Dual Role of Dysfunctional Asc-1 Transporter in Distinct Human Pathologies, Human Startle Disease, and Developmental Delay

Genomic organization of the human Asc-1 transporter and patient mutations. The SLC7A10 gene encodes the Asc-1 protein. A, The gene includes 11 exons with long intronic sequences between exons 1–2 and 2–3. B, Screening of 51 patient samples revealed three nucleotide exchanges including one missense mutation in exon 7 c.919G>A; G307R identified in patient 147 and two synonymous mutations c.1158G>A; T386T in exon 9 (patients 10 and 99) and c.1374G>T; T458T in exon 10 (patients 109, 115, 116).

Reduced whole-cell expression of Asc-1^G307R does not result in inability to traffic. Asc-1 WT and the variant Asc-1^G307R were co-expressed in HEK293 cells with the heavy chain 4F2hc and fusion protein of a neuromodulin sequence (GAP-43) and the fluorescent protein DsRed (DsRed-GAP-43, magenta). A, Forty-eight hours post-transfection, cells were either permeabilized or (B) used nonpermeabilized to show membrane expression of Asc-1 WT and the Asc-1 variant Asc-1^G307R (green) colocalized with DsRed-GAP-43. Scale bar: 30 μm. White arrow heads point to colocalization of Asc-1 with the membrane marker DsRed-GAP-43. C, Images from a parallel immunostaining at a lower magnification (scale bar: 200 μm) to demonstrate similar transfection efficiencies of the Asc-1 WT and mutant protein. Co-transfected GFP (shown in white) was used as an internal transfection efficiency control.

Asc-1^G307R is transported highly efficiently toward the cellular surface. A, Asc-1 is specifically stained as monomeric (50 kDa) and dimeric (110 kDa) protein isoform. GAPDH served as housekeeping protein. Asc-1 WT and Asc-1^G307R protein level were quantified following biotinylation of surface expressed proteins. B, C, Representative Western blots from whole-cell protein (B) and surface-expressed proteins (C) immunostained with either the pan-Cad antibody (pan-cadherin; membrane marker) or the Asc-1 antibody. D, Quantification of the Asc-1 whole-cell protein (WC). The expression of Asc-1 was correlated to the marker protein pan-Cad. Asc-1 Mon refers to the Asc-1 monomer (50 kDa), Asc-1 Dim to 110 kDa and both protein bands together (Asc-1 Mon+Dim). E, Quantification of the surface-expressed Asc-1 normalized to the pan-Cad expression. The Asc-1 WT (black) is always compared with the mutant Asc-1^G307R (red).

The startle disease mutation Asc-1^G307R is highly impaired in transport activity. Glycine uptake assays were used to estimate the transport capacity of the Asc-1^G307R variant. A, [³H] glycine was used in a concentration series of 0, 25, 50, 100, 250, 500, 750, 1000 nm. Glycine uptake was measured after 10 min. The single expressed 4F2hc heavy chain was used a negative control (gray circle) and compared with Asc-1 WT (black circles) and Asc-1^G307R (red circles). B, Glycine uptake at 100 nm glycine. C, Magnification of the glycine uptake values (pmol/mg × min) of the mutant Asc-1^G307R (red) and the heavy chain 4F2hc (gray) demonstrating that the mutant although highly impaired in glycine uptake is still significantly different from background level of glycine transport in HEK293 cells. D, Glycine uptake in the presence of the transportable inhibitor D-isoleucine (D-ile). Asc-1 WT transport capability is reduced (gly = black circles; gly+D-ile = black squares) in the presence of the inhibitor while D-ile seems not to affect the mutant variant Asc-1^G307R (gly = red circles; gly+D-ile = red squares). E, Comparison of glycine uptake in the presence and absence of the inhibitor D-ile at 100 nm glycine. F, Magnification of the glycine transport in the presence of D-ile at 100 nm glycine. Statistical significance was estimated using the ANOVA test for multiple comparisons with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All experiments were performed seven times (n = 7).

The positively charged side chain of the amino acid at position 307 is responsible for impaired Asc-1 transport activity. A, Protein lysates from cultures co-transfected with Asc-1 variants G307K, G307W (SNV present in the human population, single case described), G307A or Asc-1^WT together with 4F2hc. GAPDH was used as positive loading control protein (36 kDa). GFP transfected cells were used as MOCK control. Cells transfected with 4F2hc were used as negative control. B, Glycine uptake assays for artificial Asc-1 variants G307W, G307R, and G307A. Note that the Asc-1^G307K is unable to transport glycine while Asc-1^G307W and Asc-1^G307A retain WT capabilities of glycine transport. C, Glycine uptake a 100 nm glycine demonstrating the significant reduction in glycine transport for mutant Asc-1^G307K. D, Comparison of the endogenous glycine uptake by the cells without expression of the Asc-1 transporter compared with Asc-1^G307K. Significance values *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Five experiments have been performed (n = 5), all probes were run in duplicates.

D-isoleucine is unable to inhibit Asc-1 glycine transport capacity in Asc-1 mutants at residue 307. A–D, Glycine uptake in the presence and absence of 4 mm D-isoleucine (D-ile) for Asc-1 WT, and Asc-1 variants G307K, G307W, and G307A. In contrast to Asc-1 WT, the transport capacity was unchanged for all Asc-1 variants in the presence of D-ile. E, Comparison of glycine uptake in the absence and presence of D-ile at low (100 nm) and (F) high glycine (1000 nm) concentration. G, H, Inhibition of glycine transport by D-ile (4 mm) at glycine concentrations 25 nm (G) and 50 nm (H) for Asc-1^G307K. Four experiments have been performed (n = 4). Significance values: *p < 0.05, **p < 0.01.

Modeling of the SLC7A10 variants. A, Overall architecture of the model predicted for human SLC7A10 as predicted by AlphaFold2. In the model, sequences from 1 to 34 and 494 to 523 are not shown for easier representation. B–F, Enlarged views of the environment surrounding of G307 in the unmodified WT sequence compared with G307R, G307K, G307W, and G307A.