Role for N-glycans and calnexin-calreticulin chaperones in SARS-CoV-2 Spike maturation and viral infectivity.

Functional and epidemiological data suggest that N-linked glycans on the SARS-CoV-2 Spike protein may contribute to viral infectivity. To investigate this, we created a panel of N-to-Q mutations at N-glycosylation sites proximal to the Spike S1-S2 (N61, N603, N657, and N616) and S2' (N603 and N801) proteolysis sites. Some of these mutations, particularly N61Q and N801Q, reduced Spike incorporation into Spike-pseudotyped lentivirus and authentic SARS-CoV-2 virus-like particles (VLPs). These mutations also reduced pseudovirus and VLP entry into ACE2-expressing cells by 80 to 90%. In contrast, glycan mutations had a relatively minor effect on cell surface expression of Spike, ACE2 binding, and syncytia formation. A similar dichotomy in function was observed when virus was produced in host cells lacking ER chaperones, calnexin and calreticulin. Here, while both chaperones regulated pseudovirus function, only VLPs produced in calnexin KOs were less infectious. Overall, Spike N-glycans are likely critical for SARS-CoV-2 function and could serve as drug targets for COVID-19.

Forward Genetic Screens of Human Glycosylation Pathways Using the GlycoGene CRISPR Library.

CRISPR-Cas9-based forward genetic screens represent a powerful discovery platform to uncover genes regulating specific biological processes. This article describes a method for utilizing a freely available GlycoGene CRISPR library to knock out any gene participating in human glycosylation in arbitrary cell types. The end product is a stable GlycoGene CRISPR knockout cell library, where each cell contains one or more sgRNA and lacks corresponding function. The cell library can be screened using various lectin/antibody reagents. It can also be applied in functional assays to establish glycan structure-glycogene-glycopathway relationships. This is a powerful systems glycobiology strategy for dissecting glycosylation pathways and processes. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Scale-up and NGS validation of the GlycoGene CRISPR plasmid library Basic Protocol 2: Preparation of a GlycoGene CRISPR lentivirus pool and an isogenic cell line stably expressing Cas9 nuclease Basic Protocol 3: Preparation of a GlycoGene CRISPR cell library, self-inactivation of Cas9, and library validation by NGS Basic Protocol 4: Enrichment of lectin-binding or non-binding cells and related multiplex NGS data acquisition Basic Protocol 5: Bioinformatics pathway analysis.

Local complement factor H protects kidney endothelial cell structure and function.

Factor H (FH) is a critical regulator of the alternative complement pathway and its deficiency or mutation underlie kidney diseases such as dense deposit disease. Since vascular dysfunction is an important facet of kidney disease, maintaining optimal function of the lining endothelial cells is important for vascular health. To investigate the molecular mechanisms that are regulated by FH in endothelial cells, FH deficient and sufficient mouse kidney endothelial cell cultures were established. Endothelial FH deficiency resulted in cytoskeletal remodeling, increased angiogenic potential, loss of cellular layer integrity and increased cell proliferation. FH reconstitution prevented these FH-dependent proliferative changes. Respiratory flux analysis showed reduced basal mitochondrial respiration, ATP production and maximal respiratory capacity in FH deficient endothelial cells, while proton leak remained unaltered. Similar changes were observed in FH deficient human glomerular endothelial cells indicating the translational potential of these studies. Gene expression analysis revealed that the FH-dependent gene changes in mouse kidney endothelial cells include significant upregulation of genes involved in inflammation and the complement system. The transcription factor nuclear factor-kB, that regulates many biological processes, was translocated from the cytoplasm to the nucleus in the absence of FH. Thus, our studies show the functional relevance of intrinsic FH in kidney endothelial cells in man and mouse.

A GlycoGene CRISPR-Cas9 lentiviral library to study lectin binding and human glycan biosynthesis pathways.

