Introduction

Autism spectrum disorder (ASD) is a prevalent neurodevelopmental disorder characterized by impaired social interaction and behaviors. Congenital genetic defect is one of the causes of ASD. To date, many genes associated with ASD have been identified by several genome-wide association studies1,2,3,4,5. Mostly, these mutations are found in the genes encoding synaptic proteins, transcription factors, chromatin modifiers, and protein metabolisms. Hence, dysfunctions of these proteins caused by both de novo and inherited mutations are highly linked to the pathogenesis of ASD.

An aberrant ubiquitin–proteasome system is one of the main causes of ASD pathogenesis. The deubiquitinating enzyme, USP15, which regulates various signaling pathways and cellular processes, is involved in neuronal disorders6,7,8. Moreover, de novo mutation in the Usp15 gene is closely associated with ASD2,9,10. Previously, we have reported that USP15 deubiquitinates U6 snRNA-specific terminal uridylyl transferase 1 (TUT1) and regulates the spliceosome cascade11. A defect in Usp15 increases the probability of splicing errors and produces various abnormal variants. We also found that impaired USP15 induces the endoplasmic reticulum (ER) stress, although the mechanism linking splicing error to ER stress has not yet been clarified11.

Proper subcellular localization of the synaptic proteins is crucial for regulating neural circuit formation and brain homeostasis. The amino acid substitutions in synaptic proteins result in their accumulation in the ER, and cause abnormal trafficking to the proper sites. It is also known that these substitutions impair protein folding and frequently activate the unfolded protein response (UPR). For instance, the ASD-associated mutant Neuroligin 4 (NL4) , in which Arg87 is replaced with Trp, accumulates in the ER and does not transport to neuronal dendrites12. The ASD-associated γ-aminobutyric acid transporter 1 (GAT-1) mutant, in which Pro361 is replaced with Thr, is prone to localize in the ER13. Moreover, missense mutations of novel ASD-associated transmembrane proteins, a cell adhesion molecule-1 (CADM1), and contactin-associated protein-like 2 (CASPR2) are also localized in the ER and upregulates ER stress14,15. Therefore, abnormal amino acid substitutions of the ASD-related synaptic proteins are predisposed to accumulate in the ER and hamper their trafficking to the proper sites, attenuating innate synaptic functions.

Hevin, also known as secreted protein acidic and rich in cysteine-like 1 (Sparcl1), is a secreted protein. The C-terminus of Hevin has an amino acid sequence with approximately 60% identity to Sparc. It has been reported that Hevin and its family proteins are involved in metastasis, inflammation, angiogenesis, and apoptosis16. In the central nervous system, Hevin is predominantly expressed in astrocytes and is expressed in neurons to a lesser extent17. During brain development, Hevin is expressed in the radial glia and controls a detachment process that terminates neuronal migration in the pial surface of the cerebral cortex18. Hevin is also secreted from neural stem cells (NSCs) in the lateral ventricle subventricular zone (SVZ) and enhances glioma cell invasion19. Thus, it appears likely that Hevin is involved in cell migration and invasion in a context-dependent manner. Importantly, it has been reported that Hevin reinforces the synaptic connectivity in the cerebral cortex20. On this occasion, Hevin interacts with both Neurexin 1α (NRX1α) and Neuroligin 1B (NL1B) and promotes a trans-synaptic bridge at thalamocortical synaptic connections in the cerebral cortex. Hevin has also been reported to enhance the NMDAR-mediated synapse response21. Strikingly, genome-wide association studies have revealed that multiple mutations in the SPARCL1 gene increase the risk of ASD5, indicating that Hevin plays an important role in regulating synaptic connectivity and the brain environment.

A recent structural analysis reported that Hevin promotes the interaction between NRXs and NLs22. Hevin is composed of a flexible acidic region at the N-terminal region, a follistatin-like (FS) domain at the center region, and an extracellular calcium (EC) binding domain, including two EF-hand motifs (EF-hand 1: His586-Ala618 and EF-hand 2: His625-Phe651 amino acid in humans, EF-hand 1: His572-Ala604 amino acid and EF-hand 2: His611-Phe637 amino acid in mice) at the C-terminal region. Interestingly, the structure of the FS/EC region of Hevin differs from that of Sparc, although Sparc shares high sequence identity with Hevin. The FS-domain is crucial for promoting the bridge between NRXs and NLs. Hevin and Sparc compete to interact with NLs via the FS-domain, and thereby Sparc can thus antagonize the synaptogenic functions of Hevin. In contrast, the EC-domain in both Hevin and Sparc interacts with the extracellular matrix, such as Collagen I and V, in a Ca2+-dependent manner22,23. Therefore, analyzing the functions of the EC domain is key to understanding the full functions of Hevin.

In this study, we analyzed the importance of the EF-hand motif in Hevin. We found that the integrity of the EF-hand motif is necessary for proper trafficking to the extracellular space. In addition, a single amino acid substitution in the EF-hand motif, which is associated with ASD, causes Hevin to accumulate in the ER and activates the UPR pathway. These findings suggest the possibility that protein accumulation due to improperly folded Hevin contributes to ASD pathogenesis.

