tetano
Editor, Senior Moderator
J Biol Chem. 2014 Oct 27. pii: jbc.M114.586180. [Epub ahead of print]
Site Specific S-acylation of Influenza Virus Hemagglutinin: the Location of the Acylation Site Relative to the Membrane Border is the Decisive Factor for Attachment of Stearate.
Brett K1, Kordyukova LV2, Serebryakova MV2, Mintaev RR2, Alexeevski AV2, Veit M3.
Author information
Abstract
S-acylation of hemagglutinin (HA), the main glycoprotein of Influenza viruses, is an essential modification required for virus replication. Using mass spectrometry we have previously demonstrated specific attachment of acyl chains to individual acylation sites. Whereas the two cysteines in the cytoplasmic tail of HA contain only palmitate, stearate is exclusively attached to a cysteine positioned at the end of the transmembrane region (TMR). Here we analyzed recombinant viruses containing HA with exchange of conserved amino acids adjacent to acylation sites or with a TMR cysteine shifted to a cytoplasmic location to identify the molecular signal that determines preferential attachment of stearate. We first developed a new protocol for sample preparation that requires less material and might thus be also suitable to analyze cellular proteins. We observed cell-type specific differences in HA`s fatty acid pattern; more stearate was attached if human viruses were grown in mammalian compared to avian cells. No underacylated peptides were detected in the mass spectra and even mutations that prevented generation of infectious virus particles did not abolish acylation of expressed HA as demonstrated by metabolic labeling experiments with 3H-palmitate. Exchange of conserved amino acids in the vicinity of an acylation site had a moderate effect on the stearate content. In contrast, shifting the TMR cysteine to a cytoplasmic location virtually eliminated attachment of stearate. Thus, the location of an acylation site relative to the transmembrane span is the main signal for stearate attachment, but the sequence context and the cell type modulate the fatty acid pattern.
Copyright ? 2014, The American Society for Biochemistry and Molecular Biology.
KEYWORDS:
fatty acid; glycoprotein; hemagglutinin; influenza virus; mass spectrometry (MS); palmitate; protein acylation; protein palmitoylation; stearate; transmembrane domain
PMID:
25349209
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/25349209
Site Specific S-acylation of Influenza Virus Hemagglutinin: the Location of the Acylation Site Relative to the Membrane Border is the Decisive Factor for Attachment of Stearate.
Brett K1, Kordyukova LV2, Serebryakova MV2, Mintaev RR2, Alexeevski AV2, Veit M3.
Author information
Abstract
S-acylation of hemagglutinin (HA), the main glycoprotein of Influenza viruses, is an essential modification required for virus replication. Using mass spectrometry we have previously demonstrated specific attachment of acyl chains to individual acylation sites. Whereas the two cysteines in the cytoplasmic tail of HA contain only palmitate, stearate is exclusively attached to a cysteine positioned at the end of the transmembrane region (TMR). Here we analyzed recombinant viruses containing HA with exchange of conserved amino acids adjacent to acylation sites or with a TMR cysteine shifted to a cytoplasmic location to identify the molecular signal that determines preferential attachment of stearate. We first developed a new protocol for sample preparation that requires less material and might thus be also suitable to analyze cellular proteins. We observed cell-type specific differences in HA`s fatty acid pattern; more stearate was attached if human viruses were grown in mammalian compared to avian cells. No underacylated peptides were detected in the mass spectra and even mutations that prevented generation of infectious virus particles did not abolish acylation of expressed HA as demonstrated by metabolic labeling experiments with 3H-palmitate. Exchange of conserved amino acids in the vicinity of an acylation site had a moderate effect on the stearate content. In contrast, shifting the TMR cysteine to a cytoplasmic location virtually eliminated attachment of stearate. Thus, the location of an acylation site relative to the transmembrane span is the main signal for stearate attachment, but the sequence context and the cell type modulate the fatty acid pattern.
Copyright ? 2014, The American Society for Biochemistry and Molecular Biology.
KEYWORDS:
fatty acid; glycoprotein; hemagglutinin; influenza virus; mass spectrometry (MS); palmitate; protein acylation; protein palmitoylation; stearate; transmembrane domain
PMID:
25349209
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/25349209