Bioinformatic Analyses to Uncover Genes Involved in Trehalose Metabolism in the Polyploid Sugarcane

Trehalose-6-phosphate (T6P) is an intermediate of trehalose biosynthesis that plays an essential role in plant metabolism and development. Here, we comprehensively analyzed sequences from enzymes of trehalose metabolism in sugarcane, one of the main crops used for bioenergy production. We identified protein domains, phylogeny, and in silico expression levels for all classes of enzymes. However, post-translational modifications and residues involved in catalysis and substrate binding were analyzed only in trehalose-6-phosphate synthase (TPS) sequences. We retrieved 71 putative full-length TPS, 93 trehalose-6-phosphate phosphatase (TPP), and 3 trehalase (TRE) of sugarcane, showing all their conserved domains, respectively. Putative TPS (Classes I and II) and TPP sugarcane sequences were categorized into well-known groups reported in the literature. We measured the expression levels of the sequences from one sugarcane leaf transcriptomic dataset. Furthermore, TPS Class I has specific N-glycosylation sites inserted in conserved motifs and carries catalytic and binding residues in its TPS domain. Some of these residues are mutated in TPS Class II members, which implies loss of enzyme activity. Our approach retrieved many homo(eo)logous sequences for genes involved in trehalose metabolism, paving the way to discover the role of T6P signaling in sugarcane.

Cryo-EM structure of native human thyroglobulin

The thyroglobulin (TG) protein is essential to thyroid hormone synthesis, plays a vital role in the regulation of metabolism, development and growth and serves as intraglandular iodine storage. Its architecture is conserved among vertebrates. Synthesis of triiodothyronine (T3) and thyroxine (T4) hormones depends on the conformation, iodination and post-translational modification of TG. Although structural information is available on recombinant and deglycosylated endogenous human thyroglobulin (hTG) from patients with goiters, the structure of native, fully glycosylated hTG remained unknown. Here, we present the cryo-electron microscopy structure of native and fully glycosylated hTG from healthy thyroid glands to 3.2 Å resolution. The structure provides detailed information on hormonogenic and glycosylation sites. We employ liquid chromatography–mass spectrometry (LC-MS) to validate these findings as well as other post-translational modifications and proteolytic cleavage sites. Our results offer insights into thyroid hormonogenesis of native hTG and provide a fundamental understanding of clinically relevant mutations.

Genetic Diversity and Molecular Epidemiology of Circulating Respiratory Syncytial Virus in Central Taiwan, 2008–2017

In this study, we investigated the molecular evolution and phylodynamics of respiratory syncytial virus (RSV) over 10 consecutive seasons (2008–2017) and the genetic variability of the RSV genotypes ON1 and BA in central Taiwan. The ectodomain region of the G gene was sequenced for genotyping. The nucleotide and deduced amino acid sequences of the second hypervariable region of the G protein in RSV ON1 and BA were analyzed. A total of 132 RSV-A and 81 RSV-B isolates were obtained. Phylogenetic analysis revealed that the NA1, ON1, and BA9 genotypes were responsible for the RSV epidemics in central Taiwan in the study period. For RSV-A, the NA1 genotype predominated during the 2008–2011 seasons. The ON1 genotype was first detected in 2011 and replaced NA1 after 2012. For RSV-B, the BA9 and BA10 genotypes cocirculated from 2008 to 2010, but the BA9 genotype has predominated since 2012. Amino acid sequence alignments revealed the continuous evolution of the G gene in the ectodomain region. The predicted N-glycosylation sites were relatively conserved in the ON1 (site 237 and 318) and BA9 (site 296 and 310) genotype strains. Our results contribute to the understanding and prediction of the temporal evolution of RSV at the local level.

The Action of Recombinant Human Lysosomal α-Glucosidase (rhGAA) on Human Liver Glycogen: Pathway to Complete Degradation

