173 amino acids which had a strongly hydrophobic leader sequence of 15 residues. The mature protein sequence of 158 amino acid residues was rich in charged amino acids (32%), including 8 oppositely charged pairs of amino acids. The protein sequence contained no half-cystine residues and no potential N-glycosylation sites. Unlike 2 other fully characterized ES components which are expressed only in the parasitic stages, mRNA coding for the 20-kDa component was present in both the parasitic and free-living stages of T. colubriformis. The parasite protein had approximately 20% identity with globins from human and from the larvae of the insect Chironomus thummi thummi. The homology included the invariant distal histidine and phenylalanine, and a number of other residues highly conserved in globins.

Characterization, cloning and host-protective activity of a 30-kilodalton glycoprotein secreted by the parasitic stages of Trichostrongylus colubriformis.

The helminth Trichostrongylus colubriformis is a parasitic nematode infecting the small intestine of sheep. We report the isolation and characterization of a 30-kDa glycoprotein capable of partially protecting guinea-pigs against the parasite. This glycoprotein is secreted by the L4 and adult parasitic stages of the worm. The sequence of three separate cDNA clones predicts the polypeptide to be about 15 kDa, with four N-linked carbohydrate chains and an internal disulphide bond. The clones also indicate the existence of sequence variability in this antigen. Limited sequence homology to a porcine intestinal peptide suggests an influence on host gut physiology.

Amino acid sequence and oligosaccharide distribution of the haemagglutinin from an early Hong Kong influenza virus variant A/Aichi/2/68 (X-31).

The amino acid sequence and oligosaccharide distribution for the haemagglutinin from the early Hong Kong influenza virus A/Aichi/2/68 (X-31) was investigated. The two polypeptide chains, HA1 and HA2, were fragmented by CNBr and enzymic digestion, and the amino acid sequence of each small peptide was deduced by comparing its chromatographic behaviour, electrophoretic mobility, amino acid composition and N-terminus with that of the corresponding peptide of the haemagglutinin of known structure from the influenza-virus variant A/Memphis/102/72. Those peptides in which changes were detected were sequenced fully. The complete amino acid sequence of the haemagglutinin HA1 chain (328 residues) and 188 of the 221 residues of the HA2 chain were established by this approach, and revealed only twelve differences between the amino acid sequences of variant-A/Aichi/68 and -A/Memphis/72 haemagglutinins. These occurred at positions 2, 3, 122, 144, 155, 158, 188, 207, 242 and 275 in the HA1 chain and 150 and 216 in the HA2 chain. The highly aggregated hydrophobic region (residues 180-121) near the C-terminal end of the HA2 chain was not resolved by peptide sequencing. The oligosaccharide distribution in variant-A/Aichi/68 haemagglutinin was identical with that found in that of A/Memphis/72, with sugar units attached at asparagine residues 8, 22 38, 81, 165 and 285 in the HA1 chain and 154 on the HA2 chain. The monosaccharide compositions of the individual carbohydrate units on variant-A/Aichi/68 haemagglutinin differed from those of the corresponding units in variant-A/Memphis/72 haemagglutinin, and evidence was found for heterogeneity in the oligosaccharide units attached at single glycosylation sites.

The location of the bromelain cleavage site in a Hong Kong influenza virus Haemagglutinin.

The site of bromelain cleavage in the haemagglutinin of the Hong Kong influenza virus A/Memphis/102/72 has been determined by using a diagonal peptide mapping procedure on the thermolytic digest of amidated BHA. The data show that bromelain cleavage removes the C-terminal 46 residues from HA2, and that the new carboxyl-terminal residue of BHA2 is Gly 175. This is close to the beginning of the hydrophobic membrane-interacting sequence that starts at residue 183.

The amino acid sequence of a Hong Kong influenza haemagglutinin light (HA2) chain.

The amino acid sequence of the Hong Kong haemagglutinin light chang (HA2:222 residues) is nearly complete, lacking only the definition of a highly aggregated region near the carboxyl terminal end of the chain. This unsequenced area of approx. 25 residues occurs near the carboxyl terminal end of cyanogen bromide peptide CN-I, whose structure determination is discussed in this paper. All 1/2-cystine residues present in HA2 occur in CN-I, as a proximal cluster involving residues 137, 144 and 148, and as a distal cluster involving four other 1/2-cystine within peptides. The single glycosylated asparagine in HA2 also occurs in CN-I; the carbohydrates moiety is complex. The structure of HA2 is discussed in terms of its properties, and compared with published data from haemagglutinins from other influenza strains.

Antigenic determinants of influenza virus hemagglutinin. V. Antigenicity of the HA2 chain.

All the polypeptide fragments obtained by cyanogen bromide cleavage of the hemagglutinin from A/Memphis/102/72 influenza virus were examined for their ability to bind to IgG raised against purified virus. Within the hemagglutinin heavy chain the only fragment displaying antigenicity is HA1CN1, which comprises the amino-terminal 168 amino acid residues. By the use of a sensitive radioimmunoassay in which the antigen is unlabeled, it was shown that the light chain is also antigenic. Inhibition studies have localized the activity to the HA2CN1 region, which comprises the carboxy-terminal 90 amino acids. The determinant on HA2 is shown to be subtype specific.

Amino acid sequence of cyanogen bromide fragment CN2 from Hong Kong influenza haemagglutinin heavy chain.

The amino acid sequence of cyanogen bromide peptide CN2 from the heavy chain (HA1) of the haemagglutinin of the Hong Kong variant A/Memphis/102/72 has been obtained by direct, automated sequence analysis on the whole fragment and by manual dansyl-Edman degradation of tryptic, peptic and chymotryptic peptides. It was found to contain 92 amino acid residues, including a large, insoluble, tryptic core peptide (residues 62-87). It did not contain any half-cystine residues or carbohydrate. The determination of its structure was complicated by the presence of an Asn-Ile bond at positions 48-49 which was readily cleaved by both trypsin and chymotrypsin.

Amino acid sequence changes in the haemagglutinin of A/Hong Kong (H3N2) influenza virus during the period 1968--77.

Haemagglutinin molecules from nine strains of A/Hong Kong/68 (H3N2) influenza virus, isolated between 1968 and 1977, were examined for changes in amino acid sequences. At least 18 changes, 9 of which were located precisely, occurred in the soluble tryptic peptides of the large haemagglutinin polypeptide (HA1) during this period. These peptides contained 262 residues (82% of HA1). In HA2, only two changes in 129 residues (58% of HA2) were detected. Sequential changes at a particular locus were not found; and as far as we can tell, once an amino acid changed, it did not change again in any subsequent variant examined.