Glycan biosynthesis on cell surface proteins and lipids is orchestrated by different classes of enzymes and proteins including the following: i. glycosyltransferases that add saccharides; ii. glycosidases that trim glycans; iii. conserved oligomeric golgi complex members that regulate intracellular transport; iv. enzymes aiding the biosynthesis of sugar-nucleotides; and v. sulfotransferases. This manuscript describes a pooled "glycoGene CRISPR" lentiviral library that targets 347 human genes involved in the above processes. Approximately 10 single-guide RNA (sgRNA) are included against each glycogene, with the putative editing site spanning the length of the target. A data analysis scheme is presented in order to determine glycosylation pathways regulating biological processes. As proof of principle, forward genetic screen results are presented to identify penetrating glycogenes that regulate the binding of P-/E-selectin, anti-sialyl Lewis-X monoclonal antibody HECA-452 and selected lectins (phaseolus vulgaris leucoagglutinin, vicia villosa lectin, peanut agglutinin) to HL-60 promyelocytic cells. Besides validating previously established biology, the study identifies three enzymes, PAPSS1, SLC35B2 and TPST2, as key molecules regulating sulfation of the major P-selectin glycoprotein ligand-1 in leukocytes. Approximately 80-90% of the sgRNA used in this study displayed high editing efficiency, and the CRISPR library picked up entire gene sets regulating specific biosynthetic pathways rather than only isolated genes. These data suggest that the glycoGene CRISPR library contains high-efficiency sgRNA. Further, this resource could be useful for the rapid screening of glycosylation-related genes and pathways that control lectin recognition in a variety of contexts.

Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration.

The Spike protein of SARS-CoV-2, its receptor-binding domain (RBD), and its primary receptor ACE2 are extensively glycosylated. The impact of this post-translational modification on viral entry is yet unestablished. We expressed different glycoforms of the Spike-protein and ACE2 in CRISPR-Cas9 glycoengineered cells, and developed corresponding SARS-CoV-2 pseudovirus. We observed that N- and O-glycans had only minor contribution to Spike-ACE2 binding. However, these carbohydrates played a major role in regulating viral entry. Blocking N-glycan biosynthesis at the oligomannose stage using both genetic approaches and the small molecule kifunensine dramatically reduced viral entry into ACE2 expressing HEK293T cells. Blocking O-glycan elaboration also partially blocked viral entry. Mechanistic studies suggest multiple roles for glycans during viral entry. Among them, inhibition of N-glycan biosynthesis enhanced Spike-protein proteolysis. This could reduce RBD presentation on virus, lowering binding to host ACE2 and decreasing viral entry. Overall, chemical inhibitors of glycosylation may be evaluated for COVID-19.

COVID-19 is an infectious disease caused by the virus SARS-CoV-2. To access the internal machinery necessary for its replication, the virus needs to latch onto and then enter host cells. Such processes rely on specific 'glycoproteins' that carry complex sugar molecules (or glycans), and can be found at the surface of both viruses and host cells. In particular, the viral 'Spike' glycoprotein can attach to human proteins called ACE2, which coat the cells that line the inside of the lungs, heart, kidney and brain. Yet the roles played by glycans in these processes remains unclear. To investigate the role of Spike and ACE-2 glycans, Yang et al. designed a form of SARS-CoV-2 that could be handled safely in the laboratory. How these viruses infect human kidney cells that carry ACE2 was then examined, upon modifying the structures of the sugars on the viral Spike protein as well as the host ACE2 receptor. In particular, the sugar structures displayed by the virus were modified either genetically or chemically, using a small molecule that disrupts the formation of the glycans. Similar methods were also applied to modify the glycans of ACE2. Together, these experiments showed that the sugars present on the Spike protein play a minor role in helping the virus stick to human cells.However, they were critical for the virus to fuse and enter the host cells. These findings highlight the important role of Spike protein sugars in SARS-CoV-2 infection, potentially offering new paths to treat COVID-19 and other coronavirus-related illnesses. In particular, molecules designed to interfere with Spike-proteins and the viral entrance into cells could be less specific to SARS-CoV-2 compared to vaccines, allowing treatments to be efficient even if the virus changes.

Doxycycline-Dependent Self-Inactivation of CRISPR-Cas9 to Temporally Regulate On- and Off-Target Editing.

Exome and deep sequencing of cells treated with a panel of lentiviral guide RNA demonstrate that both on- and off-target editing proceed in a time-dependent manner. Thus, methods to temporally control Cas9 activity would be beneficial. To address this need, we describe a "self-inactivating CRISPR (SiC)" system consisting of a single guide RNA that deactivates the Streptococcus pyogenes Cas9 nuclease in a doxycycline-dependent manner. This enables defined, temporal control of Cas9 activity in any cell type and also in vivo. Results show that SiC may enable a reduction in off-target editing, with less effect on on-target editing rates. This tool facilitates diverse applications including (1) the timed regulation of genetic knockouts in hard-to-transfect cells using lentivirus, including human leukocytes for the identification of glycogenes regulating leukocyte-endothelial cell adhesion; (2) genome-wide lentiviral sgRNA (single guide RNA) library applications where Cas9 activity is ablated after allowing pre-determined editing times. Thus, stable knockout cell pools are created for functional screens; and (3) temporal control of Cas9-mediated editing of myeloid and lymphoid cells in vivo, both in mouse peripheral blood and bone marrow. Overall, SiC enables temporal control of gene editing and may be applied in diverse application including studies that aim to reduce off-target genome editing.