Glycogen is present in all tissues, but it is primarily stored in the liver and in muscle. As a branched chain carbohydrate, it is broken down by phosphorylase and debrancher enzymes, which are cytoplasmic. It is also degraded by a lysosomal α-glucosidase (GAA) also known as acid α-glucosidase and lysosomal acid α-glucosidase. The deficiency of GAA in patients is known as Pompe disease, and the phenotypes as infantile, juvenile and later onset forms. Pompe disease is treated by enzyme replacement therapy (ERT) with a recombinant form of rhGAA. Following ERT in Pompe mice and human patients there is residual carbohydrate material present in the cytoplasm of cells. The goal of this work is to improve ERT and attempt to identify and treat the residual cytoplasmic carbohydrate. Initial experiments were to determine if rhGAA can completely degrade glycogen. The enzyme cannot completely degrade glycogen. There is a residual glycosylated protein as well as a soluble glycosylated protein, which is a terminal degradation product of glycogen and as such serves as a biomarker for lysosomal glycogen degradation. The glycosylated protein has a very unusual carbohydrate composition for a glycosylated protein: m-inositol, s-inositol and sorbitol as the major carbohydrates, as well as mannitol, mannose, glucose and galactose. This work describes the residual material which likely contains the same protein as the soluble glycosylated protein. The biomarker is present in serum of control and Pompe patients on ERT, but it is not present in the serum of Pompe mice not on ERT. Pompe mice not on ERT have another glycosylated protein in their serum which may be a biomarker for Pompe disease. This protein has multiple glycosylation sites, each with different carbohydrate components. These glycosylated proteins as well as the complexity of glycogen structure are discussed, as well as future directions to try to improve the outcome of ERT for Pompe patients by being able to monitor the efficacy of ERT in the short term and possibly to adjust the timing and dose of enzyme infusions.

Computational Saturation Mutagenesis of SARS-CoV-1 Spike Glycoprotein: Stability, Binding Affinity, and Comparison With SARS-CoV-2

Severe Acute respiratory syndrome coronavirus (SARS-CoV-1) attaches to the host cell surface to initiate the interaction between the receptor-binding domain (RBD) of its spike glycoprotein (S) and the human Angiotensin-converting enzyme (hACE2) receptor. SARS-CoV-1 mutates frequently because of its RNA genome, which challenges the antiviral development. Here, we per-formed computational saturation mutagenesis of the S protein of SARS-CoV-1 to identify the residues crucial for its functions. We used the structure-based energy calculations to analyze the effects of the missense mutations on the SARS-CoV-1 S stability and the binding affinity with hACE2. The sequence and structure alignment showed similarities between the S proteins of SARS-CoV-1 and SARS-CoV-2. Interestingly, we found that target mutations of S protein amino acids generate similar effects on their stabilities between SARS-CoV-1 and SARS-CoV-2. For example, G839W of SARS-CoV-1 corresponds to G857W of SARS-CoV-2, which decrease the stability of their S glycoproteins. The viral mutation analysis of the two different SARS-CoV-1 isolates showed that mutations, T487S and L472P, weakened the S-hACE2 binding of the 2003–2004 SARS-CoV-1 isolate. In addition, the mutations of L472P and F360S destabilized the 2003–2004 viral isolate. We further predicted that many mutations on N-linked glycosylation sites would increase the stability of the S glycoprotein. Our results can be of therapeutic importance in the design of antivirals or vaccines against SARS-CoV-1 and SARS-CoV-2.

Machupo virus with mutations in the transmembrane domain and glycosylation sites is attenuated and immunogenic in animal models of Bolivian Hemorrhagic Fever

Several highly pathogenic mammarenaviruses cause severe hemorrhagic and neurologic disease in humans, for which vaccines and antivirals are limited or unavailable. New World (NW) mammarenavirus Machupo virus (MACV) infection causes Bolivian hemorrhagic fever in humans. We previously reported that the disruption of specific N-linked glycan sites on the glycoprotein (GPC) partially attenuate MACV in an IFN-αβ/γ receptor knockout mouse model. However, some capability to induce neurological pathology still remained. Highly pathogenic Junin virus (JUNV) is another NW arenavirus closely related to MACV. A F427I substitution in the GPC transmembrane domain (TMD) rendered JUNV attenuated in a lethal mouse model after intracranial inoculation. In this study, we rationally designed and rescued a MACV containing mutations at two glycosylation sites and the corresponding F438I substitution in GPC TMD. The MACV mutant is fully attenuated in IFN-αβ/γ receptor knockout mice and outbred guinea pigs. Furthermore, inoculation with this mutant MACV fully protected guinea pigs from wild-type MACV lethal challenge. Lastly, we found the GPC TMD F438I substitution greatly impaired MACV growth in neuronal cell lines of mouse and human origins. Our results highlight the critical roles of the glycans and the TMD on the GPC in arenavirus virulence, which informs the rational design of potential vaccine candidates for highly pathogenic arenaviruses.ImportanceFor arenaviruses, the only vaccine available is the live-attenuated Candid#1 vaccine, a JUNV vaccine approved in Argentina. We and others have found that the glycans on GPC and the F427 residue in the GPC TMD are important for virulence of JUNV. Nevertheless, mutating either of them is not sufficient for full and stable attenuation of JUNV. Using reverse genetics, we disrupted specific glycosylation sites on MACV GPC, and also introduced the corresponding F438I substitution in the GPC TMD. This MACV mutant is fully attenuated in two animal models and protects animals from lethal infection. Thus, our studies highlight the feasibility of rational attenuation of highly pathogenic arenaviruses for vaccine development. Another important finding from this study is that the F438I substitution in GPC TMD could substantially affect MACV replication in neurons. Future studies are warranted to elucidate the underlying mechanism and the implication of this mutation in arenavirus neural tropism.

