Revised platform for functional characterization of IDUA variants

In an effort to streamline functional evaluation of IDUA variants identified through NBS, we generated an IDUA-null HEK293 cell line. IDUA-null cells were transfected with either wild-type or variant-expressing IDUA DNA vectors and IDUA-expressing cell populations enriched using G418 selection. In cases where enzyme activity or abundance was too low to reliably measure in the selected population, we isolated single cell clones with higher enzyme expression. Our prior study showed single clones yield more consistent protein abundance and activity measurements than enriched populations7. Despite this, many of the variant α-iduronidase enzymes studied here were reliably characterized from pools of G418-selected cell populations.

Enzyme activity was measured in the lysates of selected cell populations or single cell clones using the fluorescent simple sugar substrate, 4-methylumbelliferyl-α-iduronide (4MUI). The activity values were compared to those obtained from an equivalent amount of total protein present in control lysates generated from cells expressing WT-IDUA. The same lysates were analyzed by western blot with an anti-human α-iduronidase antibody. Relative amount of the WT or variant α-iduronidase protein was assigned using densitometry software. The relative specific activity (RSA) of each α-iduronidase variant was calculated by dividing the relative activity of lysate by the relative amount of enzyme in the same lysate. An overview of the workflow is shown in Fig. 1.

Fig. 1: Overview of the IDUA platform and experimental workflow.

HEK293 IDUA KO cells are transfected with WT or variant-bearing IDUA expression vectors followed by antibiotic selection and, in some cases, single-cell cloning. Activity measurements using a fluorescent substrate for α-iduronidase are performed in the resulting cells along with quantitative western blot analysis to determine enzyme amount. These values are determined relative to the WT enzyme and combined to provide the relative specific activity (RSA) for each variant enzyme. This value represents the primary readout for each variant enzyme studied, but other secondary analyses can be performed to probe aggregation potential and ability to clear GAG accumulation.

Relative specific activity of variant α-iduronidases expressed in HEK293 cells can be used to estimate the pathogenicity of IDUA variants

A variety of IDUA variants were evaluated using this platform. For the purpose of validation, we included 8 variants associated with either severe or attenuated MPS I, and 4 known benign variants. We further analyzed variants classified as pseudodeficiency alleles (reduced enzyme activity but clinically benign), and multiple variants of uncertain clinical significance (VUS) identified in infants with an abnormal newborn screen for MPS I. The RSA value of each variant α-iduronidase plotted on the graph in Fig. 2 represents the average value from a minimum of three independent biological replicates. The amino acid position within the α-iduronidase protein structure is shown for each of the variants studied in Fig. 3, and the amino acids comprising the active site are shown in Supplementary Fig. 114. All four benign variants yielded RSA values that were indistinguishable from the wild-type enzyme. Nearly all of the variants associated with attenuated MPS I exhibited RSA values below 1%, while the variants associated with severe MPS I exhibited values up to 2 orders of magnitude lower than 1%. Of note, the RSA values of the three known pseudodeficiency variants tested (p.Gly409Arg, p.His82Gln, and p.Ala79Thr) were highly variable. The p.Ala79Thr variant exhibited an RSA of 5.4%, which is similar to the activity level detected for multiple variants present in patients with attenuated MPS I. The RSA values observed for p.Gly409Arg and p.His82Gln (70% and 24%, respectively) are in very close agreement with previously published results15,16. Although the values fall within an activity range not thought to cause MPS disease, these data demonstrate that the pseudodeficiency variants do alter enzyme function.

Fig. 2: Summary of relative specific activity values for IDUA variants tested in this study.

Graph showing the relative specific activity (% RSA) values (plotted on a log 10 scale) for every variant characterized in this study. Replicate analysis (n = 3–6) was performed on either cell populations after initial antibiotic selection, or 2-3 different single cell clones. Average RSA values are shown within the bars and standard error of the mean is depicted with the error bars. Bar colors denote the following classifications: dark gray – WT; light gray – VUS; blue – benign; green – known pseudodeficiency; yellow – associated with attenuated MPS I; orange – associated with attenuated or severe MPS I; red – associated with severe MPS I.