Results

Hevin mutants lacking the EF-hand motif are generated in the Usp15-deficient brain

Previously, we have reported that USP15 influences the spliceosome cascade and UPR pathway despite the unclear relevance11 (Fig. 1A). Thus, we looked for ASD-associated abnormal variants that changed splicing upon Usp15 deficiency and are involved in the UPR pathway. In this context, we checked the expression profile dataset obtained from mice brains11 and found that the 3'-region of the hevin transcript tends to be lacking in the Usp15-deficient brains (Fig. 1B, Supplementary Table S1). Thus, we speculated that the 3'-region of the hevin transcript is susceptible to abnormalities in the spliceosome cascade regulated by USP15. We determined which base sequence of the hevin transcript was altered in the Usp15-deficient brain. To do this, we conducted a 3'-rapid amplification of cDNA ends (3'-RACE) and identified two 3'-fragments lacking the EF-hand motif. One was the EF-hand deletion mutant and the other was the trans-splicing variant, which fused with a fragment of NADH: ubiquinone oxidoreductase subunit A11 (Ndufa11) (Fig. 1C–G). Because the EF-hand motif is pivotal for regulating protein structure and functions24, we hypothesized that EF-hand deletion mutants show abnormal structure, resulting in a decrease of secretion efficiency.

Figure 1
figure 1

Hevin mutants lacking the EF-hand motif are generated in the Usp15-deficient brain. (A) The relationship between the splicing error and ER stress in the Usp15-deficient brain has not yet clarified. (B) Schematic structure of the hevin transcript in the Usp15-deficient mouse brain. The 3'-region of the hevin transcript (PSR/Junction ID: 4,776,772) is prone to be missing in the Usp15-deficient brain. (C) The putative schematic structures of Hevin mutants. (D–G) Sequence analyses of the 3'-regions of hevin transcripts. (D) DNA sequencing chromatogram of the 3'-region of hevin WT and ΔEF transcripts. (E) The 3'-region of hevin sequence. Black box indicates the lacking sequence in ΔEF mutant. Cyan indicates the EF-hand motif. (F) DNA sequencing chromatogram of the 3'-region of hevin WT and Ndufa11. (G) The 3'-region of hevin sequence. Black box indicates the lacking sequence in the Hevin-N11 mutant. Cyan indicates the EF-hand motif.

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Hevin mutants lacking the EF-hand motif activate the UPR signaling caused by abnormal trafficking

To investigate whether these mutants are secreted to the extracellular space, we constructed the expression plasmids of full-length Hevin mutants and transfected them into culture cells. Although Hevin WT was efficiently secreted to the extracellular space, the deletion mutant of EF-hand 2 (Hevin ΔEF) and the fusion protein between Hevin lacking the EF-hand and Ndufa11 C-terminal region (Hevin-N11) showed reduced secretion efficiency compared to Hevin WT (Fig. 2A). These data suggest that the integrity of the EF-hand motifs is crucial for the secretion of Hevin. We next examined whether Hevin mutants accumulate in the ER. To do this, we introduced each plasmid into HeLa cells and stained using an anti-Hevin antibody. Consequently, Hevin mutants were found to be prone to accumulate in the ER compared to Hevin WT (Fig. 2B,C). These data demonstrate that an abnormal EF-hand region suppresses a Hevin export from the ER. Next, we examined whether the accumulation of Hevin mutants activates the UPR pathway. The expression of both Hevin ΔEF and Hevin-N11 increased the mRNA of the UPR markers, Bip and Chop (Fig. 2D,E). Moreover, the protein level of BIP, which is the ER chaperone protein, was increased when Hevin mutants were expressed. Importantly, the BIP expression level was similar to that of thapsigargin-stimulated cells (Fig. 2F), suggesting that Hevin EF-hand deletion mutants accumulate in the ER and activate the UPR pathway.

Figure 2
figure 2

Hevin mutants lacking the EF-hand motif activate the UPR signaling caused by abnormal trafficking. (A) Immunoblotting of Hevin WT and mutants (ΔEF and N11). Neuro-2a cells were transfected for 48 h with expression vectors for Hevin WT or mutants (ΔEF or N11). The cell lysates and conditioned medium were subjected to immunoblot analysis using anti-Hevin and Tubulin antibodies. (B) Immunostaining of Hevin and mutants (ΔEF and N11). HeLa cells were transfected for 24 h with expression vectors for Hevin and mCherry-ER and subjected to immunocytochemistry. Scale bar: 10 μm. (C) Quantification of Pearson’s correlation coefficient as the degree of colocalization in the panel B. n = 20 cells, mean ± standard error of the mean (SEM), **p < 0.01, ***p < 0.001 versus Hevin WT, one-way analysis of variance (ANOVA) Dunnett’s test. (D, E) Total RNAs isolated from the Hevin-overexpressed Neuro-2a cells were conducted with qPCR analysis for measurement of Bip and Chop mRNAs. 5S rRNAs were used for normalization. n = 5; mean ± SEM; *p < 0.05, **p < 0.01 versus Hevin WT, one-way ANOVA Dunnett’s test calculated using the ΔCt value. (F) Immunoblotting of BIP, Hevin, and Tubulin. HEK293T cells were transfected for 72 h with expression vectors for Hevin WT or mutants (ΔEF or N11). Cells were stimulated with 500 nM Thapsigarging (Tg) for 24 h. The cells were then subjected to immunoblot analysis using anti-BIP, Hevin and Tubulin antibodies.

