A synonymous variant is unmasked in thalassaemia.

The genetic underpinnings of beta-thalassaemia encompass a myriad of molecular mechanisms. The ability of synonymous mutations, an often-overlooked category of variants, to influence ß-globin expression and phenotypic disease is highlighted by this report by Gorivale et al. Commentary on: Gorivale et al. When a synonymous mutation breaks the silence in a thalassaemia patient. Br J Haematol 2024;204:677-682.

Angiotensin II-mediated hippocampal hypoperfusion and vascular dysfunction contribute to vascular cognitive impairment in aged hypertensive rats.

INTRODUCTION: Chronic hypertension increases the risk of vascular cognitive impairment (VCI) by ∼60%; however, how hypertension affects the vasculature of the hippocampus remains unclear but could contribute to VCI. METHODS: Memory, hippocampal perfusion, and hippocampal arteriole (HA) function were investigated in male Wistar rats or spontaneously hypertensive rats (SHR) in early (4 to 5 months old), mid (8 to 9 months old), or late adulthood (14 to 15 months old). SHR in late adulthood were chronically treated with captopril (angiotensin converting enzyme inhibitor) or apocynin (antioxidant) to investigate the mechanisms by which hypertension contributes to VCI. RESULTS: Impaired memory in SHR in late adulthood was associated with HA endothelial dysfunction, hyperconstriction, and ∼50% reduction in hippocampal blood flow. Captopril, but not apocynin, improved HA function, restored perfusion, and rescued memory function in aged SHR. DISCUSSION: Hippocampal vascular dysfunction contributes to hypertension-induced memory decline through angiotensin II signaling, highlighting the therapeutic potential of HAs in protecting neurocognitive health later in life. HIGHLIGHTS: Vascular dysfunction in the hippocampus contributes to vascular cognitive impairment. Memory declines with age during chronic hypertension. Angiotensin II causes endothelial dysfunction in the hippocampus in hypertension. Angiotensin II-mediated hippocampal arteriole dysfunction reduces blood flow. Vascular dysfunction in the hippocampus impairs perfusion and memory function.

Gene variants of coagulation related proteins that interact with SARS-CoV-2.

Thrombosis is a recognized complication of Coronavirus disease of 2019 (COVID-19) and is often associated with poor prognosis. There is a well-recognized link between coagulation and inflammation, however, the extent of thrombotic events associated with COVID-19 warrants further investigation. Poly(A) Binding Protein Cytoplasmic 4 (PABPC4), Serine/Cysteine Proteinase Inhibitor Clade G Member 1 (SERPING1) and Vitamin K epOxide Reductase Complex subunit 1 (VKORC1), which are all proteins linked to coagulation, have been shown to interact with SARS proteins. We computationally examined the interaction of these with SARS-CoV-2 proteins and, in the case of VKORC1, we describe its binding to ORF7a in detail. We examined the occurrence of variants of each of these proteins across populations and interrogated their potential contribution to COVID-19 severity. Potential mechanisms, by which some of these variants may contribute to disease, are proposed. Some of these variants are prevalent in minority groups that are disproportionally affected by severe COVID-19. Therefore, we are proposing that further investigation around these variants may lead to better understanding of disease pathogenesis in minority groups and more informed therapeutic approaches.

Translational and transcriptional responses in human primary hepatocytes under hypoxia.

The liver is the primary source of a large number of plasma proteins and plays a critical role in multiple biological processes. Inadequate oxygen supply characterizing various clinical settings such as liver transplantation exposes the liver to hypoxic conditions. Studies assessing hypoxia-induced global translational changes in liver are lacking. Here, we employed a recently developed ribosome-profiling technique to assess global translational responses of human primary hepatocytes exposed to acute hypoxic stress (1% O2) for the short term. In parallel, transcriptome profiling was performed to assess mRNA expression changes. We found that translational responses appeared earlier and were predominant over transcriptional responses. A significant decrease in translational efficiency of several ribosome genes indicated translational inhibition of new ribosome protein synthesis in hypoxia. Pathway enrichment analysis highlighted altered translational regulation of MAPK signaling, drug metabolism, oxidative phosphorylation, and nonalcoholic fatty liver disease pathways. Gene Ontology enrichment analysis revealed terms related to translation, metabolism, angiogenesis, apoptosis, and response to stress. Transcriptional induction of genes encoding heat shock proteins was observed within 30 min of hypoxia. Induction of genes encoding stress response mediators, metabolism regulators, and proangiogenic proteins was observed at 240 min. Despite the liver being the primary source of coagulation proteins and the implicated role of hypoxia in thrombosis, limited differences were observed in genes encoding coagulation-associated proteins. Overall, our study demonstrates the predominance of translational regulation over transcription and highlights differentially regulated pathways or biological processes in short-term hypoxic stress responses of human primary hepatocytes. NEW & NOTEWORTHY The novelty of this study lies in applying parallel ribosome- and transcriptome-profiling analyses to human primary hepatocytes in hypoxia. To our knowledge, this is the first study to assess global translational responses using ribosome profiling in hypoxic hepatocytes. Our results demonstrate the predominance of translational responses over transcriptional responses in early hepatic hypoxic stress responses. Furthermore, our study reveals multiple pathways and specific genes showing altered regulation in hypoxic hepatocytes.

