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 haemagglutinin chains HA1 and HA2 from the avian influenza virus A/duck/Ukraine/1/63 (Hav7, Neq2) have been subjected to amino acid analysis and N-terminal sequencing. Automated sequenator analysis of HA1 (40 cycles), after enzymic removal of the N-terminal pyroglutamic acid blocking group, and HA2 (43 cycles) showed that the Hav7 haemagglutinin closely resembled the human Hong Kong (H3) haemagglutinins including the presence of the characteristic extended 10 residue sequence at the N-terminus of HA1. These findings, together with the amino acid compositions for both chains, demonstrate that the Hav7 haemagglutinin is structurally similar to the Hong Kong (H3) haemagglutinins.

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 relationship between the haemagglutinin from the influenza virus A/duck/Ukraine/1/63 (Hav 7) and the human Hong Kong variants (H3) has been investigated. Amino-acid-sequence analysis shows that the Hav 7 haemagglutinin closely resembles the 1968 human H3 haemagglutinin in structure. However, the number of amino-acid-sequence differences (23) suggest that the Hong Kong haemagglutinin gene did not come directly from A/duck/Ukraine/1/63 but from a virus derived from it by antigenic drift during the period 1963-1968.

Predictions of secondary structure for the two chains HA1 and HA2 of the haemagglutinin from the Hong Kong influenza virus A/Memphis/102/72 reveal a striking contrast between the potential conformations of the two chains. HA1 is predicted to be rich in beta-structure while HA2 is highly helical. The predictions further suggest that coiled-coil type interactions between the central helical segments of the HA2 chains may hold the haemagglutinin monomers together in the virus.

FEBS LettersVolume 110, Issue 2 p. 181-183 Full-length articleFree Access The disulphide bonds of a Hong Kong influenza virus hemagglutinin Theo A. Dopheide, Theo A. Dopheide Division of Protein Chemistry, CSIRO, Parkville (Melbourne), VIC 3052, AustraliaSearch for more papers by this authorColin W. Ward, Colin W. Ward Division of Protein Chemistry, CSIRO, Parkville (Melbourne), VIC 3052, AustraliaSearch for more papers by this author Theo A. Dopheide, Theo A. Dopheide Division of Protein Chemistry, CSIRO, Parkville (Melbourne), VIC 3052, AustraliaSearch for more papers by this authorColin W. Ward, Colin W. Ward Division of Protein Chemistry, CSIRO, Parkville (Melbourne), VIC 3052, AustraliaSearch for more papers by this author First published: February 11, 1980 https://doi.org/10.1016/0014-5793(80)80067-6Citations: 5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1 R. Drzeniek, J.T. Seto, R. Rott, Biochim. Biophys. Acta, 128, (1966), 547– 558. 2 W.G. Laver, E.D. Kilbourne, Virology, 30, (1966), 493– 501. 3 G.K. Hirst, J. Exp. Med., 75, (1942), 49– 64. 4 C.W. Ward, T.A. Dopheide, FEBS Lett., 65, (1976), 365– 368. 5 W.G. Laver, Virology, 45, (1971), 275– 288. 6 I.P. Griffith, B.W.J. Mahy R.D. Barry Negative Strand Viruses 2, (1975), Academic Press London 741– 754. 7 D.C. Wiley, J.J. Skehel, M. Waterfield, Virology, 79, (1977), 446– 448. 8 D.O. White, Curr. Top. Microbiol. Immunol., 63, (1974), 1– 48. 9 Ward, C. W. and Dopheide, T. A. (1980) submitted. 10 Dopheide, T. A. and Ward, C. W. (1980) in preparation. 11 T.A. Dopheide, C.W. Ward, Virology, 92, (1979), 230– 235. 12 J.R. Brown, B.S. Hartley, Biochem. J., 101, (1966), 214– 228. 13 E. Mendez, C.Y. Lai, Anal. Biochem., 65, (1975), 281– 292. 14 C.J. Bruton, B.S. Hartley, Biochem. J., 108, (1968), 281– 288. 15 O. Smithies, D. Gibson, E.M. Fanning, R.M. Goodfliesh, J.G. Gilman, D.L. Ballantyne, Biochemistry, 10, (1971), 4912– 4921. 16 C.M. Brand, J.J. Skehel, Nature New. Biol., 238, (1972), 145– 147. 17 M.D. Waterfield, K. Espelie, K. Elder, J.J. Skehel, Brit. Med. Bull., 35, (1979), 57– 63. 18 C.W. Ward, T.A. Dopheide, Brit. Med. Bull., 35, (1979), 51– 56. Citing Literature Volume110, Issue2February 11, 1980Pages 181-183 ReferencesRelatedInformation