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ASD-associated W647R mutation of Hevin activates the UPR signaling

Both SPARCL1 and Usp15 mutations are associated with ASD2,5,9,10. Importantly, previous transcriptome analyses have identified single amino acid substitutions in SPARCL1/Hevin that are linked to ASD5. One of these mutants in which Trp647 is replaced with Arg is in the EF-hand motif that is also perturbed in Usp15 knockouts5 (Fig. 3A, Supplementary Fig. 1). Strikingly, this amino acid is highly conserved among aves, rodents, and primates (Fig. 3B). Therefore, we postulated that this mutant [human W647R (hW647R)] could exhibit a similar cellular phenotype to the truncated Hevin mutants that we observed in Usp15 KO mice in Fig. 2. First, to investigate whether this amino acid substitution in the EF-hand motif affects Hevin trafficking, we constructed an expression plasmid using the mouse Hevin sequence. Since human Trp647 corresponds to mouse Trp633 (Fig. 3B), we replaced mouse Trp633 with Arg [mouse W633R (mW633R)]. As we expected, the secretion efficiency in Hevin mW633R was attenuated compared to that in Hevin WT (Fig. 3C). In addition, the Hevin mW633R mutant was mostly localized in the ER, as well as Hevin ΔEF and Hevin-N11 (Fig. 3D and E) and was not transported to the Golgi apparatus efficiently (Fig. 3F and G). Furthermore, the expression of Hevin mW633R increased the mRNA level of the UPR markers, Bip and Chop, and the protein level of BIP (Fig. 3H–J), suggesting that a single amino acid substitution in the EF-hand causes Hevin accumulation in the ER, followed by activation of the UPR signaling. Next, we investigated the mechanisms through with the Hevin mutant increases ER stress. Since unfolded proteins promote dissociation of BIP from the ER stress sensors such as IRE1, leading to interaction with freed BIP, we examined whether Hevin mW633R exhibits unfolded protein-like behavior. We found that the binding affinity of Hevin mW633R to BIP is higher than that of WT (Fig. 3K and L). Importantly, Ca2+ existence has little effect on this binding. These data suggest that the Hevin mW633R mutant induces improper folding regardless of Ca2+ existence and supports our idea that the Hevin mutant exhibits a different structure compared to WT.

Figure 3
figure 3

ASD-associated W647R mutant of Hevin activates the UPR signaling. (A) Location of ASD-associated Hevin mutations. (B) Amino acid of the EF-hand motif in each species. The human Trp647 is highly conserved. Cyan indicates the EF-hand motif, Magenta indicates non-conserved amino acid. (C) Immunoblotting of Hevin WT and mW633R mutant. Neuro-2a cells were transfected with expression vectors and collected at the indicated times. The upper panel indicates the experimental schedule. The transfection efficiencies were confirmed by observing co-expressed EGFP fluorescence. The cell lysates and conditioned medium were subjected to immunoblot analysis using anti-Hevin and Tubulin antibodies. The secretion rate of the Hevin WR mutant was delayed more than that of WT. CM; conditioned medium, TCL: total cell lysate (D) Immunostaining of Hevin WT and mW633R mutant. HeLa cells were transfected for 24 h with expression vectors for Hevin and mCherry-ER and subjected to immunocytochemistry. Scale bar: 10 μm. (E) Quantification of Pearson’s correlation coefficient as the degree of colocalization in the panel D. n = 15 cells, mean ± SEM, ***p < 0.001 by Student’s t-test. (F) Immunostaining of Hevin and mW633R mutant. HeLa cells were transfected for 24 h with expression vectors for Hevin and subjected to immunocytochemistry. Scale bar: 10 μm. (G) Quantification of Manders’ coefficient as the degree of colocalization of Hevin with GM130 in the panel F. n = 20 cells, mean ± SEM, ***p < 0.001 by Student’s t-test. (H, I) Total RNAs isolated from the Hevin-overexpressed Neuro-2a cells were conducted with qPCR analysis for measurement of Bip and Chop mRNAs. 5S rRNA were used for normalization. n = 5; **p < 0.01, ***p < 0.001 versus Hevin WT, one-way ANOVA Dunnett’s test calculated p-value using the ΔCt value. (J) Immunoblotting of BIP, Hevin, and Tubulin. Neuro-2a cells were transfected for 72 h with expression vectors for Hevin WT or mW633R mutants. The cell lysates were then subjected to immunoblot analysis using anti-BIP, Hevin, and Tubulin antibodies. (K) Schematic structure of GST-Hevin. (L) Pull down of GST-Hevin and endogenous BIP in HEK293T cells. GST-Hevin was precipitated with Glutathione Sepharose beads and immunoblotted with anti-GST, BIP, and Tubulin antibodies.