A mechanistic investigation of thrombotic microangiopathy associated with IV abuse of Opana ER.

Since 2012, a number of case reports have described the occurrence of thrombotic microangiopathy (TMA) following IV abuse of extended-release oxymorphone hydrochloride (Opana ER), an oral opioid for long-term treatment of chronic pain. Here, we present unique clinical features of 3 patients and investigate IV exposure to the tablet's inert ingredients as a possible causal mechanism. Guinea pigs were used as an animal model to understand the hematopathologic and nephrotoxic potential of the inert ingredient mixture (termed here as PEO+) which primarily contains high-molecular-weight polyethylene oxide (HMW PEO). Microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury were found in a group of 3 patients following recent injection of adulterated extended-release oxymorphone tablets. Varying degrees of cardiac involvement and retinal ischemia occurred, with TMA evident on kidney biopsy. A TMA-like state also developed in guinea pigs IV administered PEO+. Acute tubular and glomerular renal injury was accompanied by nonheme iron deposition and hypoxia-inducible factor-1α upregulation in the renal cortex. Similar outcomes were observed following dosing with HMW PEO alone. IV exposure to the inert ingredients in reformulated extended-release oxymorphone can elicit TMA. Although prescription opioid abuse shows geographic variation, all physicians should be highly inquisitive of IV drug abuse when presented with cases of TMA.

A Single Synonymous Variant (c.354G>A [p.P118P]) in ADAMTS13 Confers Enhanced Specific Activity.

Synonymous variants within coding regions may influence protein expression and function. We have previously reported increased protein expression levels ex vivo (~120% in comparison to wild-type) from a synonymous polymorphism variant, c.354G>A [p.P118P], of the ADAMTS13 gene, encoding a plasma protease responsible for von Willebrand Factor (VWF) degradation. In the current study, we investigated the potential mechanism(s) behind the increased protein expression levels from this variant and its effect on ADAMTS13 physico-chemical properties. Cell-free assays showed enhanced translation of the c.354G>A variant and the analysis of codon usage characteristics suggested that introduction of the frequently used codon/codon pair(s) may have been potentially responsible for this effect. Limited proteolysis, however, showed no substantial influence of altered translation on protein conformation. Analysis of post-translational modifications also showed no notable differences but identified three previously unreported glycosylation markers. Despite these similarities, p.P118P variant unexpectedly showed higher specific activity. Structural analysis using modeled interactions indicated that subtle conformational changes arising from altered translation kinetics could affect interactions between an exosite of ADAMTS13 and VWF resulting in altered specific activity. This report highlights how a single synonymous nucleotide variation can impact cellular expression and specific activity in the absence of measurable impact on protein structure.

Depletion of haptoglobin and hemopexin promote hemoglobin-mediated lipoprotein oxidation in sickle cell disease.

Intravascular sickling and lysis of red blood cells, a hallmark feature of sickle cell disease (SCD), releases hemoglobin (Hb) into the circulation. Increased cell-free Hb has been linked to vasculopathy and in vitro lipid oxidation. Scavenger plasma proteins haptoglobin (Hp) and hemopexin (Hpx) can attenuate cell-free Hb and total plasma heme lipid-oxidative capacity but are depleted in SCD. Here, we isolated lipids from BERK-SS mice, guinea pigs (GP) infused with heme-albumin, and patients with SCD undergoing regular exchange transfusion therapy and evaluated the level of lipid oxidation. Malondialdehyde formation, an end product of lipid peroxidation, was increased in BERK-SS mice, purified lipid fractions of the heme-albumin infused GP, and patients with SCD compared with controls. In humans, the extent of lipid oxidation was associated with the absence of Hp as well as decreased Hpx in plasma samples. Postmortem pulmonary tissue obtained from patients with SCD demonstrated oxidized LDL deposition in the pulmonary artery. The relationship between no Hp and low Hpx levels with greater LDL and HDL oxidation demonstrates the loss of protection against cell-free Hb and total plasma heme-mediated lipid oxidation and tissue injury in SCD. Strategies to protect against plasma lipid oxidation by cell-free Hb and total plasma heme (e.g., therapeutic Hp and Hpx replacement) may diminish the deleterious effects of cell-free Hb and total plasma heme toward the vascular system in SCD.

