Dear Editor,
Recombinant protein therapeutics and gene therapies can help rescue monogenic disease phenotypes (
Ebrahimi and Samanta, 2023). Sufficient/high expression of a therapeutic product is an important consideration in this regard, which is usually achieved by gene redesign, focused on altering synonymous codon usage/codon context, aimed at enhancing translational rates (
Alexaki et al., 2019). Different approaches exist to adjust synonymous codon usage and boost protein expression levels (
Katneni et al., 2022). As these strategies do not alter the primary sequence of a protein (
Liu et al., 2021) they are not expected to alter protein structure and function. However, many recent studies have demonstrated that codon optimization can influence mRNA and protein biogenesis (
Hunt and Kimchi-Sarfaty, 2022).
Studies in the field additionally revealed that orientation-specific codon-pairs can alter the translational elongation rate, separately from the effects of modifying individual codon usage biases (
Gamble et al., 2016) and optimizing genetic sequences through “codon-pair biases” (codon-pair optimization) became a novel strategy to boost protein expression (
Huang et al., 2021).
In this study, we examined the effects of gene recoding through codon (CO) and codon-pair (CPO) optimization strategies on human ADAMTS13 (a disintegrin-like and metalloproteinase with a thrombospondin type 1 motif, member 13) properties and evaluated the cellular mechanisms underlying biogenesis of CO and CPO ADAMTS13 variants in Flp-In single-copy targeted integration system. We found that synonymous gene recoding alters numerous protein and cellular attributes, which were not uncovered for this protein earlier, including protein structure, function, immunogenicity, and cellular bioenergetics.
To evaluate the effects of gene recoding, we compared wild-type (WT), the previously described single synonymous (P118P) variant (
Hunt et al., 2019) used in some experiments and CO and CPO variants of
ADAMTS13 designed using different algorithms (detailed in Supplementary Materials; sequences in Supplementary Data File 1) that overall introduced more frequent and faster-translating codons throughout the ADAMTS13 sequence, predicted to enhance protein expression (Figs. 1A,1B, and S1; Table S1). The enrichment of common codons throughout the optimized sequences was reflected in the increase of codon adaptation index (CAI) %MinMax, relative synonymous codon usage, and relative synonymous codon-pair usage values (Figs. 1A, 1B, S1A and S1B). However, while overall patterns in expected translational rates based on %MinMax remained similar between CO and CPO, CPO uniquely retained many trough-like patterns (present in WT), suggesting that CPO may maintain the co-translational folding pathway similar to WT (Table S1). Regions that involve post-translational modifications (PTMs) exhibited more negative %MinMax values for WT than for optimized variants (Fig. 1B). We also found that CO variant has slightly higher mRNA minimum free folding energy levels (−1,903.70 kcal/mol) (being thus less stable) compared to CPO (−2,140.10 kcal/mol) and WT (−2,051.90 kcal/mol) variants (Figs. S1C–E).
Cell-free
in vitro translation experiments (Figs. 1C, 1D, S2A and S2B) revealed that synonymous mutations had a significant impact on ADAMTS13 translation kinetics: translation rate constants of CPO and P118P (shown previously to have elevated expression levels compared to WT) (
Hunt et al., 2019) were twice that of CO (Fig. 1C). Consequently, translation yields were significantly lower in CO and higher in CPO (Fig. 1D). In addition, analysis of ADAMTS13 solubility by ultracentrifugation showed that all variants had relatively high solubility (Fig. S2B), and minimal aggregation-prone misfolding.
We then performed cellular expression experiments using Flp-In HEK293 cell lines. We determined that intracellular mRNA levels were not significantly different between WT, CO, and CPO (Fig. 1E), however, extracellular expression of CO was significantly higher and CPO significantly lower than WT (Fig. 1F), suggesting that intracellular environment affects protein expression and biogenesis in a different way compared to the in vitro system. Apparent specific activity measured, using the fluorogenic fluorescence resonance energy transfer-von Willebrand factor (FRETS-VWF73) assay, appeared to be similar for CPO and WT variants (Figs. 1G and S2C). To analyze ADAMTS13 activity in more detail we performed subsequent substrate binding kinetic studies using biolayer interferometry (BLI) and additional enzymatic assays. We further found that WT and CPO ADAMTS13 had similar Vmax and Km rates, while CO ADAMTS13 had showed significantly higher Vmax and Km values (Figs. 1H, 1I, and S2D) indicating a lower affinity to VWF. We further determined that CO had a smaller dissociation constant (Kd), compared to WT (Figs. 1J, 1K, and S2E), and a significantly higher association rate, kon and non-significantly lower dissociation rate, koff compared to WT, while CPO had a similar binding affinity compared to WT (Fig. 1L and 1M).
As altered enzymatic kinetics and activities have been reported to be associated with altered co-translational folding (
Jiang et al., 2022), we thus further used circular dichroism (CD) to evaluate the ADAMTS13 structure. While CD (Figs. 2A, 2B, and S3) followed by structure determination using BestSel software predicted similar secondary structure composition of variants, denaturation and refolding experiments (
Greenfield, 2006) nevertheless revealed some differences in folding dynamics (Figs. 2B and S3), suggesting also potential differences in protein stabilities.
We further performed cycloheximide-chase (CHX) assays and tracked ADAMTS13 intracellular stability over a 6 h period. We observed that the CO variant was significantly more stable compared to WT and CPO, which both were barely detectable after 6 h (Fig. 2C and 2D). Conformational differences in recoded ADAMTS13 were also probed through recombinant VWF (rVWF) digestion assays (Fig. S4) and an inhibitory antibody assay (Fig. S5), but both assays demonstrated no statistically significant differences.