DeepNGlyPred: A Deep Neural Network-Based Approach for Human N-Linked Glycosylation Site Prediction

Protein N-linked glycosylation is a post-translational modification that plays an important role in a myriad of biological processes. Computational prediction approaches serve as complementary methods for the characterization of glycosylation sites. Most of the existing predictors for N-linked glycosylation utilize the information that the glycosylation site occurs at the N-X-[S/T] sequon, where X is any amino acid except proline. Not all N-X-[S/T] sequons are glycosylated, thus the N-X-[S/T] sequon is a necessary but not sufficient determinant for protein glycosylation. In that regard, computational prediction of N-linked glycosylation sites confined to N-X-[S/T] sequons is an important problem. Here, we report DeepNGlyPred a deep learning-based approach that encodes the positive and negative sequences in the human proteome dataset (extracted from N-GlycositeAtlas) using sequence-based features (gapped-dipeptide), predicted structural features, and evolutionary information. DeepNGlyPred produces SN, SP, MCC, and ACC of 88.62%, 73.92%, 0.60, and 79.41%, respectively on N-GlyDE independent test set, which is better than the compared approaches. These results demonstrate that DeepNGlyPred is a robust computational technique to predict N-Linked glycosylation sites confined to N-X-[S/T] sequon. DeepNGlyPred will be a useful resource for the glycobiology community.

N-glycosylation profiles of the SARS-CoV-2 spike D614G mutant and its ancestral protein characterized by advanced mass spectrometry

N-glycosylation plays an important role in the structure and function of membrane and secreted proteins. The spike protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is heavily glycosylated and the major target for developing vaccines, therapeutic drugs and diagnostic tests. The first major SARS-CoV-2 variant carries a D614G substitution in the spike (S-D614G) that has been associated with altered conformation, enhanced ACE2 binding, and increased infectivity and transmission. In this report, we used mass spectrometry techniques to characterize and compare the N-glycosylation of the wild type (S-614D) or variant (S-614G) SARS-CoV-2 spike glycoproteins prepared under identical conditions. The data showed that half of the N-glycosylation sequons changed their distribution of glycans in the S-614G variant. The S-614G variant showed a decrease in the relative abundance of complex-type glycans (up to 45%) and an increase in oligomannose glycans (up to 33%) on all altered sequons. These changes led to a reduction in the overall complexity of the total N-glycosylation profile. All the glycosylation sites with altered patterns were in the spike head while the glycosylation of three sites in the stalk remained unchanged between S-614G and S-614D proteins.

O-glycosylation site prediction for Homo sapiens by combining properties and sequence features with support vector machine

O-glycosylation is a protein posttranslational modification important in regulating almost all cells. It is related to a large number of physiological and pathological phenomena. Recognizing O-glycosylation sites is the key to further investigating the molecular mechanism of protein posttranslational modification. This study aimed to collect a reliable dataset on Homo sapiens and develop an O-glycosylation predictor for Homo sapiens, named Captor, through multiple features. A random undersampling method and a synthetic minority oversampling technique were employed to deal with imbalanced data. In addition, the Kruskal–Wallis (K–W) test was adopted to optimize feature vectors and improve the performance of the model. A support vector machine, due to its optimal performance, was used to train and optimize the final prediction model after a comprehensive comparison of various classifiers in traditional machine learning methods and deep learning. On the independent test set, Captor outperformed the existing O-glycosylation tool, suggesting that Captor could provide more instructive guidance for further experimental research on O-glycosylation. The source code and datasets are available at https://github.com/YanZhu06/Captor/ .