von Willebrand factor self-association is regulated by the shear-dependent unfolding of the A2 domain.

von Willebrand factor (VWF) self-association results in the homotypic binding of VWF upon exposure to fluid shear. The molecular mechanism of this process is not established. In this study, we demonstrate that the shear-dependent unfolding of the VWF A2 domain in the multimeric protein is a major regulator of protein self-association. This mechanism controls self-association on the platelet glycoprotein Ibα receptor, on collagen substrates, and during thrombus growth ex vivo. In support of this, A2-domain mutations that prevent domain unfolding due to disulfide bridging of N- and C-terminal residues ("Lock-VWF") reduce self-association and platelet activation under various experimental conditions. In contrast, reducing assay calcium concentrations, and 2 mutations that destabilize VWF-A2 conformation by preventing coordination with calcium (D1498A and R1597W VWD type 2A mutation), enhance self-association. Studies using a panel of recombinant proteins that lack the A1 domain ("ΔA1 proteins") suggest that besides pure homotypic A2 interactions, VWF-A2 may also engage other protein domains to control self-association. Addition of purified high-density lipoprotein and apolipoprotein-A1 partially blocked VWF self-association. Overall, similar conditions facilitate VWF self-association and ADAMTS13-mediated proteolysis, with low calcium and A2 disease mutations enhancing both processes, and locking-A2 blocking them simultaneously. Thus, VWF appears to have evolved 2 balancing molecular functions in a single A2 functional domain to dynamically regulate protein size in circulation: ADAMTS13-mediated proteolysis and VWF self-association. Modulating self-association rates by targeting VWF-A2 may provide novel methods to regulate the rates of thrombosis and hemostasis.

Role of calcium in regulating the intra- and extracellular cleavage of von Willebrand factor by the protease ADAMTS13.

von Willebrand factor (VWF) and the metalloprotease a disintegrin and metalloprotease with thrombospondin type 1 motif 13 (ADAMTS13) are present both within endothelial cells (ECs) and in peripheral blood. Calcium concentrations are lower in intracellular compartments (80-400 µM) compared with the extracellular milieu (∼1.25 mM). Because low calcium favors VWF A2-domain proteolysis by ADAMTS13, the dependence of proteolysis rates on calcium was assayed both within ECs and in blood. Confocal microscopy studies demonstrate partial perinuclear colocalization of VWF with ADAMTS13 in human umbilical vein ECs (HUVECs). Consequently, low levels (5%-10%) of VWF cleavage products were detected in HUVEC lysates and also culture-supernatant following EC stimulation. This proteolysis occurred before disulfide bond formation. Compared with wild-type VWF A2-domain, calcium-binding mutants including the common von Willebrand disease (VWD) type 2A R1597W mutant were expressed in an open conformation in ECs and were highly susceptible to intracellular proteolysis. Fluorescence resonance energy transfer measurements demonstrate strong calcium-dependent VWF-A2 conformation changes at concentrations <500 µM, with unfolding rates being fourfold higher for monomeric VWF A2-domain compared with multimeric, full-length VWF. Under shear, physiological levels of ADAMTS13 did not cleave VWF strings on HUVECs, unless platelets were attached to stretch these strings under flow. Further, VWF-platelet string cleavage under shear proceeded with equal efficiency in the absence and presence of calcium at shear stress ≥1 dyn/cm2. Overall, low calcium levels may promote intracellular VWF proteolysis particularly during VWD type 2A disease. Calcium has a negligible effect on VWF-platelet string proteolysis under physiologically relevant fluid shear.

Platelet GpIba binding to von Willebrand Factor under fluid shear:contributions of the D'D3-domain, A1-domain flanking peptide and O-linked glycans.

