scholarly journals Site-Specific O-Glycosylation Analysis of SARS-CoV-2 Spike Protein Produced in Insect and Human Cells

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 551
Author(s):  
Ieva Bagdonaite ◽  
Andrew J. Thompson ◽  
Xiaoning Wang ◽  
Max Søgaard ◽  
Cyrielle Fougeroux ◽  
...  
Keyword(s):  
Insect Cell ◽  
Surface Protein ◽  
Large Family ◽  
Protein S ◽  
Distinct Pattern ◽  
Surface Proteins ◽  
Variable Expression ◽  
Glycosylation Sites ◽  
Variable Extent ◽  
Viral Surface

Enveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence is difficult to predict. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S—insect cell or human cell-produced ectodomains, or insect cell derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complementary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2.

scholarly journals Site-specific O-glycosylation analysis of SARS-CoV-2 spike protein produced in insect and human cells

2021 ◽  
Author(s):  
Ieva Bagdonaite ◽  
Andrew J. Thompson ◽  
Xiaoning Wang ◽  
Max Søgaard ◽  
Cyrielle Fougeroux ◽  
...  
Keyword(s):  
Insect Cell ◽  
Surface Protein ◽  
Large Family ◽  
Protein S ◽  
Distinct Pattern ◽  
Surface Proteins ◽  
Variable Expression ◽  
Glycosylation Sites ◽  
Variable Extent ◽  
Viral Surface

AbstractEnveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence difficult to predict. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S – insect cell or human cell-produced ectodomains, or insect cell derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complementary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2.

Adhesion properties of potentially probioticLactobacillus kefirito gastrointestinal mucus

2013 ◽  
Vol 81 (1) ◽  
pp. 16-23 ◽  
Author(s):  
Paula Carasi ◽  
Nicolás M. Ambrosis ◽  
Graciela L. De Antoni ◽  
Philippe Bressollier ◽  
María C. Urdaci ◽  
...  
Keyword(s):  
Bacterial Adhesion ◽  
Significant Proportion ◽  
Surface Protein ◽  
Protein S ◽  
Surface Proteins ◽  
Binding Properties ◽  
Added Sugar ◽  
Adhesion Properties ◽  
Probiotic Strains ◽  
Lactobacillus Kefiri

We investigated the mucus-binding properties of aggregating and non-aggregating potentially probiotic strains of kefir-isolatedLactobacillus kefiri, using different substrates. All the strains were able to adhere to commercial gastric mucin (MUCIN) and extracted mucus from small intestine (SIM) and colon (CM). The extraction of surface proteins from bacteria using LiCl or NaOH significantly reduced the adhesion of three selected strains (CIDCA 8348, CIDCA 83115 and JCM 5818); although a significant proportion (up to 50%) of S-layer proteins were not completely eliminated after treatments. The surface (S-layer) protein extracts from all the strains ofLb. kefiriwere capable of binding to MUCIN, SIM or CM, and no differences were observed among them. The addition of their own surface protein extract increased adhesion of CIDCA 8348 and 83115 to MUCIN and SIM, meanwhile no changes in adhesion were observed for JCM 5818. None of the seven sugars tested had the ability to inhibit the adhesion of whole bacteria to the three mucus extracts. Noteworthy, the degree of bacterial adhesion reached in the presence of their own surface protein (S-layer) extract decreased to basal levels in the presence of some sugars, suggesting an interaction between the added sugar and the surface proteins. In conclusion, the ability of these food-isolated bacteria to adhere to gastrointestinal mucus becomes an essential issue regarding the biotechnological potentiality ofLb. kefirifor the food industry.

scholarly journals 207.Oolemmal proteomics: identification of oocyte cell surface protein complexes involved in sperm - egg interaction

2004 ◽  
Vol 16 (9) ◽  
pp. 207
Author(s):  
J. W. Paul ◽  
E. A. McLaughlin ◽  
R. J. Aitken
Keyword(s):  
Fetal Calf Serum ◽  
Protein Complexes ◽  
Calf Serum ◽  
Biological Significance ◽  
Germinal Vesicle ◽  
Surface Protein ◽  
Protein S ◽  
Surface Proteins ◽  
Middle Range ◽  
Significant Difference