The haemagglutinin from the Hong Kong influenza virus A/Memphis/102/72 contains seven oligosaccharide units attached to asparagine residues 8, 22, 38, 81, 165 and 285 in the heavy chain (HA1) and to residue 154 in the light chain (HA2). The single oligosaccharide unit in HA2 and four of the oligosaccharide units of HA1 (at residues 8, 22, 38 and 81) contain the four monosaccharides N-acetylglucosamine, mannose, galactose and fucose and are of the N-acetyllactosamine (or ‘complex’) type. The two other oligosaccharide units on HA1 are of the oligomannoside (or ‘simple’) type and contain only two residues of N-acetylglucosamine and five or six residues of mannose. The data are discussed in relation to the differences in the carbohydrate compositions of other influenza haemagglutinins.

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.

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.

Journal Article PRIMARY STRUCTURE OF THE HONG KONG (H3) HAEMAGGLUTININ Get access COLIN W WARD, BSc PhD, COLIN W WARD, BSc PhD Division of Protein Chemistry, Commonwealth Scientific and Industrial Research OrganizationParkville, Victoria, Australia Search for other works by this author on: Oxford Academic PubMed Google Scholar THEO A DOPHEIDE, BSc PhD THEO A DOPHEIDE, BSc PhD Division of Protein Chemistry, Commonwealth Scientific and Industrial Research OrganizationParkville, Victoria, Australia Search for other works by this author on: Oxford Academic PubMed Google Scholar British Medical Bulletin, Volume 35, Issue 1, January 1979, Pages 51–56, https://doi.org/10.1093/oxfordjournals.bmb.a071542 Published: 01 January 1979

Gel filtration of a cyanogen bromide digest of pure intact hemagglutinin from A/Memphis/102/72 influenza virus allowed the isolation of a variety of fragments. One of these fragments consists of three cyanogen bromide peptides (CNl and CN3 from HA1, and CNl from HA2) which remain linked together by disulphide bonds. This fragment was found to be antigenically active, as it was able to form antigen-antibody complexes (detected by affinity chromatography of radioiodinated peptide-IgG mixtures on protein A-Sepharose) with IgG directed against the protein moiety of viral hemagglutinin. The three cyanogen bromide peptides present in this disulphide-linked fragment were separated by gel filtration, carried out under reducing conditions, and tested for antigenic activity after controlled reoxidation of the individual peptides. Only one cyanogen bromide peptide, CNl from HA1, showed significant binding to antibody. The results indicate that antigenic activity of A/Mem/1O2/72 hemagglutinin resides within the N-terminal 170 amino acid residues of the hemagglutinin heavy chain.

Immunoglobulin G prepared from rabbit antisera raised against three strains of influenza virus was adsorbed with suitable recombinant viruses to yield IgG directed only against the polypeptide moiety of the hemagglutinin. Immune complexes formed by incubation of these antibodies with purified hemagglutinin heavy chain, or its derivatives, were detected by affinity chromatography using protein A-Sepharose. It was found that reduction and alkylation (carboxymethylation) of hemagglutinin heavy chain did not affect the antigenicity of the molecule. Peptides derived from the heavy chain by cyanogen bromide cleavage still possessed substantial antigenic activity. Carboxymethylation of these peptides, however, caused a more dramatic decrease in the binding of antibody.

The reduced carboxymethylated heavy chain (HA1) of a Hong Kong influenza haemagglutinin (H3) was digested with cyanogen bromide to give five peptides. The sequences of three of these peptides, together with the sequences of two methionine overlap peptides were combined to give the C-terminal 68-residue sequence of the heavy chain. Threonine is the C-terminal residue of the heavy chain, an unexpected result in view of the generation of the heavy and light chains by the proteolytic cleavage of a pro-haemagglutinin during virus assembly. Three half-cystine residues occur close together at positions 24, 48 and 52 residues in from the C-terminus. A carbohydrate group is attached to an asparagine residue 44 residues from the C-terminus; it contains 5 residues mannose and 2 residues glucosamine.

The amino acid sequence of apovitellenin I from emu (Dromaius novae-hollandiae) egg yolk has been determined. Difficulties were encountered during sequencing, due to a labile Tyr-Val bond, which was hydrolysed readily by trypsin, chymotrypsin and pepsin. By use of a sequenator, this bond was easily characterized. The protein contains 84 residues and is devoid of half-cystine and histidine. Hydrophobic residues occur in clusters; two very hydrophobic sequences of 12 and 13 residues are present. A very hydrophilic sequence of seven residues contains nearly one-third of all side-chain charges in the molecule; the remainder of the polar residues are scattered throughout the sequence. In a number of instances, residues with opposite charges occur in adjacent positions.