BACKGROUND: Von Willebrand Factor (VWF) A1-domain binding to platelet receptor GpIbα is an important fluid-shear dependent interaction that regulates both soluble VWF binding to platelets, and platelet tethering onto immobilized VWF. We evaluated the roles of different structural elements at the N-terminus of the A1-domain in regulating shear dependent platelet binding. Specifically, the focus was on the VWF D'D3-domain, A1-domain N-terminal flanking peptide (NFP), and O-glycans on this peptide. METHODS AND RESULTS: Full-length dimeric VWF (ΔPro-VWF), dimeric VWF lacking the D'D3 domain (ΔD'D3-VWF), and ΔD'D3-VWF variants lacking either the NFP (ΔD'D3NFP(─)-VWF) or just O-glycans on this peptide (ΔD'D3OG(─)-VWF) were expressed. Monomeric VWF-A1 and D'D3-A1 were also produced. In ELISA, the apparent dissociation constant (KD) of soluble ΔPro-VWF binding to immobilized GpIbα (KD≈100 nmol/L) was 50- to 100-fold higher than other proteins lacking the D'D3 domain (KD~0.7 to 2.5 nmol/L). Additionally, in surface plasmon resonance studies, the on-rate of D'D3-A1 binding to immobilized GpIbα (kon=1.8±0.4×10(4) (mol/L)(-1)·s(-1); KD=1.7 µmol/L) was reduced compared with the single VWF-A1 domain (kon=5.1±0.4×10(4) (mol/L)(-1)·s(-1); KD=1.2 µmol/L). Thus, VWF-D'D3 primarily controls soluble VWF binding to GpIbα. In contrast, upon VWF immobilization, all molecular features regulated A1-GpIbα binding. Here, in ELISA, the number of apparent A1-domain sites available for binding GpIbα on ΔPro-VWF was ≈50% that of the ΔD'D3-VWF variants. In microfluidics based platelet adhesion measurements on immobilized VWF and thrombus formation assays on collagen, human platelet recruitment varied as ΔPro-VWF<ΔD'D3-VWF<ΔD'D3NFP(─)-VWF<ΔD'D3OG(─)-VWF. CONCLUSIONS: Whereas VWF-D'D3 is the major regulator of soluble VWF binding to platelet GpIbα, both the D'D3-domain and N-terminal peptide regulate platelet translocation and thrombus formation.

Identification of novel genes that modify phenotypes induced by Alzheimer's beta-amyloid overexpression in Drosophila.

Sustained increases in life expectancy have underscored the importance of managing diseases with a high incidence in late life, such as various neurodegenerative conditions. Alzheimer's disease (AD) is the most common among these, and consequently significant research effort is spent on studying it. Although a lot is known about the pathology of AD and the role of beta-amyloid (Abeta) peptides, the complete network of interactions regulating Abeta metabolism and toxicity still eludes us. To address this, we have conducted genetic interaction screens using transgenic Drosophila expressing Abeta and we have identified mutations that affect Abeta metabolism and toxicity. These analyses highlight the involvement of various biochemical processes such as secretion, cholesterol homeostasis, and regulation of chromatin structure and function, among others, in mediating toxic Abeta effects. Several of the mutations that we identified have not been linked to Abeta toxicity before and thus constitute novel potential targets for AD intervention. We additionally tested these mutations for interactions with tau and expanded-polyglutamine overexpression and found a few candidate mutations that may mediate common mechanisms of neurodegeneration. Our data offer insight into the toxicity of Abeta and open new areas for further study into AD pathogenesis.

A model for studying Alzheimer's Abeta42-induced toxicity in Drosophila melanogaster.

Alzheimer's disease is a neurological disorder resulting in the degeneration and death of brain neurons controlling memory, cognition and behavior. Although overproduction of Abeta peptides is widely considered a causative event in the disease, the mechanisms by which Abeta peptides cause neurodegeneration and the processes of Abeta clearance and degradation remain unclear. To address these issues, we have expressed the Abeta peptides in Drosophila melanogaster. We show that overexpression of Abeta42 peptides in the nervous system results in phenotypes associated with neuronal degeneration in a dose- and age-dependent manner. We further show that a mutation in a Drosophila neprilysin gene suppresses the Abeta42 phenotypes by lowering the levels of the Abeta42 peptide, supporting the role of neprilysin in the catabolism of Abeta peptides in vivo. We propose that our Drosophila model is suitable for the study and elucidation of Abeta metabolism and toxicity at the genetic level.

Drosophila checkpoint kinase 2 couples centrosome function and spindle assembly to genomic integrity.

In syncytial Drosophila embryos, damaged or incompletely replicated DNA triggers centrosome disruption in mitosis, leading to defects in spindle assembly and anaphase chromosome segregation. The damaged nuclei drop from the cortex and are not incorporated into the cells that form the embryo proper. A null mutation in the Drosophila checkpoint kinase 2 tumor suppressor homolog (DmChk2) blocks this mitotic response to DNA lesions and also prevents loss of defective nuclei from the cortex. In addition, DNA damage leads to increased DmChk2 localization to the centrosome and spindle microtubules. DmChk2 is therefore essential for a "mitotic catastrophe" signal that disrupts centrosome function in response to genotoxic stress and ensures that mutant and aneuploid nuclei are eliminated from the embryonic precursor pool.