scholarly journals Identification of the Pseudomonas aeruginosa 1244 Pilin Glycosylation Site

2002 ◽  
Vol 70 (6) ◽  
pp. 2837-2845 ◽  
Author(s):  
Jason E. Comer ◽  
Mark A. Marshall ◽  
Vincent J. Blanch ◽  
Carolyn D. Deal ◽  
Peter Castric
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Epitope ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Peptide Epitope ◽  
B Cell Epitope ◽  
Terminal Amino ◽  
Carboxy Terminal

ABSTRACT Previous work (P. Castric, F. J. Cassels, and R. W. Carlson, J. Biol. Chem. 276:26479-26485, 2001) has shown the Pseudomonas aeruginosa 1244 pilin glycan to be covalently bound to a serine residue. N-terminal sequencing of pilin fragments produced from endopeptidase treatment and identified by reaction with a glycan-specific monoclonal antibody indicated that the glycan was present between residue 75 and the pilin carboxy terminus. Further sequencing of these peptides revealed that serine residues 75, 81, 84, 105, 106, and 108 were not modified. Conversion of serine 148, but not serine 118, to alanine by site-directed mutagenesis, resulted in loss of the ability to carry out pilin glycosylation when tested in an in vivo system. These results showed the pilin glycan to be attached to residue 148, the carboxy-terminal amino acid. The carboxy-proximal portion of the pilin disulfide loop, which is adjacent to the pilin glycan, was found to be a major linear B-cell epitope, as determined by peptide epitope mapping analysis. Immunization of mice with pure pili produced antibodies that recognized the pilin glycan. These sera also reacted with P. aeruginosa 1244 lipopolysaccharide as measured by Western blotting and enzyme-linked immunosorbent assay.

scholarly journals Anti-CD3 Antibody Treatment Reduces Scar Formation in a Rat Model of Myocardial Infarction

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 295 ◽  
Author(s):  
Bernhard Wernly ◽  
Vera Paar ◽  
Achim Aigner ◽  
Patrick M Pilz ◽  
Bruno K Podesser ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
T Cell ◽  
Rat Model ◽  
Mirna Expression ◽  
Cell Epitope ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Treatment ◽  
Scar Size

Introduction: Antibody treatment with anti-thymocyte globulin (ATG) has been shown to be cardioprotective. We aimed to evaluate which single anti-T-cell epitope antibody alters chemokine expression at a level similar to ATG and identified CD3, which is a T-cell co-receptor mediating T-cell activation. Based on these results, the effects of anti-CD3 antibody treatment on angiogenesis and cardioprotection were tested in vitro and in vivo. Methods: Concentrations of IL-8 and MCP-1 in supernatants of human peripheral blood mononuclear cell (PBMC) cultures following distinct antibody treatments were evaluated by Enzyme-linked Immunosorbent Assay (ELISA). In vivo, anti-CD3 antibodies or vehicle were injected intravenously in rats subjected to acute myocardial infarction (AMI). Chemotaxis and angiogenesis were evaluated using tube and migration assays. Intracellular pathways were assessed using Western blot. Extracellular vesicles (EVs) were quantitatively evaluated using fluorescence-activated cell scanning, exoELISA, and nanoparticle tracking analysis. Also, microRNA profiles were determined by next-generation sequencing. Results: Only PBMC stimulation with anti-CD3 antibody led to IL-8 and MCP-1 changes in secretion, similar to ATG. In a rat model of AMI, systemic treatment with an anti-CD3 antibody markedly reduced infarct scar size (27.8% (Inter-quartile range; IQR 16.2–34.9) vs. 12.6% (IQR 8.3–27.2); p < 0.01). The secretomes of anti-CD3 treated PBMC neither induced cardioprotective pathways in cardiomyocytes nor pro-angiogenic mechanisms in human umbilical vein endothelial cell (HUVECs) in vitro. While EVs quantities remained unchanged, PBMC incubation with an anti-CD3 antibody led to alterations in EVs miRNA expression. Conclusion: Treatment with an anti-CD3 antibody led to decreased scar size in a rat model of AMI. Whereas cardioprotective and pro-angiogenetic pathways were unaltered by anti-CD3 treatment, qualitative changes in the EVs miRNA expression could be observed, which might be causal for the observed cardioprotective phenotype. We provide evidence that EVs are a potential cardioprotective treatment target. Our findings will also provide the basis for a more detailed analysis of putatively relevant miRNA candidates.