Single synonymous mutation in factor IX alters protein properties and underlies haemophilia B.

BACKGROUND: Haemophilia B is caused by genetic aberrations in the F9 gene. The majority of these are non-synonymous mutations that alter the primary structure of blood coagulation factor IX (FIX). However, a synonymous mutation c.459G>A (Val107Val) was clinically reported to result in mild haemophilia B (FIX coagulant activity 15%-20% of normal). The F9 mRNA of these patients showed no skipping or retention of introns and/or change in mRNA levels, suggesting that mRNA integrity does not contribute to the origin of the disease in affected individuals. The aim of this study is to elucidate the molecular mechanisms that can explain disease manifestations in patients with this synonymous mutation. METHODS: We analyse the molecular mechanisms underlying the FIX deficiency through in silico analysis and reproducing the c.459G>A (Val107Val) mutation in stable cell lines. Conformation and non-conformation sensitive antibodies, limited trypsin digestion, activity assays for FIX, interaction with other proteins and post-translation modifications were used to evaluate the biophysical and biochemical consequences of the synonymous mutation. RESULTS: The Val107Val synonymous mutation in F9 was found to significantly diminish FIX expression. Our results suggest that this mutation slows FIX translation and affects its conformation resulting in decreased extracellular protein level. The altered conformation did not change the specific activity of the mutated protein. CONCLUSIONS: The pathogenic basis for one synonymous mutation (Val107Val) in the F9 gene associated with haemophilia B was determined. A mechanistic understanding of this synonymous variant yields potential for guiding and developing future therapeutic treatments.

Exposing synonymous mutations.

Synonymous codon changes, which do not alter protein sequence, were previously thought to have no functional consequence. Although this concept has been overturned in recent years, there is no unique mechanism by which these changes exert biological effects. A large repertoire of both experimental and bioinformatic methods has been developed to understand the effects of synonymous variants. Results from this body of work have provided global insights into how biological systems exploit the degeneracy of the genetic code to control gene expression, protein folding efficiency, and the coordinated expression of functionally related gene families. Although it is now clear that synonymous variants are important in a variety of contexts, from human disease to the safety and efficacy of therapeutic proteins, there is no clear consensus on the approaches to identify and validate these changes. Here, we review the diverse methods to understand the effects of synonymous mutations.

Chronic hypertension alters the relationship between collateral blood flow, cortical cerebral blood flow, and brain tissue oxygenation.

This study measured the relationship between pial collateral (leptomeningeal anastomoses, LMA) flow, intraparenchymal cortical cerebral blood flow (cCBF) and brain tissue oxygenation (btO2) during acute ischemic stroke to investigate how pial flow translates to downstream cCBF and btO2 and examined how this relationship is altered in hypertension. Proximal transient middle cerebral artery occlusion (tMCAO) was performed in male Wistar (n = 8/group) and Spontaneously Hypertensive Rats (SHR, n = 8/group). A combination laser Doppler-oxygen probe was placed within the expected cortical peri-infarct in addition to a surface laser doppler probe which measured LMA flow. Phenylephrine (PE) was infused 30 minutes into tMCAO to increase blood pressure (BP) by 30% for 10 minutes and assessed CBF autoregulation. During the initial 30-minute period of tMCAO, btO2 and cCBF were lower in SHR compared to Wistar rats (btO2: 11.5 ± 10.5 vs 17.5 ± 10.8 mmHg and cCBF: -29.7 ± 23.3% vs -17.8 ± 41.9%); however, LMA flow was similar between groups. The relationship between LMA flow, cCBF and btO2 were interdependent in Wistar rats. However, this relationship was disrupted in SHR rats and partially restored by induced hypertension. This study provides evidence that cCBF and btO2 were diminished during tMCAO in chronic hypertension, and that induced hypertension was beneficial regardless of hypertensive status.