Fig. 3: Position of amino acids within the α-iduronidase protein structure for which variants were characterized.

The location of specific amino acid residues corresponding to the variants we studied in this paper is shown. Domains within the crystal structure (PDB: 3W82; RSCB PDB)14,26 of the enzyme are also labeled.

Two other missense variants (p.Arg100Gly and p.Arg100Lys) exhibited RSA values indistinguishable from the WT enzyme, however, both of the corresponding nucleotide changes (c.298A>G, c.299G>A, occurring at the end of exon 2) have high SpliceAI scores, indicating that these variants may affect splicing rather than having a missense associated effect on enzyme activity. In light of the fact that the platform utilizes only the coding sequence, additional studies will be needed to fully resolve the pathogenicity of these variants. The SpliceAI scores for all variants investigated in this study are shown in Supplementary Table 1, along with predictions on the resulting impact of the splice effects. Only the nucleotide changes corresponding to the missense variants at amino acid position 100 (p.Arg100Gly and p.Arg100Lys) predicted a significant effect on splicing. We propose that all IDUA nucleotide variants selected for functional studies going forward be initially screened for possible effects on splicing as the current design of the platform can only gauge the effects of different amino acid substitutions on enzyme function.

Altered expression/stability and processing were observed for many variant α-iduronidases

In addition to their utility for gauging enzyme amount, western blot analysis also highlighted multiple differences in the proteolytic processing of the variant α-iduronidase enzymes. The wild-type enzyme undergoes a series of proteolytic processing steps within the secretory pathway that include removal of the signal peptide and digestion of peptide fragments from both the N- and C-terminus17. Much of this processing occurs within the lysosomal compartment. Overexpression of WT-IDUA enzyme primarily yielded three different protein species (~75 kDa, ~70 kDa, ~62 kDa). In cells treated with bafilomycin A, an inhibitor of lysosomal proton transport that neutralizes the pH in this organelle, the highest molecular weight species (75 kDa) accumulated, reducing the abundance of the two lower molecular weight species (Fig. 4A). These data support the likelihood that each of the protein species detected on western blot result from processing events in the lysosome. As expected, each of the benign α-iduronidase variants analyzed exhibited normal patterns of protein processing (Fig. 4B). This was more variable in the other forms of IDUA analyzed, with many variants showing limited enzymatic processing (Fig. 4C-F). The extent of processing, along with the RSA values and allele frequencies of each variant enzyme is summarized in Table 1. Our data suggest that increased levels of enzyme processing generally correlate with higher RSA values. One explanation for poor enzymatic processing may be inefficient lysosomal targeting. Alternatively, it is possible that lysosomal targeting is normal but certain variant forms of α-iduronidase are unstable within the low pH environment of the lysosome. This latter hypothesis is difficult to interrogate, as modulating lysosomal pH would itself prevent enzyme processing.

Fig. 4: Impaired lysosomal processing is observed in certain variant α-iduronidase enzymes.

Representative western blots for variant α-iduronidases in this study. A WT IDUA-expressing cells were treated with bafilomycin A (BafA), or the protease inhibitors, E64d or pepstatin A (PepA) prior to analysis by western blot. Note that only bafilomycin A blocked proteolytic processing of the enzyme. B Comparison of WT to the four known benign variants in this study. Note all four variant α-iduronidases are processed similarly to the WT enzyme. C Western blot analysis of various single-cell clones expressing the variant enzymes. Despite differences in enzyme amount within the different clones, the extent of proteolytic processing is comparable. D, E, F Representative blots for different variant-expressing selected cell populations showing variable impact on the proteolytic processing of the enzymes.

