Mass spectrometric sequencing and acylation character analysis of C-terminal anchoring segment from Influenza A hemagglutinin.
Influenza A virus hemagglutinin (HA) is a major envelope glycoprotein mediating viral and cell membrane fusion. HA is anchored in the viral envelope by a light HA(2) chain containing one transmembrane domain and a cytoplasmic tail. Three cysteine residues in the C-terminal region, one in the transmembrane domain and two in the cytoplasmic tail, are highly conserved and potentially palmitoylated in all HA subtypes. The HA(2) C- terminal anchoring segments were extracted to organic phase from the bromelain-digested viruses (subviral particles) of three strains: A/X-31 (H3 subtype), A/Puerto Rico/8/34 (H1 subtype) and A/FPV/Weybridge/34 (H7 subtype). Their primary structures were assessed by matrix-assisted laser desorption/ionization time-of-flight time-of- flight mass spectrometry (MALDI-ToF-ToF MS). Trypsin-type protease-cleaved peptides prevailed over bromelain- cleaved ones in the peptide mixtures. All of them included transmembrane domains. Several distinctive features of the C-terminal HA(2) peptides acylation character were discovered by MALDI-ToF MS: 1) the peptides isolated from the viruses, which were digested by bromelain in the absence of beta-mercaptoethanol, were predominantly triply acylated; 2) the peptides were acylated not only by palmitic, but also by stearic acid residues; 3) the palmitate/stearate ratio was different for the three strains studied; 4) the A/FPV/Weybridge/34 strain has a priority to stearate binding. This fatty acid residue was discovered at the first of three conservative cysteine residues located in the transmembrane domain. It was found that presence of thiol reagent during preparation of subviral particles led to the appearence of the C-terminal HA(2) peptides acylated to different degrees. Triply, doubly, mono- and even unacylated peptides were detected. It was demonstrated that the thioester bond in the isolated acylpeptides was extremely sensitive to thiol reagents.
Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Moscow.
Marina V
MV
Serebryakova
Larisa V
LV
Kordyukova
Ludmila A
LA
Baratova
Stanislav G
SG
Markushin
D000215:Acylation; D000595:Amino Acid Sequence; D001963:Bromelains / Q000737:chemistry; D005227:Fatty Acids / Q000378:metabolism; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000032:analysis*; D009980:Influenza A virus / Q000737:chemistry*; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry
D005227:Fatty Acids; D019267:Hemagglutinin Glycoproteins, Influenza Virus; C483468:hemagglutinin, human influenza A virus; D001963:Bromelains; D014357:Trypsin