The impact of collateral therapeutics on stroke hemodynamics in normotensive and hypertensive rats: a step toward translation.

Introduction: Stroke interventions that increase collateral flow have the potential to salvage penumbral tissue and increase the number of patients eligible for reperfusion therapy. We compared the efficacy of two different collateral therapeutics during transient middle cerebral artery occlusion (tMCAO) in normotensive and hypertensive rats. Methods: The change in collateral and core perfusion was measured using dual laser Doppler in response to either a pressor agent (phenylephrine, 10 mg/kg iv or vehicle) or a collateral vasodilator (TM5441, 5 mg/kg iv or vehicle) given 30 min into tMCAO in male Wistar and spontaneously hypertensive rats (SHRs). Results: Pressor therapy increased collateral flow in the Wistar rats but was ineffective in the SHRs. The increase in collateral flow in the Wistar rats was associated with impaired cerebral blood flow autoregulation (CBFAR) that was intact in the SHRs. TM5441 caused a decrease in collateral perfusion in the Wistar rats and a modest increase in the SHRs. The pressor therapy reduced early infarction in both groups but increased edema in the SHRs, whereas TM5441 did not have any beneficial effects in either group. Conclusions: Thus, the pressor therapy was superior to a collateral vasodilator in increasing collateral flow and improving outcomes in the Wistar rats, likely due to pial collaterals that were pressure passive; the lack of CBF response in the SHRs to pressor therapy was likely due to intact CBFAR that limited perfusion. While TM5441 modestly increased CBF in the SHRs but not in the Wistar rats, it did not have a beneficial effect on stroke outcomes. These results suggest that collateral therapies may need to be selected for certain comorbidities.

Cyclosporin A impairs the secretion and activity of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat).

The protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder thrombotic thrombocytopenic purpura (TTP). It has been observed that cyclosporin A (CsA) treatment, particularly in transplant patients, may sometimes be linked to the development of TTP. Until now, the reason for such a link was unclear. Here we provide evidence demonstrating that cyclophilin B (CypB) activity plays an important role in the secretion of active ADAMTS13. We found that CsA, an inhibitor of CypB, reduces the secretion of ADAMTS13 and leads to conformational changes in the protein resulting in diminished ADAMTS13 proteolytic activity. A direct, functional interaction between CypB (which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone functions) and ADAMTS13 is demonstrated using immunoprecipitation and siRNA knockdown of CypB. Finally, CypB knock-out mice were found to have reduced ADAMTS13 levels. Taken together, our findings indicate that cyclophilin-mediated activity is an important factor affecting secretion and activity of ADAMTS13. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization in the proper folding of this protein. These results altogether provide a novel mechanistic explanation for CsA-induced TTP in transplant patients.

Differential effect of gold nanoparticles on cerebrovascular function and biomechanical properties.

Human stroke serum (HSS) has been shown to impair cerebrovascular function, likely by factors released into the circulation after ischemia. 20 nm gold nanoparticles (GNPs) have demonstrated anti-inflammatory properties, with evidence that they decrease pathologic markers of ischemic severity. Whether GNPs affect cerebrovascular function, and potentially protect against the damaging effects of HSS on the cerebral circulation remains unclear. HSS obtained 24 h poststroke was perfused through the lumen of isolated and pressurized third-order posterior cerebral arteries (PCAs) from male Wistar rats with and without GNPs (~2 × 109 GNP/ml), or GNPs in vehicle, in an arteriograph chamber (n = 8/group). All vessels were myogenically reactive ≥60 mmHg intravascular pressure; however, vessels containing GNPs had significantly less myogenic tone. GNPs increased vasoreactivity to small and intermediate conductance calcium activated potassium channel activation via NS309; however, reduced vasoconstriction to nitric oxide synthase inhibition. Hydraulic conductivity and transvascular filtration, were decreased by GNPs, suggesting a protective effect on the blood-brain barrier. The stress-strain curves of PCAs exposed to GNPs were shifted leftward, indicating increased vessel stiffness. This study provides the first evidence that GNPs affect the structure and function of the cerebrovasculature, which may be important for their development and use in biomedical applications.

Algal polysaccharides for 3D printing: A review.