While the molecular basis of sperm-oolemma interaction is of considerable biological significance, the protein(s) involved in this process are yet to be identified. In particular, the point at which developing mammalian oocytes acquire the capacity to bind to, and fuse with, capacitated, acrosome-reacted spermatozoa is yet to be clearly defined. Previous reports suggested that mature metaphase II (MII) mouse oocytes possessed the capacity to bind and fuse with spermatozoa while, in contrast, immature germinal vesicle (GV) phase oocytes did not. In this study oocytes were cultured in α-MEM containing 5% fetal calf serum. For GV oocytes, α-MEM was also supplemented with 1 μM Milrinone to prevent GV breakdown. Standard murine IVF was performed in 100 μL α-MEM media droplets containing 10 to 15 oocytes and 2.5×104 capacitated spermatozoa. These studies found that no significant difference existed in the levels of sperm-oocyte binding or fusion between freshly isolated GV oocytes (14 sperm/egg bound, 7 sperm/egg fused), cultured arrested GV oocytes (16 sperm/egg bound, 7 sperm/egg fused) or cultured MII phase oocytes (17 sperm/egg bound, 8 sperm/egg fused). Interestingly, upon fusion MII oocytes commenced sperm nuclear decondensation whereas arrested GV oocytes did not. Comparison of biotinylated oolemmal surface proteins revealed markedly different protein profiles between the GV oolemma and the MII oolemma. The MII oolemma revealed a multitude of proteins ranging in size from approximately 20 to 200 kDa, whilst the GV oolemma revealed only six middle range molecular weight proteins ranging from approximately 50 to 90 kDa. The protein(s) implicated in sperm-egg interaction must be one or more of those proteins found in both oocyte populations. Comparison of these two profiles has greatly reduced the number of possible candidates, allowing possible identification of the proposed GPI-linked sperm receptor.

scholarly journals Structure and Glycosylation Patterns of Surface Proteins from Woodchuck Hepatitis Virus

1998 ◽  
Vol 72 (12) ◽  
pp. 9978-9985 ◽  
Author(s):  
Tanja K. Tolle ◽  
Dieter Glebe ◽  
Monica Linder ◽  
Dietmar Linder ◽  
Sigrid Schmitt ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Hepatitis Virus ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Viral Proteins ◽  
Surface Proteins ◽  
Woodchuck Hepatitis Virus ◽  
Amino Terminal ◽  
Glycosylation Sites ◽  
B Virus

ABSTRACT Woodchucks chronically infected with woodchuck hepatitis virus (WHV) are a valuable model for human hepatitis B virus (HBV) in studies of pathogenesis, immunity, and antiviral therapy. For this reason, substantial efforts to characterize both the similarities and the differences between HBV and WHV are being made. The structure of the WHV surface proteins (WHs proteins) has not yet been adequately elucidated. The bands that would be expected for glycosylated and nonglycosylated small (S) WHs protein are found by sodium dodecyl sulfate gel electrophoresis of purified WHs protein, but the bands corresponding to the middle (M) and large (L) WHs proteins of HBV are not seen at the expected sizes, even though the sequences of the WHV and HBV surface protein genes are 60% homologous. By amino-terminal sequencing we have identified two bands at 41 and 45 kDa as the MWHs proteins, 8 kDa larger than expected. We have also confirmed that two bands at 24 and 27 kDa are SWHs proteins. A protein of 49 kDa was blocked at the N terminus, which using immunoblotting with an antiserum against WHV pre-S1 (positions 126 to 146) was identified, together with a part of the 45-kDa protein, as glycosylated and nonglycosylated LWHs protein of the expected size. Sialidase and O-glycosidase digestion showed that the larger size of MWHs protein results from the presence of O glycoside groups which are probably in the pre-S2 domain of MWHs protein. Since the pre-S2 domains of HBV and WHV have similar numbers of potential O glycosylation sites, it appears to be likely that the glycosyltransferases act differently on the viral proteins in woodchucks and humans.

scholarly journals Structure-Based Vaccine Antigen Design

2019 ◽  
Vol 70 (1) ◽  
pp. 91-104 ◽  
Author(s):  
Barney S. Graham ◽  
Morgan S.A. Gilman ◽  
Jason S. McLellan
Keyword(s):  
Vaccine Development ◽  
Structural Information ◽  
Protein Structures ◽  
Surface Protein ◽  
Surface Proteins ◽  
Vaccine Antigen ◽  
Viral Fusion ◽  
Emerging Pathogens ◽  
Syncytial Virus ◽  
Viral Surface

Enabled by new approaches for rapid identification and selection of human monoclonal antibodies, atomic-level structural information for viral surface proteins, and capacity for precision engineering of protein immunogens and self-assembling nanoparticles, a new era of antigen design and display options has evolved. While HIV-1 vaccine development has been a driving force behind these technologies and concepts, clinical proof-of-concept for structure-based vaccine design may first be achieved for respiratory syncytial virus (RSV), where conformation-dependent access to neutralization-sensitive epitopes on the fusion glycoprotein determines the capacity to induce potent neutralizing activity. Success with RSV has motivated structure-based stabilization of other class I viral fusion proteins for use as immunogens and demonstrated the importance of structural information for developing vaccines against other viral pathogens, particularly difficult targets that have resisted prior vaccine development efforts. Solving viral surface protein structures also supports rapid vaccine antigen design and application of platform manufacturing approaches for emerging pathogens.