The observed differences of recoded ADAMTS13 variant features may result from altered protein folding and secretion, thus altering cellular fitness. We therefore evaluated whether cellular phenotypes were altered depending on the expressed variant. To evaluate bioenergetics, we performed Seahorse respiration assays. We observed that cells expressing the WT variant had similar respiration rates and ATP production as control HEK293 cell lines, while CO and CPO-expressing cells had higher ATP production and maximal oxygen consumption rate compared to WT (Fig. 2E and 2F).
We hypothesized that higher ADAMTS13 variant expression levels could incur greater stress on the secretory system. We note that Flp-In cells express higher levels of CO ADAMTS13, while CPO levels are not significantly different, compared to WT (Fig. 2G and 2H). Consequently, we further found that the intracellular levels of endoplasmic reticulum (ER) stress markers, BiP, and phosphorylated-eIF2α are higher in cells expressing the CO variant (Fig. 2G, 2H, and S6). BiP is an ATP-dependent (ER-resident) chaperone, and thus increased chaperone surveillance may be required to support protein folding challenges associated with recoded proteins. CHX administration, which reduces protein translation stress, resulted in greater secretion of CO ADAMTS13 (Figs. 2G, 2H, and S6). In addition, greater ADAMTS13-BiP interaction with CO ADAMTS13 was found via immunoprecipitation assays (Fig. 2I). The ratio of BiP:ADAMTS13 binding was ~3–5-fold greater with CO ADAMTS13 than WT (Fig. 2J). These experiments clearly suggest altered cellular fitness during the expression of recoded proteins.
As recoded ADAMTS13 variants revealed differences in protein conformation, we hypothesized that the proteolytic processing of the variants within antigen-presenting cells could also differ. Major histocompatibility complex (MHC)-associated peptide proteomics (MAPPs) assay, using monocyte-derived dendritic cells (MoDCs) from 12 donors revealed distinct sets of MHC-II peptides presented for all three ADAMTS13 variants (Fig. S8A). Although most donors showed similar peptide presentations, subtle differences were observed in certain peptides (Fig. 2K). For example, peptides 4 and 9 were presented by MoDCs in response to CO, but not to WT. However, small total numbers of peptides precluded any exhaustive statistical analyses. As a result, to further explore immunogenicity differences, we investigated whether recoded ADAMTS13 could elicit CD4+ T-cell proliferation, and statistically significant differences in T-cell stimulation were found between WT and CO and CPO proteins (Fig. S8B and S8C; Table S2).
We also evaluated glycosylation profiles of the ADAMTS13 variants (Figs. 2L–N and S9; Tables S3 and S4) since glycosylation is known to alter secretory protein expression levels (
Ricketts et al., 2007;
Sorvillo et al., 2014). Out of 10 reported N-linked glycopeptides (
Verbij et al., 2016), we identified seven. The three missing N-linked glycopeptides (N142, N146, and N828) were outside the detection limits by mass spectrometry. As shown in Fig. 2L, all ADAMTS13 proteins were glycosylated at similar levels. However, a slight increase in glycosylation abundance were observed in CO and CPO at the N614 site, while a decrease in glycosylation abundance were seen at N1235 for CO and at N1354 for CO and CPO. Further quantitative determination of site-specific N-glycopeptides revealed distinct glycosylation profiles for each ADAMTS13 variant. Comparing all glycans identified across all N-linked sites, we found that WT was less glycosylated than CO and CPO (Fig. 2M and 2N).
Changes to PTM may be caused by protein structural differences, which alter the degree of accessibility of potential PTM sites (
Esmail and Manolson, 2021). Interestingly, we found (Figs. 2N and S9A; Table S5) that CO and CPO variants had more complex glycans, from which the most abundant ones were the fucosylated species. The N614 position was the most highly glycosylated site across all variants (Fig. 2L; Table S3). Glycan structures and abundance levels were clearly distinct (Figs. 2N and S9A; Tables S3 and S5) and O-fucosylation differences were also observed between the CO and CPO variants, having different modifications at the S863 and S1027 sites (Fig. S9B; Table S4). Moreover, fucosylation of additional O-sites (S886, T889, and S1288) were uniquely detected in CO and CPO. They were shown to be required for ADAMTS13 secretion (
Ricketts et al., 2007). ADAMTS13 proteins were also modified by C-mannosylation. (Fig. S9B; Table S4), representing impactful determinants of protein folding. Changes to mannosylation sites, caused e.g., by W387A mutation were shown to affect ADAMTS13 secretion and activity (
Ling et al., 2013). The observed differences in PTMs could thus explain differences in expression/secretion (Fig. S10), and ADAMTS13 properties.
In this study, we carried out a systematic evaluation of the biochemical and biophysical consequences of gene recoding using WT, CO, and CPO ADAMTS13 variants. We revealed multiple changes in protein expression, stability, conformation, PTMs, and immunogenicity of recoded variants.
Our studies indicate that careful evaluation of the recoded transgene within the context of the host cell characteristics is required to inform the optimal design of protein therapeutics. We demonstrate that recoding can result in undesirable protein properties that could affect drug safety. Understanding the molecular and cellular mechanisms that underlie changes in the biophysical and biochemical properties of proteins following recoding is important to circumvent the limitations of gene recoding.
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