Algae hold particular promise as a feedstock for biomaterials, as they are capable of producing a wide variety of polymers with the properties required for 3D printing. However, the use of algal polymers has been limited to alginate, agar, carrageenan, and ulvan extracted from seaweeds. Diverse algal taxa beyond seaweeds have yet to be explored. In this comprehensive review, we discuss available algal biomaterials, their properties, and emerging applications in 3D printing techniques. We also identify elite algal strains to be used in 3D printing and comment on both advantages and limitations of algal biomass as a printing material. Global 3D printing market trends and material demands are also critically analyzed. Finally, the future prospects, opportunities, and challenges for using algal polymers in 3D printing market for a sustainable economy are discussed. We hope this review will provide a foundation for exploring the 3D printable biomaterials from algae.

Contribution of ADAMTS13-independent VWF regulation in sickle cell disease.

BACKGROUND: Von Willebrand factor (VWF) is elevated in sickle cell disease (SCD) and contributes to vaso-occlusion through its thrombogenic properties. VWF is regulated by ADAMTS13, a plasma protease that cleaves VWF into less bioactive multimers. Independent investigations have shown VWF to be elevated in SCD, whereas measurements of ADAMTS13 have been variable. OBJECTIVES: We assessed ADAMTS13 activity using multiple activity assays and measured levels of alternative VWF-cleaving proteases in SCD. METHODS/ PATIENTS: Plasma samples were collected from adult patients with SCD (n = 20) at a single institution when presenting for routine red cell exchange transfusion therapy. ADAMTS13 activity was measured by FRETS-VWF73, Technozym ADAMTS-13 Activity ELISA kit and a full-length VWF digestion reaction. Alternative VWF-cleaving proteases were identified by ELISA. A cell culture model was used to study the impact of SCD stimuli on endothelial ADAMTS13 and alternative VWF-cleaving proteases. RESULTS: ADAMTS13 activity was found to be moderately deficient across the SCD cohort as assessed by activity assays using a VWF A2 domain peptide substrate. However, SCD plasma showed preserved ability to digest full-length VWF, suggesting assay-discrepant results. Neutrophil and endothelial-derived proteases were found to be elevated in SCD plasma. Matrix metalloproteinase 9 specifically showed preferential cleavage of full-length VWF. Upregulation of alternative VWF-cleaving proteases occurred in endothelial cells exposed to SCD stimuli such as heme and hypoxia. CONCLUSIONS: This is the first demonstration of accessory plasma enzymes contributing to the regulation of VWF in a specific disease state and may have implications for assessing the VWF/ADAMTS13 axis in other settings.

Techno-economic assessment for the production of algal fuels and value-added products: opportunities for high-protein microalgae conversion.

BACKGROUND: Microalgae possess numerous advantages for use as a feedstock in producing renewable fuels and products, with techno-economic analysis (TEA) frequently used to highlight the economic potential and technical challenges of utilizing this biomass in a biorefinery context. However, many historical TEA studies have focused on the conversion of biomass with elevated levels of carbohydrates and lipids and lower levels of protein, incurring substantial burdens on the ability to achieve high cultivation productivity rates relative to nutrient-replete, high-protein biomass. Given a strong dependence of algal biomass production costs on cultivation productivity, further TEA assessment is needed to understand the economic potential for utilizing potentially lower-cost but lower-quality, high-protein microalgae for biorefinery conversion. RESULTS: In this work, we conduct rigorous TEA modeling to assess the economic viability of two conceptual technology pathways for processing proteinaceous algae into a suite of fuels and products. One approach, termed mild oxidative treatment and upgrading (MOTU), makes use of a series of thermo-catalytic operations to upgrade solubilized proteins and carbohydrates to hydrocarbon fuels, while another alternative focuses on the biological conversion of those substrates to oxygenated fuels in the form of mixed alcohols (MA). Both pathways rely on the production of polyurethanes from unsaturated fatty acids and valorization of unconverted solids for use as a material for synthesizing bioplastics. The assessment found similar, albeit slightly higher fuel yields and lower costs for the MA pathway, translating to a residual solids selling price of $899/ton for MA versus $1033/ton for MOTU as would be required to support a $2.50/gallon gasoline equivalent (GGE) fuel selling price. A variation of the MA pathway including subsequent upgrading of the mixed alcohols to hydrocarbon fuels (MAU) reflected a required solids selling price of $975/ton. CONCLUSION: The slight advantages observed for the MA pathway are partially attributed to a boundary that stops at oxygenated fuels versus fungible drop-in hydrocarbon fuels through a more complex MOTU configuration, with more comparable results obtained for the MAU scenario. In either case, it was shown that an integrated algal biorefinery can be economical through optimal strategies to utilize and valorize all fractions of the biomass.