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Hevin W647R mutant shows improper folding of the hydrophobic core

Because BIP tends to recognize the hydrophobic sites of misfolded proteins25,26, we speculate that the Hevin WR mutant exhibits structural instability. To verify this hypothesis, a set of μs-order all-atom molecular dynamics (MD) simulations was performed for the EC domain (515–664 amino acid) of WT and hW647R mutant (Fig. 4A). As the first analysis of both trajectories, the root-mean-square deviation (RMSD) for each initial structure during each MD simulation was calculated. As shown in the RMSD profiles, the hW647R system structurally fluctuated compared to the WT system since the hW647R RMSD value increased during the MD simulations, indicating that a replacement of Trp647 with Arg causes an structural instability (Supplementary Fig. 2A–D). To address the origin of this structural fluctuation, the solvent-accessible surface area (SASA) was measured in both systems. It is reasonable to measure the SASA values to address the mutational effect since the structural fluctuation might be caused by a protein surface deformation, in which a part of the protein surface of the WT system might be exposed to the external solvent upon this amino acid substitution. For all of the residues included within 8 Å around the mutation site, their SASA distributions significantly changed in shape, and the SASA values increased in the hW647R system compared to the WT system (Fig. 4B and C). These analyses indicate that this amino acid substitution causes structural fluctuation of the hW647R system. As the second analysis, the radial distribution function (RDF) of water was defined for all of the residues included within 8 Å around the mutation site. The RDFs around each residue allow for identifications of the essential residues that increase SASA. Among all of the calculated distributions, the RDFs of water around V520 showed characteristic profiles (Fig. 4D), where the RDF around V520 for the hW647R system significantly increased compared to that for the WT system. This increase in RDF indicates that the water density around V520 became higher due to the point mutation. Based on the difference in these RDFs, the difference in the density of water was compared among the corresponding residues as follows:

$$\mathop \sum \limits_{r} \Delta {\text{g}}\left( r \right) = \mathop \sum \limits_{r} {\text{g}}\left( r \right)_{{{\text{WT}}}} - {\text{g}}\left( r \right)_{{{\text{W}}647{\text{R}}}} .$$

From the decomposed accumulation, we focused on the top ten residues with the especially large RDF differences (Fig. 4E). Based on the structural observation in the WT system, the hydrophobic residues included in the top ten residues had showed characteristic properties, that is, they tightly contacted each other and constructed a core site. Notably, this core formed in the WT system was not formed in the hW647R system (Fig. 4F and G). To evaluate the structural changes of the hydrophobic core quantitively, the radius of gyration (Rg) of seven hydrophobic residues was measured. The Rg value of these hydrophobic residues characterizes the size of each hydrophobic core. The distributions of Rg differed between the two systems (Fig. 4H,I). Specifically, the median value of Rg measured in the WT system was 8.847 Å while that measured in the hW647R system was 9.675 Å, indicating that the hydrophobic core of the hW647R system was changed. Therefore, it is likely that Hevin hW647R has improper folding around the hydrophobic residues, resulting in structural instability.

Figure 4
figure 4

Hevin W647R mutant shows improper folding of the hydrophobic core. (A) The modeled structure of the WT Hevin system (EC domain). Each color represents the three-helix domain (green), EF-hand domain (cyan), and the N or C terminals (pink), respectively. The spheres (grey) represent Ca2+ ions. The structure is reconstituted from the data in the previous research22 (B, C) The SASA distribution of all of the residues included within 8 Å around the mutation site. (D) Characteristic RDFs of water around V520 with their standard deviations. (E) The accumulation of the difference in the RDFs of water between the WT and W647R systems, i.e., \(\mathop \sum \limits_{r} \Delta {\text{g}}\left( r \right) = \mathop \sum \limits_{r} {\text{g}}\left( r \right)_{{{\text{WT}}}} - {\text{g}}\left( r \right)_{{{\text{W}}647{\text{R}}}}\), versus the top ten residues. (F, G) A set of configurations of each hydrophobic core (D517, V520, L616, I521, F630, T628, Y588, I658, K582 and Y590), where the hydrophobic core and mutated residues are highlighted in green and magenta. (H, I) The Rg distributions of each hydrophobic core.

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Discussions

In this study, we identified that the defects in the Hevin EF-hand motif decrease secretion efficiency and accumulate in the ER. In addition, the ASD-associated Hevin mutant also shows impaired trafficking and hampered an export from the ER. Indeed, the Hevin mutant exposes a hydrophobic region, which is normally hidden inside, to the surface, inhibiting the export of Hevin from the ER and activating the UPR pathway (Fig. 5). Thus, our findings provide a potential mechanism that links an ASD mutation in the SPARCL1 gene to UPR.

Figure 5
figure 5

Model describing the dynamics of Hevin WT and WR mutant in the ER. BIP associates with Hevin WR mutant and triggers the UPR activation.

