An N-terminal motif unique to primate tau enables differential protein-protein interactions

Abstract

Compared with other mammalian species, humans are particularly susceptible to tau-mediated neurodegenerative disorders. Differential interactions of the tau protein with other proteins are critical for mediating tau's physiological functions as well as tau-associated pathological processes. Primate tau harbors an 11-amino acid-long motif in its N-terminal region (residues 18-28), which is not present in non-primate species and whose function is unknown. Here, we used deletion mutagenesis to remove this sequence region from the longest human tau isoform, followed by glutathione S-transferase (GST) pulldown assays paired with isobaric tags for relative and absolute quantitation (iTRAQ) multiplex labeling, a quantitative method to measure protein abundance by mass spectrometry. Using this method, we found that the primate-specific N-terminal tau motif differentially mediates interactions with neuronal proteins. Among these binding partners are proteins involved in synaptic transmission (synapsin-1 and synaptotagmin-1) and signaling proteins of the 14-3-3 family. Furthermore, we identified an interaction of tau with a member of the annexin family (annexin A5) that was linked to the 11-residue motif. These results suggest that primate Tau has evolved specific residues that differentially regulate protein-protein interactions compared with tau proteins from other non-primate mammalian species. Our findings provide in vitro insights into tau's interactions with other proteins that may be relevant to human disease.

Keywords: Tau protein (Tau); mass spectrometry (MS); neurochemistry; protein-protein interaction; tauopathy.

Figure 1.

A unique 11-amino acid motif in the N-terminal region of primate tau. A, alignment of mammalian tau primary sequences. Primary sequences of N-terminal regions of human, non-human primate, macaque, bovine, goat, dog, cat, and rodent tau proteins were aligned using ClustalOmega with marked up similarities (gray shading). Note the absence of a motif between amino acids 18 and 28 of primate tau in other mammalian tau sequences. Only canine tau contains a 10-amino acid motif of lesser similarity to primate tau. B, dendrogram of N-terminal mammalian tau protein sequences. Dendrogram analysis of sequences between the indicated residues in A (dashed lines) was performed using ClustalOmega. Note that canine tau clusters with primate (human, Pan troglodytes, and Macaca mulatta) tau N-terminal sequences due to its 10-amino acid motif albeit with weaker similarity. C, amino acids 18–28 were deleted in human tau40 to generate the tauΔ18 construct. D, tauΔ18 and tau full-length (tauFL) were transfected into 293T cells. Immunoblot of cell lysates from tauFL, tauΔ18, or untransfected control was detected with anti-human tau antibody. tauΔ18 shows reduced retention, consistent with deletion of amino acids 18–28. Probing for glyceraldehyde-phosphate dehydrogenase (GAPDH) served as loading control.

Figure 2.

iTRAQ-based mass spectrometry to identify tau protein–protein interactions modulated by aa 18–28. A, production of recombinant tau proteins. GST-tagged tauFL and tauΔ18–28 were produced in E. coli. Samples of washed and eluted protein were separated by SDS-PAGE and visualized by Coomassie Brilliant Blue. B, experimental setup for quantitative comparison of tau protein–protein interactions in dependence of the 11-aa motif in tau's N-terminal region using 8-plex iTRAQ. Technical replicates of GST pulldown with GST-tagged tauFL and tauΔ18–28 were subjected to tryptic digest and iTRAQ labeled (labels 113, 114, 115, 116, 117, 118, 119, and 121). Peptides were detected by mass spectrometry (Triple TOF 5600⁺ mass spectrometer, SCIEX). Cross-comparison ratios of iTRAQ-labeled peptides were determined by ProteinPilot version 4.0 software.

Figure 3.

Gene ontology (GO) and protein network analysis of differential protein–protein interactions modulated by presence of the aa 18 to 28 N-terminal tau motif. A, protein ontology was analyzed using DAVID (version 6.8) gene ontology. B, protein interaction network of deregulated protein–protein interactions with tauΔ18–28 was analyzed using STRING (v10.5). Network edges are defined by confidence indicating the strength of data support by line thickness.

Figure 4.

Validated interactions modulated by primate-specific N-terminal tau motif. A–E, selected tau protein interaction partners annexin A5/Lipocortin-IV (AnxA5) (A) 14-3-3β (B), 14-3-3η (C), synaptotagmin-1 (D), and GSK3β (E) were expressed in 293T cells together with V5-tagged human tauFL or tauΔ18–28. Protein–protein interaction was assessed by co-immunoprecipitation (IP) using a V5-specific antibody and detected by immunoblotting for HA, myc, green fluorescent protein (GFP), or GSK3. Results from 3 independent experiments are represented as mean ± S.D. (Student t test) ** p < 0.05. ns, not significant.