scholarly journals Fluorescent glycan fingerprinting of SARS2 spike proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zhengliang L. Wu ◽  
James M. Ertelt
Keyword(s):  
Sialic Acid ◽  
Gel Electrophoresis ◽  
Fluorescent Labeling ◽  
Biological Functions ◽  
Post Translational Modification ◽  
Quick Method ◽  
New Techniques ◽  
S Protein ◽  
Sds Page ◽  
Novel Coronavirus

AbstractGlycosylation is the most common post-translational modification and has myriad of biological functions. However, glycan analysis has always been a challenge. Here, we would like to present new techniques for glycan fingerprinting based on enzymatic fluorescent labeling and gel electrophoresis. The method is illustrated on SARS2 spike (S) glycoproteins. SARS2, a novel coronavirus and the causative agent of the COVID-19 pandemic, has had significant social and economic impacts since the end of 2019. To obtain the N-glycan fingerprint of an S protein, glycans released from the protein are first labeled through enzymatic incorporation of fluorophore-conjugated sialic acid or fucose, then separated by SDS-PAGE, and finally visualized with a fluorescent imager. To identify the labeled glycans of a fingerprint, glycan standards and glycan ladders are enzymatically generated and run alongside the samples as references. By comparing the mobility of a labeled glycan to that of a glycan standard, the identity of glycans maybe determined. O-glycans can also be fingerprinted. Due to the lack of an enzyme for broad O-glycan release, O-glycans on the S protein can be labeled with fluorescent sialic acid and digested with trypsin to obtain labeled glycan peptides that are then separated by gel electrophoresis. Glycan fingerprinting could serve as a quick method for globally assessing the glycosylation of a specific glycoprotein.

scholarly journals Fluorescent Glycan Fingerprinting of SARS2 Spike Proteins

2021 ◽  
Author(s):  
Zhengliang L Wu ◽  
James M Ertelt
Keyword(s):  
Sialic Acid ◽  
Causative Agent ◽  
Gel Electrophoresis ◽  
Global Assessment ◽  
Fluorescent Labeling ◽  
Biological Functions ◽  
Post Translational Modification ◽  
New Techniques ◽  
The World ◽  
Novel Coronavirus

AbstractGlycosylation is the most common post-translational modification and has myriad biological functions. However, glycan analysis and research has always been a challenge. Here, we would like to present new techniques of glycan fingerprinting based on enzymatic fluorescent labeling and gel electrophoresis. The method is illustrated on SARS-2 spike (S) glycoproteins. SARS-2, a novel coronavirus and the causative agent of COVID-19 pandemic, has devastated the world since the end of 2019. To obtain the N-glycan fingerprint of a S protein, glycans released from the protein are first labeled through enzymatic incorporation of fluorophore-conjugated sialic acid or fucose, and then separated on acrylamide gel through electrophoresis, and finally visualized with a fluorescent imager. To identify the labeled glycans of a fingerprint, glycan standards and glycan ladders that are enzymatically generated are run alongside the samples as references. By comparing the mobility of a labeled glycan to that of a glycan standard, the identity of glycans maybe determined. Due to lack of enzyme for broad O-glycans releasing, O-glycans on the RBD protein are labeled with fluorescent sialic acid and digested with trypsin to obtain labeled glycan peptides that are then separated on gel. Glycan fingerprinting could serve as a quick way for global assessment of the glycosylation of a glycoprotein.

scholarly journals Conservation analysis and screening of Ayurvedic compounds against SARS-CoV-2 Spike protein

2020 ◽  
Author(s):  
Zarrin Basharat ◽  
Muhammad Jahanzaib ◽  
Noor Rahman ◽  
Ishtiaq Ahmad Khan ◽  
Azra Yasmin
Keyword(s):  
Caspase 3 ◽  
Spike Protein ◽  
The Novel ◽  
Post Translational Modification ◽  
S Protein ◽  
Conservation Analysis ◽  
The Past ◽  
Online Tools ◽  
Novel Coronavirus ◽  
Over Time

