Theo A.A. Dopheide

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.

Completion of the amino acid sequence of a Hong Kong influenza hemagglutinin heavy chain: Sequence of cyanogen bromide fragment CN1

Abstract The amino acid sequence of cyanogen bromide fragment CN1 from the hemagglutinin heavy chain (HA 1 ) of the Hong Kong influenza virus A/Memphis/102/72 has been determined by manual Edman degradation of tryptic and chymotryptic peptides and automated sequenator analysis of whole HA 1 after removal of the N-terminal pyroglutamic acid blocking group with calf liver pyroglutamate aminopeptidase. CN1 is the N-terminal cyanogen bromide peptide of HA 1 and extends from residues 1 to 168. The elucidation of the sequence of CN1 completes the amino acid sequence of A/Memphis/102/72 HA 1 . This Hong Kong heavy chain contains a total of 328 amino acid residues and when compared with the HA 1 polypeptides of other influenza strains has an additional 10 residues at its N terminus including a glycosylated asparagine residue. It contains six glycosylated asparagine residues, five of which occur in CN1 at residues 8, 22, 38, 81, and 165 and one in CN3 at residue 285. No carbohydrate groups were found attached to serine or threonine residues. The Hong Kong HA 1 contains nine half-cystine residues, six of which are in CN1 at positions 14, 52, 64, 76, 97, and 139 and three in CN3 at positions 277, 281, and 305. The structure is discussed in relation to the available published data on the hemagglutinins from other strains of influenza virus and recent developments in the analysis of antigenic shift and drift.

Size and chemical composition of influenza virus hemagglutinin chains

Influenza viruses are filamentous or spherical particles composed of a segmented viral genome, which together with several types of protein is enclosed within a lipoprotein envelope from which two types of glycoprotein project outwards [1]. Although three virus types (A, B and C) can infect man [2], successive epidemics are only caused by the A viruses. New strains arise as a result of major (antigenic shift) or minor (antigenic drift) changes in the structure of the major spike protein, the hemagglutinin [3-51. This letter reports the molecular weight and the amino acid and carbohydrate compositions of the two chains of the hemagglutinin from a Hong Kong virus variant A2/Memphis/102/72. The results obtained confirm that the heavy (HA1) chain contains most of the carbohydrate, and show that the true tool. wt. of the heavy chain is considerably less than that estimated from standard SDS gels. We further show, that the apoprotein portion of the heavy chain is only 29% larger than the apoprotein part of the light chain (HA2).

Molecular characterisation of a protective, 11-kDa excretory-secretory protein from the parasitic stages of Trichostrongylus colubriformis

An 11-kDa protein occurring as a major component of the non-glycosylated fraction of 4th larval stage (L4) and adult Trichostrongylus colubriformis excretory-secretory (ES) fluid has been found to be highly protective in guinea pigs, an alternate host for T. colubriformis. The protein has been purified, characterised and partly sequenced. With a reverse-complement oligonucleotide based on the carboxy-terminal sequence of the protein, recombinant lambda gt11 clones were detected in an L4 cDNA library. The DNA sequence from one clone has a single extended open reading frame coding for a highly charged 11-kDa protein which lacks a leader sequence and contains a potential N-glycosylation site. Expression of the cloned DNA in Escherichia coli was detected with an antibody, raised in rabbits against gel-purified 11-kDa protein.

Characterization of the major immunogen in the excretory-secretory products of exsheathed third-stage larvae of Trichostrongylus colubriformis

The excretory-secretory products of exsheathed third-stage larvae of Trichostrongylus colubriformis conferred some protection to guinea pigs against homologous challenge. A glycoprotein with an apparent molecular mass of approximately 94 kDa was the dominant immunogen in post-exsheathment products. Immunoblots revealed IgG antibodies to this glycoprotein in sera from multiply-infected guinea pigs and some sheep, and in sera of guinea pigs after three truncated infections which had been restricted by anthelmintic treatments to development of the third parasitic stage. IgA antibodies to this protein were also found in intestinal lymph of a naturally infected sheep. Fluorescent antibody studies indicated that this 94 kDa component was associated with cells in the central body cavity of third-stage larvae, but was absent from fourth-stage larvae or adult worms. Fractionation and protection assays in guinea pigs revealed that while the native and aggregated 94 kDa protein conferred some host protection, it was not the only protective component of the excretory-secretory products of exsheathed third-stage larvae of T. colubriformis.

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 HONG KONG HEMAGGLUTININ. STRUCTURAL RELATIONSHIPS BETWEEN THE HUMAN (H3) HEMAGGLUTININS AND THE HEMAGGLUTININ FROM THE PUTATIVE PROGENITOR STRAIN A/DUCK/UKRAINE/1/63 (HAV7)

ABSTRACT The structural relationship between the hemagglutinins from the human Hong Kong variants (H3) and the putative progenitor strain A/duck/Ukraine/1/63 (Hav7) has been investigated. Amino acid composition and sequence analysis showed that the Hav7 hemagglutinin closely resembled the H3 hemagglutinin in structure. It exhibited extensive sequence homology with the H3 hemagglutinins and contained the glycosylated 10 residue extension that is characterisitc of the N-terminal end of the Hong Kong HA1. Twenty-three differences were found between the hemagglutinin of A/duck/Ukraine/1/63 and the early Hong Kong variant A/Aichi/2/68; of these fifteen occurred in HA1 (at positions 4, 25, 62, 81, 92, 135, 137, 144, 145, 182, 186, 193, 226, 228 and 309), and eight occurred in HA2 (at positions 2, 67, 71, 106, 132, 133, 154 and 161). The oligosaccharide distribution was also different with the A/duck/Ukrainehemagglutinin containing only five carbohydrate side chains at positions 8, 22, 38, 165 and 285 on HA1. It lacked the oligosaccharide units at positions 81 on HA1 and 154 on HA2. The large number of amino acid sequence differences between the Hav7 and the early H3 hemagglutinins suggest that the Hong Kong hemagglutinin gene did not come directly from A/duck/Ukraine/1/63 but from a virus derived from it by antigenic drift during the period 1963 to 1968.

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.

A Hong Kong influenza hemagglutinin light chain: amino acid sequence of cyanogen bromide fragment CN2.

Abstract The amino acid sequence of cyanogen bromide peptide CN2 from the hemagglutinin light chain (HA 2 ) of the Hong Kong influenza varient A/Memphis/102/72 has been determined by manual Edman degradation of tryptic, chymotryptic, thermolytic, and Staphylococcus aureus protease peptides. This fragment contains 98 amino acid residues and extends from residues 18 to 115 in the sequence of HA 2 . It contains no proline, half-cystine, or glycosylated residues.