N-Linked Glycans on Therapeutic Factor VIII (FVIII) Proteins Attenuate Immunogenicity Potential: Evidence from Independent HLA-Class-II/FVIII (HLAcII/FVIII) Peptidomes

The role played by N-linked glycans (NLGs) in the immunogenicity of therapeutic Factor VIII (tFVIII) proteins is poorly understood. Our study addresses this question using peptidomic profiling data on HLA-class-II (HLAcII)-presented peptides from dendritic cell (DC)-protein processing and presentation assays (PPPAs) performed across three independent experiments reported in the literature (Sorvillo et al. (2016), Peyron et al. (2018), and Diego et al. (2020)). Assuming that the number of peptides presented on HLAcII molecules is directly proportional to immunogenicity potential (IP), we asked if the NLGs on tFVIII proteins provide some degree of protection from proteolytic processing within DCs for the amino acid (AA) residues in their immediate vicinity. If NLGs are protective, then we expect an attenuated IP, which would reflect in lower counts of associated AAs. We examined the effects of NLGs both pointwise (i.e., at the single glycated asparagine residue) and at the glycosylation umbrella (GUMB), a term we defined as being −5 to +5 AAs from the glycated asparagine residue of all NLGs. Our first step in addressing these effects was to construct 2×2 contingency tables of the number of AA residues in the HLAcII bound and unbound fractions, and the number of AA residues falling either within the set of NLGs or within the set of GUMBs. We statistically evaluated our question by using Fisher's Exact tests of the hypothesis of no association, and calculated the odds ratio (OR) and its 95% confidence interval (CI). Results from the pointwise tests of the effect of NLGs reported in Figure 1 suggest that overall NLGs exert a protective effect in that non-glycated AAs were at significantly greater risk of being in the fraction of peptides bound to and presented by HLAcII molecules. The results from these statistical analyses-listed as OR (95% CI LB, 95% CI UB)-were: 7.0 (2.6, 21.9) for Diego et al.; 5.6 (0.9, 230.6) for Sorvillo et al. with 25 nM tFVIII; 6.5 (1.0, 269.0) for Sorvillo et al. with 50 nM tFVIII; 3.7 (1.1, 19.8) for Peyron et al.; and 4.4 (2.3, 9.3) for all data combined. The data from Sorvillo et al. (2016; for their experiments using 50 nM tFVIII but not for their experiments using 25 nM tFVIII), Peyron et al. (2018), and Diego et al. (2020), as well as the combined data, all revealed significantly greater risk of non-glycated AAs being in the HLAcII bound/presented fraction relative to glycated AAs. While the results from Sorvillo et al. with 25 nM tFVIII was not statistically significant, this was most likely due to the effects on power of its "small sample size" as the data was trending. The results for the GUMB-level tests reported in Figure 2 are even more robust with the data suggesting a protective effect of the GUMBs in that the AAs not located in their immediate vicinity (as defined above) were at significantly greater risk of being in the bound fraction of peptides presented on HLAcII molecules in all experiments, as well as in the combined data. The results from these statistical analyses-listed as OR (95% CI LB, 95% CI UB)-were: 6.0 (4.5, 8.2) for Diego et al.; 19.9 (6.7, 97.6) for Sorvillo et al. with 25 nM tFVIII; 7.3 (3.9, 15.6) for Sorvillo et al. with 50 nM tFVIII; 2.5 (1.8, 3.6) for Peyron et al.; and 3.8 (3.2, 4.6) for all data combined. In conclusion, these results-from the tFVIII proteins tested using peptidomic profiling of HLAcII-presented peptides from DC-PPPAs performed across three independent experiments-demonstrate NLGs provided significant protection from either proteolytic processing in DCs or peptide binding to HLAcII molecules, or both, and thus lower IP. Our results support the conclusion that NLGs play a significant role in the immunogenicity of tFVIII proteins. Disclosures Luu: Haplogenics Corporation: Current Employment. Hofmann:CSL Behring: Current Employment. Dinh:Haplogenics Corporation: Current Employment. Powell:Haplogenics Corporation: Membership on an entity's Board of Directors or advisory committees. Mead:CSL Behring: Current Employment. Escobar:Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; National Hemophilia Foundation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Maraskovsky:CSL Behring: Current Employment. Howard:Haplogenics Corporation: Membership on an entity's Board of Directors or advisory committees.

Platelets and Defective N-Glycosylation

N-glycans are covalently linked to an asparagine residue in a simple acceptor sequence of proteins, called a sequon. This modification is important for protein folding, enhancing thermodynamic stability, and decreasing abnormal protein aggregation within the endoplasmic reticulum (ER), for the lifetime and for the subcellular localization of proteins besides other functions. Hypoglycosylation is the hallmark of a group of rare genetic diseases called congenital disorders of glycosylation (CDG). These diseases are due to defects in glycan synthesis, processing, and attachment to proteins and lipids, thereby modifying signaling functions and metabolic pathways. Defects in N-glycosylation and O-glycosylation constitute the largest CDG groups. Clotting and anticlotting factor defects as well as a tendency to thrombosis or bleeding have been described in CDG patients. However, N-glycosylation of platelet proteins has been poorly investigated in CDG. In this review, we highlight normal and deficient N-glycosylation of platelet-derived molecules and discuss the involvement of platelets in the congenital disorders of N-glycosylation.