Table 1 IDUA variants analyzed in this studyp.Leu526Pro α-iduronidase is prone to aggregation

The RSA values for several variants known to be associated with attenuated MPS I indicate that most values lie below the 1–3% range. However, several of the variants that have been identified in attenuated MPS I patients exhibited RSA values that were 10–30% of WT α-iduronidase18. This indicates that the RSA value may not alone be sufficient to assign pathogenicity to certain variants. To address whether other mechanisms contribute to altered α-iduronidase function, we utilized previously isolated single-cell clones to further investigate the p.Leu526Pro VUS, as a model for variants that seem to contradict the ability of the RSA value to predict pathogenicity. This variant has an allele frequency of 3.91 × 10−4 (gnomAD v.4; total allele count of 595) in the general population and an RSA of 15%. This is three times higher than that of the pseudodeficiency variant, p.Ala79Thr, which is clinically benign.

Leu526 is located within a ß-sandwich structure, where it contributes to a series of hydrophobic interactions between two ß-strands near the surface of the enzyme (Fig. 5A). The p.Leu526Pro mutation likely disrupts the beta-sheet structure, and the corresponding disruption exposes hydrophobic residues. We hypothesized this may cause the enzyme to aggregate, impairing its trafficking in vivo. Neither effect would be evident by measuring specific activity of variant enzymes in vitro. Using native gel electrophoresis, we showed the WT and p.Ala79Thr enzyme migrate exclusively as lower MW bands (60–75 kDa), similar to previously shown under denaturing conditions (Fig. 5B). Consistent with aggregated forms of the enzyme the p.Leu526Pro enzyme migrated in two broad heterogenous bands at higher MWs (~125–300 kDa). This aggregation was much weaker for the p.Alal79Thr compared to p.Leu526Pro. To ask if the appearance of high MW bands or aggregates is exacerbated by heat, lysates were incubated at 40 °C for 15 min. As shown in Fig. 5C, with the exception of the WT enzyme, all variant forms of the enzymes tested exhibited increased aggregation when heated. This supports the conclusion that missense variants structurally compromise the enzyme making it prone to aggregation. This aggregation or structural alteration may cause the enzyme to be inefficiently trafficked to the lysosome. Since these variant-containing enzymes retain activity towards the 4-MUI sugar substrate, the aggregation potential caused by these variants is not likely associated with misfolding within the ER. This suggests the enzyme accumulates within other compartments of the secretory pathway. Attempts to localize the variant forms of α-iduronidase using confocal microscopy were not informative, as steady-state analyses of overexpressed enzyme are challenged by the wide enzyme distribution within the cell, even 12-18 hours post-transfection.

Fig. 5: p.Leu526Pro increases aggregation of the resulting variant α-iduronidase enzyme.

A Protein structure of α-iduronidase (PDB: 3W82; RSCB PDB)14,26 inset depicts the location of leucine 526. B Representative blot of WT, p.Ala79Thr and p.Leu526Pro α-iduronidase following native gel electrophoresis. C Western blot showing accumulation of high molecular weight forms of α-iduronidase with certain variants in the presence or absence of heating at 40 °C. D Western blot analysis of WT, p.Leu526Pro, and p.Leu526Met α-iduronidase following denaturing gel electrophoresis. Note that substitution of Leu526 with methionine permits proteolytic processing while proline does not. E Western blot of WT, p.Leu526Pro, and p.Leu526Met α-iduronidase on a native gel with and without prior heating at 40 °C.

To further explore the mechanism of aggregation and impaired lysosomal delivery, we engineered another variant at position 526 (p.Leu526Met) predicted to have a less severe impact on the secondary structure of the protein. The p.Leu526Met variant IDUA was introduced into the IDUA KO HEK293 cells and selected pool of cells was obtained. The relative specific activity of this variant enzyme was considerably higher (74%) than seen with the corresponding proline substitution (15%), indicating a less severe impact on enzymatic function (Fig. 5D). Comparison of the electrophoretic mobility (on native and denaturing gels) and lysosomal processing of p.Leu526Pro and p.Leu526Met enzymes demonstrated clear differences in the characteristics of the two variant proteins (Fig. 5E). Unlike p.Leu526Pro which shows little or no processing, the p.Leu526Met variant α-iduronidase was processed similar to the WT enzyme. Most noteworthy was the lack of aggregation observed with the p.Leu526Met enzyme. This supports the hypothesis that the proline substitution more substantially alters enzyme structure increasing the aggregation potential.