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Here, we found that this single amino acid substitution in the EF-hand has two cellular phenotypes. First, the expression of Hevin WR activates the UPR pathway. Second, the mutation attenuates the secretion efficiency. As such, we presume that the activation of the UPR pathway and/or the decrease of secretion efficiency are possible mechanisms that connect the Hevin dysfunctions to ASD. Previously, ER stress was proposed as a cellular mechanism related to the onset of ASD27. In fact, the UPR-related transcripts are dysregulated in the frontal cortices of ASD patients28. In most cases of regulating the UPR, three distinct pathways, the inositol-requiring enzyme 1 (IRE1)—X-box binding protein 1 (XBP1) pathway, protein kinase R-like ER kinase (PERK)—eukaryotic initiation factor 2α (eIF2α) pathway, and activating transcription factor 6 (ATF6) pathway, are upregulated by the ER stress, such as abnormal Ca2+ homeostasis in the ER and perturbed ER-Golgi trafficking. Because Hevin is predominantly expressed in astrocytes, it is likely that the perturbed the UPR pathways in astrocytes could be an important mechanism underlying ASD pathogenesis. A key question for future research is the biological relevance of how the ER stress response in astrocytes contributes to neural circuits and brain homeostasis. In Caenorhabditis elegans, overexpression of Xbp1 in astrocyte-like glial cells extends the life span via changing neuropeptide secretion machinery29. This observation demonstrates that upregulation of the UPR in astrocyte-like glial cells is pivotal for regulating ER stress resistance and longevity, exhibiting a positive effect in nematodes. Interestingly, a mutation in Xbp1 is responsible for the onset of bipolar disorder30. Hence, these results raise the interesting hypothesis that dysfunctions of the Xbp1 axis could also regulate both neurodevelopmental and psychiatric disorders. In contrast, overactivation of PERK-eIF2α signaling in astrocytes causes neuronal loss and neurodegeneration in mice31. Strikingly, impaired UPR signaling markedly changes their secretory factors from astrocytes, suggesting that each UPR signaling in astrocytes regulates the surrounding cells in a non-cell-autonomous manner. Thus, it is plausible that the upregulation of ER stress induced by Hevin WR may change the global secretome, resulting in circuit dysfunction that is causal to ASD. Another possibility is that UPR signaling controls the timing of neural and glial differentiation. For instance, OASIS, a subfamily of ATF6, controls proper UPR signaling and regulates astrocyte differentiation in the embryonic stage32. Since perturbed glial differentiation brings about developmental disorders, it is plausible that Hevin mutant-induced ER stress during the embryonic period influences glial differentiation, leading to the development of ASD. Previously, various maternal immune activations (MIAs), such as exposures to environmental irritants and infections with pathogens, have been reported to cause ASD. Intriguingly, MIA activates the PERKs-eIF2α pathway and inhibits new protein synthesis. Either genetic or pharmacological inhibition of this pathway improves MIA-induced ASD-like behavior33, supporting the idea that ER stress is related to ASD.

Hevin promotes synaptogenesis through stabilizing the interaction between NRX1α and NL1B20. The structure of Hevin is divided into three main parts, the acidic domain, FS domain, and EC domain22. The FS domain binds to NRX1α and NL1B concurrently, and the EC domain, of Hevin, including the EF-hand motif, is predicted to be essential for interacting with the collagen in a Ca2+-dependent manner22,23. We found that only a small amount of the Hevin mW633R mutant can be secreted to the extracellular space even in a mutation of the EF-hand. These data imply that exposure to the hydrophobic region of the C-terminus may result in the acquiring of novel functions that disturb innate functions in the extracellular space even in the presence of Ca2+. One hypothesis is that the binding affinity between Hevin and collagen proteins is changed and affects the functions of synaptogenesis. Ehlers-Danlos syndromes, which exhibit autism-like phenotypes, arise from mutation in Col1A and Col5A genes34. Therefore, the Hevin WR mutant could also impact the conditions of the extracellular matrix and brain microenvironment. Previous studies have reported that Sparc, a homolog of Hevin, suppresses synaptogenesis. Intriguingly, the deletion mutant lacking an acidic domain (SLF) exhibits a similar phenotype as Sparc in which SLF suppresses synaptogenesis35. Hevin is also cleaved at the center region (approximately 350 amino acids) by a metalloprotease, ADAMTS4, and produces the fragment containing both FS and EC domains36. Therefore, it is likely that Hevin hW647R mutant acquires a distinct function, like Sparc and SLF. However, further studies will be needed to clarify this possibility in the future.

Previous studies have reported that other mutations in the SPARCL1 gene associated with ASD have been revealed, and most mutations reside in the FS domain and EC domain5 (see Fig. 3A). Mutations in which Thr516 and Met587 are replaced with Met and Ile, respectively, are next to the residue (Asp517 and Tyr588) with large RDF differences. Thus, it is likely that the native hydrophobic core is unfolded when these amino acids are substituted. ASD-associated amino acid substitutions around the EF-hand motif may change the native hydrophobic core, resulting in a fluctuated structure of Hevin. Notably, Ca2+ deprivation has less effect on the binding between BIP and the Hevin WR mutant. Thus, it is plausible that exposure of the hydrophobic region covered not only by Trp647 but also by Thr516 and Met587 impairs Hevin secretion and synaptogenesis, regardless of Ca2+.

