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b1-tubulin role in platelet function
TUBB1 variants), present with a macrothrombocytopenia (107x109/L and 85x109/L respectively). Individual A:2 car- ries the GFI1B variant but is WT for TUBB1 and presents with a normal platelet count (221x109/L). Individuals A:1 and A:3 also present with significantly higher immature platelet fractions (IPF) and mean platelet volumes (MPV) when compared to their TUBB1 WT relatives (53.5% and 55.1% compared to 24.5%, MPV for A:1 and A:3 too large for measurement). This variation in count, IPF and platelet morphology in individuals with the TUBB1 R359W vari- ant may suggest that the TUBB1 variant is linked to the macrothrombocytopenia observed.
Family/patient B was an elderly gentleman (now deceased) with a G insertion and subsequent frameshift truncation of the b1-tubulin protein 19 amino acids from the site of insertion (c.1080insG, p.L361Afs*19).30,31 This patient had a severe thrombocytopenia with a platelet count of 11x109/L and an MPV above 13.4 (Figure 1B). At the time of study, IPF measurement was unavailable.
p.L361Afs*19 is absent in the latest version of the gno- mad database (accessed October 2020), while p.R359W is a rare variant with a frequency of 6.78x10-3 (gnomad accessed October 2020) with a significant pathogenicity prediction score of 25.4 using combined annotation dependent depletion (CADD), where a score of greater or equal to 20 indicates the 1% most deleterious sequence variants in the genome. Both variants were analyzed using in silico bioinformatic tools and were scored as ‘uncertain significance’ based on the current ACMG guidelines32 (Online Supplementary Figure S1). Both TUBB1 variants are positioned towards the C-terminal region of b1-tubulin as indicated in Figure 1C and D. This region is positioned away from the dimer interface with a-tubulin, and the glutamate rich C-terminal tail, not present in the model, is an established site for PTM (Figure 1C and D).1 Both affected TUBB1 sequence variants are highly conserved in mammals (Figure 1E). R359 is found within a loop region towards the C-terminus of tubulin, and we predict mutat- ing this residue would not cause dramatic misfolding within the protein secondary structure. R359 does form direct polar contacts with the N-terminal helix and remov- ing this contact through the sequence variant to the hydrophobic tryptophan may result in minor structural changes to the connected C-terminal regions, potentially affecting PTM or interactions with critical microtubule accessory proteins (MAP) (Figure 1C and D). Similarly, the G insertion and subsequent frameshift effectively deletes the C-terminus of the protein and would therefore, if the protein folds correctly, result in effects similar, or more extreme, to the substitution of R359.
Patient B demonstrated a significant reduction in surface P-selectin expression and fibrinogen binding by flow cytometry in response to all agonists tested (Online Supplementary Figure S1). Family A showed no change in the levels of surface receptor expression, but weak P- selectin and fibrinogen responses when activated with a low concentration ADP, CRP, and PAR-1, suggesting a mild functional defect (Online Supplementary Figure S1C). Patient B also showed marked reduced expression of P- selectin and fibrinogen uptake in response to the same activation agonists (Online Supplementary Figure S1D and E). Patients with C-terminal variants in this study and oth- ers previously reported by Fiore et al. present with a macrothrombocytopenia also found in individuals with a complete loss of the b1-tubulin, suggesting that the C-ter-
minal tail is likely critical to the function of TUBB1 in the roles of microtubules in MK and platelets.18–20 As this C-ter- minal tail is rich in glutamate residues which are often tar- geted for polymodification, we began to investigate the polymodification of b1-tubulin.
C-terminal variants of b1-tubulin fold correctly and demonstrate a reduction in polymodification
First, we investigated the effects of the two patient vari- ants (p.R359W and p.L361Afs*19) on the folding and potential polymodification of b1-tubulin. We designed, generated, and validated a b1-tubulin-mApple fusion con- struct (Online Supplementary Figure S2). This plasmid was further mutated to harbor each of the patient variants (p.R359W and p.L361Afs*19), and an artificial C-terminal truncation which specifically deletes the glutamate rich C- terminal tail (Figure 2A; Online Supplementary Figure S2). The WT b1-tubulin construct was transfected into Hek293T cells and co-stained for polyglutamylated and polyglycylated tubulin specifically. Transfected cells are exclusively positive for both residues indicating that b1- tubulin is indeed polymodified (Figure 2B). Expression of each of the mutated constructs show that both patient variants and the C-terminal truncation fold correctly (Figure 2C). Furthermore each of them results in a consis- tent and significant reduction in polymodification (Figure 2D). This data indicates that both patient variants result in a correctly folded b1-tubulin which has a similar effect to a truncation of the C-terminus, and is further supported by western blotting of mutant constructs (Online Supplementary Figure S3) which show a significant reduc- tion in polymodification.
Induced pluripotent stem cell-derived proplatelet forming megakaryocytes are both polyglycylated and polyglutamylated
The tubulin code posits that a highly lineage and species specific expression of modifying enzymes mediates cell specific PTM. While our transfection data successfully indicates that b1-tubulin is indeed polymodified, and that each of our patient genetic variants result in the expression of a functional b1-tubulin with a C-terminal truncation, data from both human MK and platelets is needed to dis- sect the role of polymodification in these cells.
To date, polyglycylation has not been reported in either platelets or MK. Polyglutamylation has recently been reported in a modified CHO cell line and human platelets. In order to investigate polymodification in human MK, we adapted a directed differentiation protocol previously reported by Feng et al. to generate large populations of mature, proplatelet forming cells (Online Supplementary Figure S4). iPSC-MK were stained for CD42b as a marker for mature, and hence TUBB1 expressing, MK, and both polyglutamylated and polyglycylated tubulin. CD42b+ cells were found to be positive for both polyglutamylated tubulin and polyglycylated tubulin (Figure 3A), while neighboring cells in the sample negative for CD42b did not demonstrate these polymodifications (Figure 3B). Across multiple differentiations we consistently yielded a purity of approximately 50-60% CD42b+ cells (Figure 3C), which on analysis are positive for both polyglutamylated and polyglycylated tubulin (Figure 3D). Finally, 100% of pro- platelet forming cells observed across replicates were posi- tive for both polymodifications (Figure 3E), and this was further confirmed by western blotting (Figure 3F).
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