Projected Structure of the Surface Protein of Sulfolobus Spec. B12 Determined to a Resolution of 1.0 NM by Cryo-Electronmicroscopy

1990 ◽  
Vol 48 (1) ◽  
pp. 102-103
Author(s):  
G. Lembcke ◽  
F. Zemlin
Keyword(s):  
Low Dose ◽  
Maximum Dose ◽  
Three Dimensional ◽  
Surface Protein ◽  
Protein S ◽  
Radiation Sensitivity ◽  
Specimen Preparation ◽  
Molecular Architecture ◽  
High Radiation ◽  
Fritz Haber

The thermoacidophilic archaebacterium Sulfolobus spec. B12 , which is closely related to Sulfolobus solfataricus , possesses a regularly arrayed surface protein (S-layer), which is linked to the plasma membrane via spacer elements spanning a distinct interspace of approximately 18 nm. The S-layer has p3-Symmetry and a lattice constant of 21 nm; three-dimensional reconstructions of negatively stained fragments yield a layer thickness of approximately 6-7 nm.For analysing the molecular architecture of Sulfolobus surface protein in greater detail we use aurothioglucose(ATG)-embedding for specimen preparation. Like glucose, ATG, is supposed to mimic the effect of water, but has the advantage of being less volatile. ATG has advantages over glucose when working with specimens composed exclusively of protein because of its higher density of 2.92 g cm-3. Because of its high radiation sensitivity electromicrographs has to be recorded under strict low-dose conditions. We have recorded electromicrographs with a liquid helium-cooled superconducting electron microscope (the socalled SULEIKA at the Fritz-Haber-lnstitut) with a specimen temperature of 4.5 K and with a maximum dose of 2000 e nm-2 avoiding any pre-irradiation of the specimen.

Control of Thrombin Mediated Cleavage of Protein S

1986 ◽  
Vol 56 (02) ◽  
pp. 151-154 ◽  
Author(s):  
Christina A Mitchell ◽  
Lena Hau ◽  
Hatem H Salem
Keyword(s):  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Surface Protein ◽  
Protein S ◽  
Intact Protein ◽  
Control Mechanisms ◽  
Physiological Control ◽  
Endothelial Cell Surface ◽  
Thrombin Cleavage ◽  
Cofactor Activity

SummaryThrombin has been shown to cleave the vitamin K dependent cofactor protein S with subsequent loss of its cofactor activity. This study examines the control mechanisms for thrombin cleavage of protein S.The anticoagulant activity of activated protein C (APC) is enhanced fourteen fold by the addition of protein S. Thrombin cleaved protein S is seven fold less efficient than the native protein, and this loss of activity is due to reduced affinity of cleaved protein S for APC or the lipid surface compared to the intact protein.In the absence of Ca++, protein S is very sensitive to minimal concentrations of thrombin. As little as 1.5 nM thrombin results in complete cleavage of 20 nM protein S in 10 min and loss of cofactor activity. Ca++, in concentrations greater than 0.5 mM, will inhibit this cleavage and in the presence of physiological Ca++ concentrations, no cleavage of protein S could be demonstrated in spite of high concentrations of thrombin (up to 1 μM) and prolonged incubations (up to two hours). The endothelial surface protein thrombomodulin is very efficient in inhibiting the cleavage of protein S by thrombin suggesting that any thrombin formed on the endothelial cell surface is unlikely to cleave protein S, thus allowing the intact protein to act as a cofactor to APC.We conclude that the inhibitory effects of Ca++ and thrombomodulin on thrombin mediated cleavage of protein S imply that this event, by itself, is unlikely to represent a physiological control of the activity of protein S.

Identification of ecto-PKC on surface of human platelets: role in maintenance of latent fibrinogen receptors

2000 ◽  
Vol 278 (6) ◽  
pp. H2008-H2019 ◽  
Author(s):  
Anna Babinska ◽  
Michael V. Hogan ◽  
Tomasz Sobocki ◽  
Malgorzata B. Sobocka ◽  
Yigal H. Ehrlich ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Calcium Concentration ◽  
Surface Protein ◽  
Human Platelets ◽  
Surface Proteins ◽  
Free Calcium ◽  
Platelet Surface ◽  
Inhibitory Peptides ◽  
Intracellular Free Calcium Concentration ◽  
Free Calcium Concentration