In summary, here we found that the EF-hand motif in Hevin plays an important role for proper trafficking to the extracellular spaces. A loss of this domain causes protein accumulation in the ER and activates the UPR pathway. Furthermore, we found that ASD-associated mutation in which Trp647 is replaced with Arg exhibits a similar phenotype in cells. Importantly, in the hW647R mutant Hevin the hydrophobic core of the EF hand is exposed to the surface likely causing structural instability. Taken together, our findings provide multiple molecular mechanisms linking an ASD point mutation in the SPARCL1 gene with cellular phenotypes underlying the onset of ASD.

Materials and methods

Plasmids

Mouse Hevin cDNA was amplified from pMD-mSPARCL1 (Sino Biological Inc. MG50544-M) and cloned into pCRblunt. The deletion mutant of EF-hand (Hevin ΔEF) was constructed using the inverse plymerase chain reaction (PCR) method. Briefly, Hevin ΔEF cDNA was amplified from pCRblunt-Hevin, followed by conducting a T4 polynucleotide kinase reaction (TAKARA). After phosphorylation, this fragment was self-ligated and constructed a circular plasmid as a pCRb-Hevin ΔEF. The Hevin-Ndufa11 (Hevin-N11) fragment was amplified from Hevin cDNA and Ndufa11 cDNA was obtained from the 3'-RACE method; next, these fragments were ligated using an In-Fusion system (TAKARA). The Hevin-N11 fragment was inserted into the pCR-blunt using the In-Fusion system. The gene encoding Hevin mW633R was generated by a quick-change method using KOD polymerase. The GST-Hevin proteins were generated by the SLiCE method37. Briefly, the full-length Hevin cDNA was amplified using KOD plus DNA polymerase (TOYOBO) and inserted into the BglII sites of pCS4 vector. The GST-tagged Hevin and Hevin mW633R mutant were generated using the SLiCE reaction. The GST fragment was subcloned from pGEX-6P-1 plasmid into the site between the Hevin signal-peptide and another part using the SLiCE reaction. The primers used were as follows:

  • Full-length Hevin forward, 5′-aaagatctgccaccatgaaggctgtgcttctcc-3′;

  • Full-length Hevin reverse, 5′-ggagatcttcaaaagaggaggttttcatctatatcctcctc-3′;

  • Hevin ΔEF inverse sense, 5′-tgctttggaattaaagaggaggatatagatg -3′;

  • Hevin ΔEF inverse antisense, 5′-gtgttccatgggcaccaggg-3′;

  • N-terminal Hevin forward, 5′-gctggaattcaggagtgaaggctgtgcttc-3′;

  • N-terminal Hevin reverse, 5′-gatggtctccagccaagagtctcttctcatcc-3′;

  • C-terminal Hevin-Ndufa11 mutants forward, 5′-gagactcttggctggagaccatcccattg-3′;

  • C-terminal Hevin-Ndufa11 mutants reverse, 5′-tctgcagaattcaggacaccttgggggtag-3′;

  • Hevin W633R mutant forward, 5′-gaaggaacggggccactgc-3′;

  • Hevin W633R mutant reverse, 5′-gcagtggccccgttccttc-3′;

  • Hevin (pCS4) for SLiCE forward, 5′-tcgtgacatcccgacaagtacaaggtttctctc-3′;

  • Hevin (pCS4) for SLiCE reverse, 5′-tataggggatgccacagcggttcccaag-3′;

  • GST for SLiCE forward, 5′-ctgtggcatcccctatactaggttattgg-3′;

  • GST for SLiCE reverse, 5′-ttgtcgggatgtcacgatgcgg-3′;

3′-rapid amplification of cDNA ends (3′-RACE)

Total RNAs from WT and USP15-deficient brains were isolated by ISOGEN II (NIPPON GENE) according to the manufacturer’s instructions. The cDNAs were synthesized by reverse transcriptase and 100 units of ReverTra Ace (TOYOBO) together with 25 pmol random hexamer primer (TOYOBO), 20 nmol dNTPs, and total RNAs. The 3′-region of the hevin transcript was determined using the 3′-RACE method. First-strand synthesis was conducted using the Hevin outside primer and Hevin adaptor primer. Then, the second round of PCR was conducted using the Hevin inside primer and Hevin amplification primer. The 3′-fragment amplified by 3′-RACE was inserted into the pCR-blunt and the DNA sequences were analyzed. The primers used were as follows:

  • Hevin outside primer, 5′-aaatgctgaaccttcagatgagggc-3′;

  • Hevin adaptor primer, 5′-ggccacgcgtcgactagtacttttttttttttttttt-3′;