Early detection of cerebrovascular pathology and protective antiviral immunity by MRI.

Central nervous system (CNS) infections are a major cause of human morbidity and mortality worldwide. Even patients that survive, CNS infections can have lasting neurological dysfunction resulting from immune and pathogen induced pathology. Developing approaches to noninvasively track pathology and immunity in the infected CNS is crucial for patient management and development of new therapeutics. Here, we develop novel MRI-based approaches to monitor virus-specific CD8+ T cells and their relationship to cerebrovascular pathology in the living brain. We studied a relevant murine model in which a neurotropic virus (vesicular stomatitis virus) was introduced intranasally and then entered the brain via olfactory sensory neurons - a route exploited by many pathogens in humans. Using T2*-weighted high-resolution MRI, we identified small cerebral microbleeds as an early form of pathology associated with viral entry into the brain. Mechanistically, these microbleeds occurred in the absence of peripheral immune cells and were associated with infection of vascular endothelial cells. We monitored the adaptive response to this infection by developing methods to iron label and track individual virus specific CD8+ T cells by MRI. Transferred antiviral T cells were detected in the brain within a day of infection and were able to reduce cerebral microbleeds. These data demonstrate the utility of MRI in detecting the earliest pathological events in the virally infected CNS as well as the therapeutic potential of antiviral T cells in mitigating this pathology.

Synonymous ADAMTS13 variants impact molecular characteristics and contribute to variability in active protein abundance.

The effects of synonymous single nucleotide variants (sSNVs) are often neglected because they do not alter protein primary structure. Nevertheless, there is growing evidence that synonymous variations may affect messenger RNA (mRNA) expression and protein conformation and activity, which may lead to protein deficiency and disease manifestations. Because there are >21 million possible sSNVs affecting the human genome, it is not feasible to experimentally validate the effect of each sSNV. Here, we report a comprehensive series of in silico analyses assessing sSNV impact on a specific gene. ADAMTS13 was chosen as a model for its large size, many previously reported sSNVs, and associated coagulopathy thrombotic thrombocytopenic purpura. Using various prediction tools of biomolecular characteristics, we evaluated all ADAMTS13 sSNVs registered in the National Center for Biotechnology Information database of single nucleotide polymorphisms, including 357 neutral sSNVs and 19 sSNVs identified in patients with thrombotic thrombocytopenic purpura. We showed that some sSNVs change mRNA-folding energy/stability, disrupt mRNA splicing, disturb microRNA-binding sites, and alter synonymous codon or codon pair usage. Our findings highlight the importance of considering sSNVs when assessing the complex effects of ADAMTS13 alleles, and our approach provides a generalizable framework to characterize sSNV impact in other genes and diseases.

Structural, functional, and immunogenicity implications of F9 gene recoding.

Hemophilia B is a blood clotting disorder caused by deficient activity of coagulation factor IX (FIX). Multiple recombinant FIX proteins are currently approved to treat hemophilia B, and several gene therapy products are currently being developed. Codon optimization is a frequently used technique in the pharmaceutical industry to improve recombinant protein expression by recoding a coding sequence using multiple synonymous codon substitutions. The underlying assumption of this gene recoding is that synonymous substitutions do not alter protein characteristics because the primary sequence of the protein remains unchanged. However, a critical body of evidence shows that synonymous variants can affect cotranslational folding and protein function. Gene recoding could potentially alter the structure, function, and in vivo immunogenicity of recoded therapeutic proteins. Here, we evaluated multiple recoded variants of F9 designed to further explore the effects of codon usage bias on protein properties. The detailed evaluation of these constructs showed altered conformations, and assessment of translation kinetics by ribosome profiling revealed differences in local translation kinetics. Assessment of wild-type and recoded constructs using a major histocompatibility complex (MHC)-associated peptide proteomics assay showed distinct presentation of FIX-derived peptides bound to MHC class II molecules, suggesting that despite identical amino acid sequence, recoded proteins could exhibit different immunogenicity risks. Posttranslational modification analysis indicated that overexpression from gene recoding results in suboptimal posttranslational processing. Overall, our results highlight potential functional and immunogenicity concerns associated with gene-recoded F9 products. These findings have general applicability and implications for other gene-recoded recombinant proteins.