The amino acid sequence of component 0.62, a protein derived from wool keratin, with molecular weight of 6950, rich in glycine and aromatic residues, has been determined. The protein contains no lysine, histidine, glutamic acid, isoleucine or methionine. Of the total residues, more than 50% are glycine and aromatic amino acids. The sequence contains two sections rich in glycine, (Gly‐X)3 and (Gly‐X)4, which comprise 22% of the sequence.

The complete amino acid sequence of the protein subfraction B of S-carboxymethylated high-sulfur protein from reduced and S-carboxymethylated high-sulfur protein from wool has been determined. The protein contains 156 amino acid residues; the NH2-terminal alanine residue is acetylated and the COOH-terminal residue is S-carboxymethylcysteine. The protein contains no lysine, histidine, nor methionine, and only 6 arginine residues. Only six carboxyl side chains are present in addition to the 34 residues of derived S-carboxymethylcysteine. Tryptic digestion of this protein gave four peptides containing 2, 7, 59, and 88 residues. The structures of these peptides were determined by enzymic cleavage with thermolysin, pepsin, and chymotrypsin, and the use of the Edman degradation with dansylation. Protein SCMK-B2B displays a high degree of internal homology.

By incorporating aroG, the structural gene for 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase (phe), into the genome of a heat-inducible susR60 mutant of phage lambda, it has been possible to increase the intracellular levels of DAHP synthetase (phe) in a lysogenized strain of Escherichia coli some 15-fold over levels found in the wild-type strain. By using this strain, the enzyme has been purified approximately 2,000-fold compared with wild type, and various kinetic parameters of the purified enzyme have been studied. In contrast to previous reports, the inhibition by phenylalanine was found to exhibit sigmoidal kinetics, suggestive of cooperative interactions between phenylalanine binding sites. Stimulation of enzyme activity by Co(2+) was minimal (14%).

The selectivity of 2-hydroxy-5-nitrobenzyl bromide for tryptophan, as demonstrated by Koshland and his coworkers, has been used to study some of the properties of the residues of this amino acid in pepsin. The aromaticity and absorbance of the alkylated tryptophan facilitate the isolation of peptides containing the modified residues. Pepsin freshly prepared from pepsinogen was reduced, carboxymethylated, and treated with 2-hydroxy-5-nitro-benzyl bromide. The total incorporation of reagent corresponded to 4 residues of tryptophan per molecule. Hydrolysis of pepsin with alkali or with enzymes, followed by ion exchange chromatography, gave the same result. Reduced and carboxymethylated pepsinogen, when treated with 2-hydroxy-5-nitrobenzyl bromide, also was found to contain 4 tryptophan residues per molecule. The zymogen derivative was hydrolyzed by chymotrypsin and a group separation of peptides was made by gel filtration on Sephadex G-50. Each of four groups containing alkylated tryptophan residues was further hydrolyzed by thermolysin. The mixtures of small peptides were fractionated on columns of Sephadex G-25 and G-15. The peptides containing alkylated tryptophan were retarded by adsorption on the gel and their elution could be followed by measurement of the color of the hydroxynitrobenzyl group. The amino acid compositions and sequences of all of the peptides isolated were consistent with the presence of the following four sequences in pepsin and pepsinogen: I, Val-Phe-Asp-Asn-Leu-Trp-Asp-Gln-Gly; II, Leu-Trp-Val-Pro-Ser; III, Val-Glu-Gly(Trp,Gln); IV, Leu-Asn-Trp-Val-Pro. Each of the above sequences in the protein gave rise to several peptides which were separable on the gel columns but showed identical amino acid compositions. The multiplicity of peaks is probably related to the chemistry of the alkylation, which Koshland and associates have shown may yield several isomeric, monosubstituted derivatives of the indole ring of tryptophan. When active pepsin (with the disulfide bonds intact) was treated with 2-hydroxy-5-nitrobenzyl bromide at pH 3.5, only 2 residues of the reagent were incorporated; one has been shown to be in Sequence I, above, the second in Sequence IV. The loss in specific activity against both hemoglobin and acetyl-l-phenylalanyl-l-diiodotyrosine resulting from this incorporation is only 25 to 30%, indicating that the reagent is not reacting with groups that are essential for catalytic activity. In alkali-denatured pepsin, 3 residues of tryptophan were accessible, whereas the 4th became reactive only after cleavage of the disulfide bonds of the protein by reduction and carboxymethylation. In the course of these experiments it was also observed that all 4 of the methionine residues of pepsin are unavailable to alkylation by iodoacetate or iodoacetamide at pH 2.2.