  • Hevin inside primer, 5′-agagactcttggctggagaccatc-3′;

  • Hevin amplification primer, 5′-ggccacgcgtcgactagtac-3′;

Antibodies

For immunoblotting goat anti-Hevin (1:1000, R&D Systems, Cat# AF2836), mouse anti-Tubulin (1:1000, Sigma, DM1A), rabbit anti-BIP (1:1000, Cell Signaling, CB0B12), and mouse anti-GST (1:500, Santa Cruz, B14) antibodies were used as primary antibodies. Horseradish peroxidase-conjugated anti-mouse IgG, anti-rabbit IgG, and anti-goat IgG (1:20,000, respectively, SeraCare) antibodies were used as secondary antibodies. For immunofluorescence, rabbit anti-RFP (1:500, Rockland, Cat# 600-401-379), goat anti-Hevin (1:500, R&D Systems, Cat# AF2836), and mouse anti-GM130 (1:500, MBL, Cat# M179-3) were used as primary antibodies. Alexa Fluor 488 donkey anti-goat IgG H&L (1:500, Thermo Fisher Scientific), DyLight 594 donkey anti-rabbit IgG H&L (1:500, abcam), and DyLight 594 donkey anti-mouse IgG H&L (1:500, abcam) antibodies were used as secondary antibodies.

Cell culture and transfection

HEK293T38,39 cells and HeLa cells38,39 were cultured in Dulbecco's modified Eagle's medium (high glucose) (Wako) containing 5% fetal bovine serum (FBS), 100 units penicillin, and 100 mg streptomycin (P/S Thermo Fisher Scientific). Cells were transfected using polyethyleneimine MAX (PEI Max) (Polyscience). The amounts of the plasmids and PEI Max were optimized in proportion to the relative surface area and number of cells. Cells were plated at 1.5 to 5.0 × 105 cells in 4 ml per 60 mm dish and incubated at 5% CO2 and 37 °C for 1 day. The plasmids (8.0—16.0 μg) were mixed with 500 μl of Opti-MEM (Thermo Fisher); 1.0 μg/μl of PEI Max (32.0—64.0 μl) was mixed with 500 μl of Opti-MEM in another tube. Both solutions were combined and incubated for 20 min at room temperature, followed by adding these mixtures to cells. After incubation for 1 h, the media were changed to fresh culture medium. Neuro-2a cells were cultured in Eagle's minimum essential medium (Wako) containing 10% FBS and P/S. Cells were plated at 1.5 × 105 cells on a 6-well plate and incubated at 5% CO2 and 37 °C for 1 day. The plasmids (4.0 μg) were mixed with 250 μl of Opti-MEM (Thermo Fisher); and 1.0 μg/μl of PEI Max (8.0 μl) was mixed with 250 μl of Opti-MEM in another tube. Both solutions were combined and incubated for 20 min at room temperature, followed by adding these mixtures to cells. After incubation for 4 h, the media were changed to fresh culture medium. HeLa cells and 293 T cells were obtained from American Type Culture Collection (ATCC, US). Neuro-2A cells were obtained from JCRB Cell Bank (NIBIOHN, Japan).

Immunocytochemistry

HeLa cells were plated at 5.0 × 104 cells and incubated on 15 mm coverslips on a 12-well plate for 1 day and then transfected with indicated plasmids. After the incubation for 24 h, the cells were washed in PBS and fixed with 4% paraformaldehyde (Merck KGaA) in PBS for 10 min at 4 °C. The coverslips were washed in PBS and blocked with 5% bovine serum albumin (BSA; Wako) in PBS with 0.4% Triton X-100 (MP Biomedicals), then incubated with primary antibodies diluted in blocking solution overnight at 4 °C. After washing with PBS, the cells were incubated with secondary antibodies diluted in blocking solution for 30 min at room temperature. Nuclei were stained with 10 μg/ml Hoechst 33,342 (Life Technologies). The coverslips were then mounted onto slides using FLUOROSHIELD Mounting Medium (ImmunoBioScience). Fluorescent images were obtained using confocal laser scanning fluorescence microscopy (ZEISS, LSM700) with a 63x (Plan-Apochromat 63x/1.4 Oil DIC) or 100x (αPlan-Apochromat 100x/1.46 Oil DIC) objective. The diode excitation lasers (Diode 405, Diode 488, and Diode 555) were operated and directed to a photomultiplier tube (LSM T-PMT, Carl Zeiss) through a series of band pass filters (Ch1:BP420-475 + BP500-610, Ch2:BP490-635, and Ch3:BP585-1000).

Colocalization analysis

Colocalization analysis of immunofluorescence signals was performed according to the previous study40. The colocalization values (Pearson’s co-efficients or Manders’ co-efficients) were calculated using the immunofluorescence signals of Hevin and either mCherry-ER or GM130 from representative images by Fiji plugin Coloc-2. All data were reproduced in at least two independent experiments.