Abstract Recent infections caused by the novel coronavirus (SARS-CoV-2) have led to global panic and mortality. Here, we analyzed the spike (S) protein of this virus using bioinformatics tools. We aimed to determine relative changes among different coronavirus species over the past two decades and to understand the conservation of the S-protein. Representative sequences of coronaviruses were collected from humans and other animals between 2000 and 2020. Evolutionary analyses found that the S-protein did not evolve overnight, but rather continuously over time. Post-translational modification (PTM) analysis using online tools and virtual screening of S-protein against a phytochemical database of Ayurvedic medicinal compounds (n = 2103) identified the S-protein inhibitors. Among these, top ranked were Gingerol (IUPAC name: 4'-Me ether, 3,5-di-Ac 3,5-di-Gingerdiols), 1-(5-Butyltetrahydro-2-furanyl)-2-hexacosanone and Ginsenoyne N ginseng that stimulates Caspase-3, Caspase-8, and the immune system. Gingerol is found in the fresh ginger and has reputation of being a potent antiviral. These compounds might prove useful to design drugs against COVID-19.

scholarly journals Expression of melanotransferrin isoforms in human serum: relevance to Alzheimer's disease

2003 ◽  
Vol 374 (2) ◽  
pp. 463-471 ◽  
Author(s):  
Richard R. DESROSIERS ◽  
Yanick BERTRAND ◽  
Quynh-Tran NGUYEN ◽  
Michel DEMEULE ◽  
Reinhard GABATHULER ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Human Serum ◽  
Gel Electrophoresis ◽  
Post Translational Modification ◽  
Two Dimensional Gel Electrophoresis ◽  
Control Subjects ◽  
Bivalent Cations ◽  
Sds Page ◽  
High Level

Levels of soluble melanotransferrin in serum have been reported to be higher in patients with Alzheimer's disease than in control subjects. The present study investigated melanotransferrin in human body fluids in the light of these findings. To clarify the correlation between melanotransferrin and Alzheimer's disease, the melanotransferrin content was determined by non-reducing, denaturing SDS/PAGE and Western blotting. Under these conditions, serum melanotransferrin migrated at 79 and 82 kDa. Melanotransferrin antigenicity and the relative proportions of the two forms were very sensitive to factors that altered its conformation, including disulphide bridges, pH and bivalent cations. Serum melanotransferrin levels were not significantly different between control subjects and patients with Alzheimer's disease using whole serum, EDTA-supplemented serum or serum immunoglobulin-depleted by Protein G–Sepharose and enriched by affinity precipitation with the lectin from Asparagus pea. Glycosylated forms of serum melanotransferrin bound to Asparagus lectin manifested similar patterns on two-dimensional gel electrophoresis in samples from controls and Alzheimer's disease subjects. Melanotransferrin was also present in saliva and at a high level in urine, but contents were similar in controls and patients with Alzheimer's disease. Together, these results demonstrate that serum melanotransferrin exists in various conformations depending on the binding of bivalent cations or following post-translational modification. These data also indicate that human serum melanotransferrin levels are unchanged in subjects with Alzheimer's disease.

Polymerization Sties Of Desialated Fibrinogen

1981 ◽  
Author(s):  
P M Allison ◽  
N U Bang
Keyword(s):  
Sialic Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Clotting Time ◽  
Mean Values ◽  
Fibrin Monomer ◽  
Sds Page

It has long been known (Laki and Chandrasekhar, Nature, 197:1267,1963) that the enzymatic removal of sialic acid (SA) frcm fibrinogen (F) shortens the thrccabin clotting time. Martinez et al. (J. Lab. Clin. Med., 89:367, 1977) demonstrated that this phenomenon is due to enhanced fibrin monomer polymerization. To more accurately identify the mechanisms involved we examined the binding of normal F, V. cholerae neuraminidase - treated F (NF) and their plasmic digests. Mean SA content of F of 4.5 residues / molecule decreased to unmeasurable levels in NF with a concomitant 10s shortening of the thrombin clotting time. F, NF, and plasmin digests of F (FP) and NF (NFP) were subjected to fibrin-monomer-Sepharose chromatography (FMSC) essentially according to Kudryk and Blcmback (J.Biol. Chan., 249:3322, 1974) and effluent fractions analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. Mean values for F retained on FMSC and eluted with acid urea buffer was 61%, for NF 86%, for for FP 65%, for NFP 78%. In NFP and FP 3 fragments D (D1, D2 D3) of Mr 103K, 89K, and 83K were discerned by SDS-PAGE. D1 in FP and NFP was almost quantitatively retained on FMSC, whereas little, if any, D3 was retained frcm either digest. FMSC resulted in poor retention of D2 for FP and virtually complete retention of this fragment from NFP. Fragments E were not retained on FMSC of either FP or NFP. The data suggests that desialation of F exposes additional polymerization sites present in D2 but not D3 of F.