Despite having an RSA value that falls within the benign range, our results support the conditional pathogenicity of the p.Leu526Pro variant under conditions that promote enzyme aggregation and impaired lysosomal delivery. Any variant enzyme that fails to reach the lysosomal compartment will not be available to effectively catabolize GAGs despite having sufficient residual catalytic function to degrade substrates. To address this possibility, we performed GAG analysis on single-cell clones expressing different variant forms of α-iduronidase and assayed which of the variant enzymes prevented lysosomal GAG storage (Fig. 6). A two- to three-fold elevation in GAG abundance was observed in the HEK293 IDUA null cells compared to the parental WT HEK293 cells. The elevation in total GAG abundance was effectively eliminated in both the WT IDUA-expressing clone and p.Leu526Pro-expressing cells, indicating that despite the poor apparent lysosomal delivery and processing in the latter, there is sufficient enzyme function to degrade GAGs. We estimate that the α-iduronidase is overexpressed roughly 500 times in the single cell clones compared to the parental WT HEK293 cells so with the impaired lysosomal delivery caused by the variant, enough enzyme still reaches the lysosome. It is unclear whether this would be true under endogenous expression levels.

Fig. 6: Overexpression of WT or p.Leu526Pro α-iduronidase reduces accumulation of dermatan and heparan sulfate.

Graph depicting abundance of total GAGs (dermatan and heparan sulfate) in WT parental and IDUA-KO HEK293 cells, and IDUA-KO HEK293 cells-expressing either WT or p.Leu526Pro IDUA. Measurements were made in four independent replicates. Error bars represent the standard error of the mean. Statistical analyses were performed using a Student’s t-test; *** denotes P < 0.001, n.s. denotes not significant.

Lastly, we explored the impact of the p.Leu526Pro variant further by performing the semi-quantitative analysis of endogenous non-reducing end (NRE) GAGs, specifically UA-HNAc(1S) that is elevated in MPS I patients, in urine from patients bearing this variant. A summary of the findings is shown in Fig. 7. In all cases where p.Leu526Pro is present in trans with a pathogenic or likely pathogenic variant, urine UA-HNAc(1S), while elevated compared to normal controls (2.6–48 apparent µmol/mol creatinine versus <0.50), is much lower than levels observed in patients with a confirmed clinical diagnosis of MPS I (906–5326 apparent µmol/mol creatinine). While the urine GAG elevation is not in the range expected for patients with MPSI, there is a measurable increase that does support the accumulation of some GAG storage when this variant enzyme is expressed in trans with other loss-of-function alleles. More samples from patients with atypical attenuated phenotypes, such as the patient described by Asumda and colleagues18, which had mostly a cardiac phenotype with retinopathy, will help clarify the clinical significance of mild urine NRE elevations, both in patients with the p.Leu526Pro variant and in those with other variants of uncertain clinical significance.

Fig. 7: Endogenous, non-reducing end GAG semi-quantitative analysis by tandem mass spectrometry on urine of patients bearing the p.Leu526Pro shows very modest elevation of UA-HNAc(1S).

Graph depicted NRE levels in different individuals as a function of age. Samples from two patients carrying the p.Leu526Pro were analyzed. Levels of the MPS I NRE marker UA-HNAc(1S) are expressed in apparent μmoles/mole of creatinine. Patient 1 is 27 years and 10 months old, with a genotype of p.Trp402Ter (pathogenic)/p.Leu526Pro (VUS). This patient had 48 apparent μmoles/mole of creatinine. Patient 2 (same genotype as patient 1) is 7 month-old male with 57 apparent μmoles/mole of creatinine. Their levels of UA-HNAc(1S) early are above the normal range (≤0.5 apparent μmoles/mole of creatinine), however they are not in the affected range seen for patients with MPS I (range: 906–5326 apparent μmoles/mole of creatinine).