GST pull-down assay

HEK293T cells were plated at 5.0 × 105 cells and incubated for 1 day and then transfected with indicated plasmids. After the incubation for 48 h, cells were washed with PBS and collected with lysis buffer [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 1 mM DTT, 1 mM PMSF, 3 µg/ml leupeptin, 3 µg/ml Pepstatin A, and 5 µg/ml aprotinin] in the presence or absence of 1 mM EDTA. Cell lysates were centrifuged at 14,000 rpm for 5 min. The supernatants were mixed with Glutathione Sepharose beads (GE Healthcare) and then incubated for 2 h at 4 °C with rotation. The precipitants were washed 3 times with lysis buffer and subjected to immunoblot analysis. All data were reproduced in at least two independent experiments.

Immunoblot analysis

HEK293T cells or Neuro-2a cells were plated at 0.8 or 1.5 × 105 cells on a 6-well plate and incubated at 37 °C with 5% CO2 for 1 d. Cells were transfected and then incubated for 3 days. The cells were collected with lysis buffer [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, and 1 mM DTT]. Cell lysates were centrifuged at 14,000 rpm for 5 min. The supernatant was run on SDS-PAGE for protein separation, followed by electrophoretic transfer to a polyvinylidene difluoride membrane (Pall). After 1 h blocking by 5% skim milk at room temperature, membranes were incubated with primary antibodies overnight at 4 °C. The proteins on the membranes were then detected with HRP-conjugated secondary antibodies and chemiluminescence reagents (Chemi-Lumi One Super, Nacalai Tesque). All data were reproduced in at least two independent experiments.

Quantitative real-time PCR (qPCR)

Neuro-2a cells (1.5 × 105 cells/ 6-well plate) were cultured for 24 h and then transfected with indicated plasmids. After the incubation for 48 h, the cells were washed in PBS and collected with ISOGEN II (NIPPON GENE). Total RNAs from cells were isolated according to the ISOGEN II manufacturer’s instructions. The cDNAs were synthesized by reverse transcriptase, 100 units ReverTra Ace (TOYOBO) together with 25 pmol Random Primer (nonamer; TOYOBO), 20 nmol dNTPs and 500 ng total RNAs. Quantitative (q)PCR was performed on a 96-well plate (Scientific Specialties) using THUNDERBIRD SYBR qPCR Mix (TOYOBO) in an Applied Biosystems 7900HT Fast Real Time PCR System (Applied Biosystems). The relative quantity of the target expression was calculated by 2−ΔΔCt methods using SDS Software 2.4.2 (Applied Biosystems) with the following calculation. The relative quantity = 2-ΔΔCt, ΔΔCt = (Cttarget − Ct5S)sample − (Cttarget − Ct5S)reference; Ct, threshold cycle. All data were reproduced in at least two independent experiments. The primers used were as follows:

  • 5S rRNA forward, 5′-cggccataccaccctgaac-3′;

  • 5S rRNA reverse, 5′-gcggtctcccatccaagtac-3′;

  • BiP forward, 5′-gagactgctgaggcgtattt-3′;

  • BiP reverse, 5′-cctcatgacattcagtccag-3′;

  • Chop forward, 5′-ctggaagcctggtatgaggat-3′;

  • Chop reverse, 5′-cagggtcaagagtagtgaaggt-3′;

Molecular dynamics (MD) simulation

The WT and W647R systems of Hevin were prepared to conduct an MD simulation. In each system, only the EC domain (511–663) of Hevin was considered. The WT and mutant (W647R) systems were modeled using a crystal structure (PDB code: 7KBU)22. To model the W647R system, this amino acid substitution was induced using the Pymol program (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.). In each system, the missing amino acid residues were added using the SWISS-MODEL program41. The modeled structures were solvated with the TIP3P water model42 in an orthorhombic box. The LINCS43 and SETTLE44 algorithms were adopted to extend the MD time step to 2 fs. The modified Berendsen thermostat45 and Parrinello–Rahman method46,47 controlled the temperatures and pressures of all systems, respectively. The particle mesh Ewald method48 evaluated electrostatic interactions using a real-space cutoff value of 10 Å. The cutoff value for the van der Waals interactions was set to 10 Å. All of the MD simulations were performed with the GPU version of the GROMACS 2020 package49 using the Amber 14SBonlysc force field50. Before conformational sampling, energy minimizations on the initially modeled systems removed the steric crashes of atoms. Subsequently, 100-ps NVT (T = 300 K) and 100-ps NPT (T = 300 K and P = 1 bar) MD simulations relaxed each system. Lastly, the final snapshots of the NPT simulations on each system were specified as the starting configurations of their production runs. To generate statistically reliable MD trajectories, multiple 1-μs MD simulations (1-μs × 3 runs for each system) were independently started from each relaxed configuration by regenerating their initial velocities. Finally, the first 0.4-μs trajectories were considered as the equilibration phase, and the remaining 0.6-μs trajectories were used for analyses in each system.

Statistical analysis

Prism ver.8.4.3 software (GraphPad Software, Inc.) was used for all statistical analyses. Statistical significance was analyzed using Student's t-test and analysis of variance (ANOVA) followed by Dunnett’s test.