Asparagine residue 368 is involved in Alzheimer's disease tau strain–specific aggregation

In tauopathies, tau forms pathogenic fibrils with distinct conformations (termed “tau strains”) and acts as an aggregation “seed” templating the conversion of normal tau into isomorphic fibrils. Previous research showed that the aggregation core of tau fibril covers the C-terminal region (243–406 amino acids (aa)) and differs among the diseases. However, the mechanisms by which distinct fibrous structures are formed and inherited via templated aggregation are still unknown. Here, we sought to identify the key sequences of seed-dependent aggregation. To identify sequences for which deletion reduces tau aggregation, SH-SY5Y cells expressing a series of 10 partial deletion (Del 1–10, covering 244–400 aa) mutants of tau-CTF24 (243–441 aa) were treated with tau seeds prepared from a different tauopathy patient's brain (Alzheimer's disease, progressive supranuclear palsy, and corticobasal degeneration) or recombinant tau, and then seed-dependent tau aggregation was assessed biochemically. We found that the Del 8 mutant lacking 353–368 aa showed significantly decreased aggregation in both cellular and in vitro models. Furthermore, to identify the minimum sequence responsible for tau aggregation, we systematically repeated cellular tau aggregation assays for the delineation of shorter deletion sites and revealed that Asn-368 mutation suppressed tau aggregation triggered by an AD tau seed, but not using other tauopathy seeds. Our study suggested that 353–368 aa is a novel aggregation-responsible sequence other than PHF6 and PHF6*, and within this sequence, the Asn-368 residue plays a role in strain-specific tau aggregation in different tauopathies.

A VPS13D spastic ataxia mutation disrupts the conserved adaptor-binding site in yeast Vps13

Mutations in each of the four human VPS13 (VPS13A–D) proteins are associated with distinct neurological disorders: chorea-acanthocytosis, Cohen syndrome, early-onset Parkinson’s disease and spastic ataxia. Recent evidence suggests that the different VPS13 paralogs transport lipids between organelles at different membrane contact sites. How each VPS13 isoform is targeted to organelles is not known. We have shown that the localization of yeast Vps13 protein to membranes requires a conserved six-repeat region, the Vps13 Adaptor Binding (VAB) domain, which binds to organelle-specific adaptors. Here, we use a systematic mutagenesis strategy to determine the role of each repeat in recognizing each known adaptor. Our results show that mutation of invariant asparagines in repeats 1 and 6 strongly impacts the binding of all adaptors and blocks Vps13 membrane recruitment. However, we find that repeats 5–6 are sufficient for localization and interaction with adaptors. This supports a model where a single adaptor-binding site is found in the last two repeats of the VAB domain, while VAB domain repeat 1 may influence domain conformation. Importantly, a disease-causing mutation in VPS13D, which maps to the highly conserved asparagine residue in repeat 6, blocks adaptor binding and Vps13 membrane recruitment when modeled in yeast. Our findings are consistent with a conserved adaptor binding role for the VAB domain and suggest the presence of as-yet-unidentified adaptors in both yeast and humans.

A VPS13D spastic ataxia mutation disrupts the conserved adaptor binding site in yeast Vps13

Mutations in each of the four human VPS13 (VPS13A-D) proteins are associated with distinct neurological disorders: chorea-acanthocytosis, Cohen syndrome, early-onset Parkinson’s disease and spastic ataxia. Recent evidence suggests that the different VPS13 paralogs transport lipids between organelles at different membrane contact sites. How each VPS13 isoform is targeted to organelles is not known. We have shown that the localization of yeast Vps13 protein to membranes requires a conserved six-repeat region, the Vps13 Adaptor Binding (VAB) domain, which binds to organelle-specific adaptors. Here, we use a systematic mutagenesis strategy to determine the role of each repeat in recognizing each known adaptor. Our results show that mutation of invariant asparagines in repeats 1 and 6 strongly impact the binding all adaptors and block Vps13 membrane recruitment. However, we find that repeats 5 to 6 are sufficient for localization and interaction with adaptors. This supports a model where a single adaptor binding site is found in the last two repeats of the VAB domain, while VAB domain repeat 1 may help maintain domain conformation. Importantly, a disease-causing mutation in VPS13D, which maps to the highly conserved asparagine residue in repeat 6, blocks adaptor binding and Vps13 membrane recruitment when modeled in yeast. Our findings are consistent with a conserved adaptor binding role for the VAB domain and suggests the presence of as-yet-unidentified adaptors in both yeast and humans.