scholarly journals The Effect of Age on the Glycosylation of Human Alpha-2-Macroglobulin

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2800-2800
Author(s):  
Laura S.E. Calvert ◽  
Helen M. Atkinson ◽  
Leslie R. Berry ◽  
Anthony K.C. Chan
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Western Blotting ◽  
Acid Content ◽  
Gel Electrophoresis ◽  
Ricinus Communis ◽  
Apparent Molecular Weight ◽  
Plasma Samples ◽  
Sialic Acid Content ◽  
Sds Page

Abstract Introduction: Alpha-2-macroglobulin (a2m) is a plasma glycoprotein capable of inhibiting all classes of serine proteases. Within the coagulation and fibrinolytic systems, a2m inhibits several critical factors, including thrombin, plasmin, and activated protein C. Previous studies have shown that coagulation factor concentrations are highly variable with age. Notably, a2m levels are approximately twice as high in newborns compared to adults. Elevated levels of a2m in newborns may, in part, contribute to a resistance towards thrombotic events observed in this population. Protein glycosylation is known to affect protein activity as well as pharmacodynamics. The glycosylation profile of adult a2m has previously been analyzed and shown to contain 8 potential sites of N-linked glycosylation. Information regarding glycosylation of a2m in other age groups has yet to be elucidated. Therefore, the purpose of this study is to examine the differences in the glycosylation profiles between newborn and adult a2m. Methods: Normal adult pooled platelet-poor plasma was obtained commercially. Newborn platelet-poor plasma was isolated by standard methods from umbilical cord blood obtained immediately after delivery of healthy full-term infants. Pooled newborn plasma was constructed from 30 different donors. To evaluate the degree of N-linked glycosylation, plasma samples were enzymatically deglycosylated by peptide N-glycosidase F (PNGaseF). Samples were subjected to SDS polyacrylamide (5%) gel electrophoresis (SDS PAGE) and western blotting to detect a2m in plasma. To evaluate a2m sialic acid content, plasma samples were incubated with Neuraminidase from Clostridium perfringens. Following incubation, samples were subjected to native polyacrylamide (4%) gel electrophoresis and western blotting. To detect the presence of non-sialylated terminal galactose residues, plasma samples were incubated with immobilized Ricinus communis lectin, and lectin-bound molecules were separated from unbound molecules by centrifugation. Bound and unbound fractions were subjected to SDS-PAGE and western blotting as described above to detect a2m. Results: Deglycosylation of both newborn and adult a2m with PNGaseF resulted in a change in migration and apparent molecular weight on SDS-PAGE. There was no significant difference (p=0.28, n=3) between the change in apparent molecular weight for newborn a2m (16.1 ± 0.42 kDa) versus adult a2m (14.5 ± 0.99 kDa). On native polyacrylamide gel electrophoresis, newborn a2m exhibited slightly increased migration compared to adult a2m. This difference in migration was abolished following treatment with neuraminidase, indicating the variation in migration was due to differing sialic acid content. Additionally, a lower proportion of newborn a2m was bound to Ricinus communis compared to adult a2m. This indicates newborn a2m has fewer non-sialylated galactose residues available for binding to this lectin. Conclusions: To our knowledge, this is the first study investigating potential glycan heterogeneity between newborn and adult a2m molecules. The results from PNGaseF analyses indicate that there is no macroheterogeneity in total N-glycan content apparent between newborn and adult a2m. However, the observed increase in sialic acid content of newborn a2m compared to adult a2m may be responsible for age-related differences in pharmacodynamic and pharmacokinetic properties of this important protease inhibitor. Disclosures No relevant conflicts of interest to declare.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

Molecular Forms of Human Protein C: Comparison and Distribution in Human Adult Plasma

1989 ◽  
Vol 62 (03) ◽  
pp. 902-905 ◽  
Author(s):  
Brian S Greffe ◽  
Marilyn J Manco-Johnson ◽  
Richard A Marlar
Keyword(s):  
Heavy Chain ◽  
Protein C ◽  
Molecular Forms ◽  
Post Translational Modification ◽  
Single Chain ◽  
Beta Form ◽  
Sds Page ◽  
Dependent Protein ◽  
Adult Plasma ◽  
The Difference

