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The HA tag is cleaved and loses immunoreactivity during apoptosis

Nature Methods volume 4, pages 107–108 (2007)Cite this article

To the editor: Since its introduction almost 20 years ago as a short immunoreactive tag1, the hemagglutinin epitope YPYDVPDYA (HA tag) from the influenza virus A, has been extensively used in various experimental contexts of cell biology and biochemistry to track proteins of interest within cells, to isolate them and to coprecipitate their possible partners. This holds particularly true in the field of apoptosis, where the ever-increasing number of candidates participating in apoptosis signaling pathways has prompted the community to most often rely on engineered tags—such as the HA tag—rather than embarking on the risky development of specific antibodies. As an illustration, a simple full-text search “HA tag” and “apoptosis” in the Highwire press database (http://www.highwire.org) yields over 200 publications only for the past 12 months.

Indeed, the fluorescence release from the membrane of cells expressing SNAP-25–HA-GFP is remarkably synchronous with the development of caspase 3/7 activity (Fig. 1a) as it coincided with the signal of a recombinant caspase 3/7 probe consisting in a fusion of a red fluorescent protein with a mitochondrial outer membrane anchor domain tethered together by a caspase 3/7–sensitive DEVD linker. Transfection and expression of a constitutively active form of caspase 3 induced GFP release as well, further supporting the involvement of caspase 3/7 in HA cleavage (Supplementary Fig. 3 online). Altogether, these data suggested that release of GFP from the plasma membrane could result from a direct proteolytic cleavage of SNAP-25–HA-GFP by caspase 3/7. Notably, we realized that the HA tag itself contained a DVPD sequence, which had been documented as a caspase 3/7 cleavage site in two other proteins3,4.

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Figure 1: The HA tag is cleaved by caspase 3/7.

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Authors and Affiliations

  1. INSERM E347, Institut Bergonié, 229 Cours de l'Argonne, Bordeaux, 33076, France

    Laura Schembri, Renee Dalibart, Flora Tomasello, François Ichas & Francesca De Giorgi

  2. CNRS UMR5164, Université Bordeaux 2, 146 rue Léo Saignat, Bordeaux, 33076, France

    Patrick Legembre

Authors
  1. Laura Schembri
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  2. Renee Dalibart
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  3. Flora Tomasello
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  4. Patrick Legembre
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  5. François Ichas
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  6. Francesca De Giorgi
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Corresponding author

Correspondence to Francesca De Giorgi.

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Competing interests

L.S., F.T., F.I. and F.D.G. are authors on a patent application WO, 2005/012913.

Supplementary information

Supplementary Figure 1

SNAP-HAGFP signal redistribution during apoptosis.

Supplementary Figure 2

SNAP-HAGFP cleavage occurs early during apoptosis.

Supplementary Figure 3

Active caspase 3 cleaves SNAP-HAGFP.

Supplementary Figure 4

SNAP-mutHAGFP (DVPD of HA replaced by DVPE) is not cleavable and does not react with the anti-HA antibody.

Supplementary Figure 5

HA cleavage occurs irrespective of the nature and the location of the construct.

Supplementary Video 1

Live cell imaging of HA cleavage.

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Schembri, L., Dalibart, R., Tomasello, F. et al. The HA tag is cleaved and loses immunoreactivity during apoptosis. Nat Methods 4, 107–108 (2007). https://doi.org/10.1038/nmeth0207-107

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