Human platelets express a protein phosphorylation system on their surface. A specific protein kinase C (PKC) antibody, monoclonal antibody (MAb) 1.9, which binds to the catalytic domain of PKC and inhibits its activity, causes the aggregation of intact platelets while inhibiting the phosphorylation of platelet surface proteins. Photoaffinity labeling with 100 nM 8-azido-[α32P]ATP identified this ecto-PKC as a single surface protein of 43 kDa sensitive to proteolysis by extracellular 0.0005% trypsin. Inhibition of the binding of 8-azido-[α32P]ATP to the 43-kDa surface protein by MAb 1.9 identified this site as the active domain of ecto-PKC. Covalent binding of the azido-ATP molecule to the 43-kDa surface protein inhibited the phosphorylative activity of the platelet ecto-PKC. Furthermore, PKC pseudosubstrate inhibitory peptides directly induced the aggregation of platelets and inhibited azido-ATP binding to the 43-kDa protein. Platelet aggregation induced by MAb 1.9 and by PKC inhibitory peptides required the presence of fibrinogen and resulted in an increase in the level of intracellular free calcium concentration. This increase in intracellular free calcium concentration induced by MAb 1.9 was found to be dependent on the binding of fibrinogen to activated GPIIb/IIIa integrins, suggesting that MAb 1.9 causes Ca2+flux through the fibrinogen receptor complex. We conclude that a decrease in the state of phosphorylation of platelet surface proteins caused by inhibition of ecto-PKC results in membrane rearrangements that can induce the activation of latent fibrinogen receptors, leading to platelet aggregation. Accordingly, the maintenance of a physiological steady state of phosphorylation of proteins on the platelet surface by ecto-PKC activity appears to be one of the homeostatic mechanisms that maintain fibrinogen receptors of circulating platelets in a latent state that cannot bind fibrinogen.

scholarly journals Targeting Viral Surface Proteins through Structure-Based Design

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1320
Author(s):  
Yogesh B Narkhede ◽  
Karen J Gonzalez ◽  
Eva-Maria Strauch
Keyword(s):  
Public Health ◽  
Viral Infections ◽  
Respiratory Viruses ◽  
Surface Proteins ◽  
Host Cells ◽  
Viral Fusion ◽  
Health It ◽  
Viral Particles ◽  
Pathogenic Viruses ◽  
Viral Surface

The emergence of novel viral infections of zoonotic origin and mutations of existing human pathogenic viruses represent a serious concern for public health. It warrants the establishment of better interventions and protective therapies to combat the virus and prevent its spread. Surface glycoproteins catalyzing the fusion of viral particles and host cells have proven to be an excellent target for antivirals as well as vaccines. This review focuses on recent advances for computational structure-based design of antivirals and vaccines targeting viral fusion machinery to control seasonal and emerging respiratory viruses.

scholarly journals The variant-specific surface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein

1997 ◽  
Vol 322 (1) ◽  
pp. 49-56 ◽  
Author(s):  
Philemon PAPANASTASIOU ◽  
Malcolm J. McCONVILLE ◽  
Julie RALTON ◽  
Peter KÖHLER
Keyword(s):  
Specific Surface ◽  
Acyl Chain ◽  
Giardia Duodenalis ◽  
Compositional Analysis ◽  
Surface Protein ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Surface Proteins ◽  
Phase Partitioning ◽  
Chromatography Analysis

The variant-specific surface proteins (VSPs) of the ancient protist Giardia duodenalis(syn.: Giardia intestinalis, Giardia lamblia) are cysteine- and threonine-rich polypeptides that can vary considerably in sequence and size. In the present study, we have purified a VSP (VSP4A1, formerly called CRISP-90) from a cloned Giardiaisolate, derived from a sheep, by Triton X-114 phase partitioning and anion-exchange chromatography. Analysis of the purified VSP4A1 showed that this protein is post-translationally modified with both glycans and lipid. The glycans of VSP4A1 were detected and partially characterized by (1) compositional analysis, which indicated the presence of GlcNAc and Glc (0.5 and 1.0 mol/mol of protein respectively), and (2) the specific labelling of VSP4A1 with galactosyltransferase/UDP-[3H]Gal. The glycans were released by β-elimination, suggesting that they are O-linked to the protein. Bio-Gel P4 chromatography of the released galactosylated glycans and further compositional analysis suggested that the major glycan on the VSP is a trisaccharide with Glc at the reducing terminus. These and other results indicate the absence of any N-linked glycans on the VSP and suggest instead that it is elaborated with a novel type of short O-linked glycan. Compositional analysis and radiolabelling experiments also indicated that VSP4A1 is modified with covalently linked palmitate (1 mol/mol of protein). Hydroxylamine treatment at neutral pH of [3H]palmitate-labelled VSP4A1 indicated that the acyl chain may be attached by a thioester linkage. A likely location for the lipid modification appears to be in the region of the C-terminal domain where it may facilitate association of the protein with the plasma membrane.

Sign in / Sign up

Export Citation Format

Share Document