SummaryProtein C (PC) is a vitamin K-dependent protein which functions as both an anticoagulant and profibrinolytic. It is synthesized as a single chain protein (SC-PC) and post-transla-tionally modified into a two chain form (2C-PC). Two chain PC consists of a light chain (LC) and a heavy chain (HC). The present study was undertaken to determine the composition of the molecular forms of PC in plasma. PC was immunoprecipitated, subjected to SDS-PAGE and Western blotting. The blots were scanned by densitometry to determine the distribution of the various forms. The percentage of SC-PC and 2C-PC was found to be 10% and 90% respectively. This is in agreement with previous work. SC-PC and the heavy chain of 2C-PC consisted of three molecular forms (“alpha”, “beta”, and “gamma”). The “alpha” form of HC is the standard 2C form with a MW of 40 Kd. The “beta” form of HC has also been described and has MW which is 4 Kd less than the “alpha” form. The “gamma” species of the SC and 2C-PC has not been previously described. However, its 3 Kd difference from the “beta” form could be due to modification of the “beta” species or to a separate modification of the alpha-HC. The LC of PC was shown to exist in two forms (termed form 1 and form 2). The difference between these two forms is unknown. The molecular forms of PC are most likely due to a post-translational modification (either loss of a carbohydrate or a peptide) rather than from plasma derived degradation.

Identifikasi Daging Tikus Pada Produk Baso Dengan Metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

2018 ◽  
Vol 26 (2) ◽  
pp. 058
Author(s):  
Anna P. Roswiem ◽  
Triayu Septiani
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Sds Page

<em>Bahan<strong> </strong>baku untuk membuat baso adalah daging hewan, pada umumnya dari daging sapi, ayam, ikan dan babi. Di beberapa daerah di Indonesia terjadi kasus baso tikus. Tujuan penelitian ini adalah menguji ada tidaknya kandungan daging tikus pada produk baso yang dijual di pasar Cempaka Putih-Kecamatan Kramat Jakarta Pusat dan di pedagang baso atau mie baso di sekitar kampus Universitas YARSI Jakarta. Daging adalah protein salah satu metode untuk mengidentifikasi protein adalah metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE).<strong> </strong>Hasil penelitian menunjukkan bahwa dari 6 sampel baso terindikasi ada 2 sampel baso dengan nomor 1 dan 5 yang dibuat dari campuran daging sapi dan tikus; ada 1 sampel baso dengan nomor 6 yang terbuat dari daging tikus; dan 2 sampel baso dengan nomor 2 dan 3 yang terbuat dari campuran sapi  dan babi, dan hanya 1 sampel baso dengan nomor sampel 4 yang benar-benar terbuat dari daging sapi.</em>

scholarly journals Natural variants in SARS-CoV-2 Spike protein pinpoint structural and functional hotspots with implications for prophylaxis and therapeutic strategies

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Suman Pokhrel ◽  
Benjamin R. Kraemer ◽  
Scott Burkholz ◽  
Daria Mochly-Rosen
Keyword(s):  
Amino Acid ◽  
Severe Disease ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Severe Respiratory Distress ◽  
S Protein ◽  
Individual Sequences ◽  
Natural Variants ◽  
Novel Coronavirus ◽  
The Individual

AbstractIn December 2019, a novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the cause of pneumonia with severe respiratory distress and outbreaks in Wuhan, China. The rapid and global spread of SARS-CoV-2 resulted in the coronavirus 2019 (COVID-19) pandemic. Earlier during the pandemic, there were limited genetic viral variations. As millions of people became infected, multiple single amino acid substitutions emerged. Many of these substitutions have no consequences. However, some of the new variants show a greater infection rate, more severe disease, and reduced sensitivity to current prophylaxes and treatments. Of particular importance in SARS-CoV-2 transmission are mutations that occur in the Spike (S) protein, the protein on the viral outer envelope that binds to the human angiotensin-converting enzyme receptor (hACE2). Here, we conducted a comprehensive analysis of 441,168 individual virus sequences isolated from humans throughout the world. From the individual sequences, we identified 3540 unique amino acid substitutions in the S protein. Analysis of these different variants in the S protein pinpointed important functional and structural sites in the protein. This information may guide the development of effective vaccines and therapeutics to help arrest the spread of the COVID-19 pandemic.

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