Characterization of monoclonal antibodies recognizing each extracellular loop domain of occludin

2019 ◽  
Vol 166 (4) ◽  
pp. 297-308 ◽  
Author(s):  
Yoshimi Shimizu ◽  
Yoshitaka Shirasago ◽  
Takeru Suzuki ◽  
Tomoyuki Hata ◽  
Masuo Kondoh ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Transmembrane Protein ◽  
Site Directed Mutagenesis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Intact Cells ◽  
Flow Cytometric ◽  
Junction Protein ◽  
Fcm Analysis

Abstract The tight junction protein occludin (OCLN) is a four-pass transmembrane protein with two extracellular loops (ELs), and also functions as a co-receptor for hepatitis C virus (HCV). Recently, we reported the establishment of monoclonal antibodies (mAbs) recognizing each intact EL domain of OCLN that can strongly prevent HCV infection in vitro and in vivo, and these mAbs were applicable for flow cytometric (FCM) analysis, immunocytochemistry (ICC) and cell-based enzyme-linked immunosorbent assay. In the present study, we further examined the application of these anti-OCLN mAbs and characterized their binding properties. All four mAbs were available for immunoprecipitation. The three first EL (EL1)-recognizing mAbs were applicable for immunoblotting, but the second EL (EL2)-recognizing one was not. Using site-directed mutagenesis, we also determined residues of OCLN critical for recognition by each mAb. Our findings showed that the small loop between two cysteines of the EL2 domain is essential for the binding to one EL2-recognizing mAb and that the recognition regions by three EL1-recognizing mAbs overlap, but are not the same sites of EL1. To obtain a deeper understanding of OCLN biology and its potential as a therapeutic target, specific mAbs to detect or target OCLN in intact cells should be powerful tools for future studies.

scholarly journals Mutation of N-glycosylation Sites in Salmonid Alphavirus (SAV) Envelope Proteins Attenuate the Virus in Cell Culture

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1071
Author(s):  
Ida Aksnes ◽  
Turhan Markussen ◽  
Stine Braaen ◽  
Espen Rimstad
Keyword(s):  
Cell Culture ◽  
Infectious Clone ◽  
Envelope Proteins ◽  
Glycosylation Site ◽  
Viral Envelope ◽  
Site Directed Mutagenesis ◽  
Complete Inactivation ◽  
Immunization Strategies ◽  
Viral Virulence

Salmonid alphavirus (SAV) is the cause of pancreas disease and sleeping disease in farmed salmonid fish in Europe. The spread of these diseases has been difficult to control with biosecurity and current vaccination strategies, and increased understanding of the viral pathogenesis could be beneficial for the development of novel vaccine strategies. N-glycosylation of viral envelope proteins may be crucial for viral virulence and a possible target for its purposed attenuation. In this study, we mutated the N-glycosylation consensus motifs of the E1 and E2 glycoproteins of a SAV3 infectious clone using site-directed mutagenesis. Mutation of the glycosylation motif in E1 gave a complete inactivation of the virus as no viral replication could be detected in cell culture and infectious particles could not be rescued. In contrast, infectious virus particles could be recovered from the SAV3 E2 mutants (E2319Q, E2319A), but not if they were accompanied by lack of N-glycosylation in E1. Compared to the non-mutated infectious clone, the SAV3-E2319Q and SAV3-E2319A recombinant viruses produced less cytopathic effects in cell culture and lower amounts of infectious viral particles. In conclusion, the substitution in the N-linked glycosylation site in E2 attenuated SAV3 in cell culture. The findings could be useful for immunization strategies using live attenuated vaccines and testing in fish will be desirable to study the clone’s properties in vivo.

scholarly journals The Overlapping angB and angG Genes Are Encoded within the trans-Acting Factor Region of the Virulence Plasmid in Vibrio anguillarum: Essential Role in Siderophore Biosynthesis

2000 ◽  
Vol 182 (23) ◽  
pp. 6762-6773 ◽  
Author(s):  
Timothy J. Welch ◽  
Sunghee Chai ◽  
Jorge H. Crosa
Keyword(s):  
Iron Transport ◽  
Vibrio Anguillarum ◽  
Site Directed Mutagenesis ◽  
Virulence Plasmid ◽  
Dihydroxybenzoic Acid ◽  
Lyase Activity ◽  
Carboxy Terminal ◽  
Regulatory Functions ◽  
Maximal Expression

Products encoded in the trans-acting factor (TAF) region are necessary for the biosynthesis of anguibactin and for maximal expression of iron transport and biosynthesis genes in the plasmid-encoded iron-scavenging system of Vibrio anguillarum. Here we identify angB, a locus located in the TAF region, which encodes products essential for anguibactin biosynthesis. We demonstrate that a 287-amino-acid polypeptide, encoded by angB and designated AngB, has an isochorismate lyase activity necessary for the synthesis of 2,3-dihydroxybenzoic acid, an anguibactin biosynthesis intermediate. Complementation of variousangB mutations provided evidence that an additional, overlapping gene exists at this locus. This second gene, designatedangG, also has an essential biosynthetic function. TheangG gene directs the expression of three polypeptides when overexpressed in Escherichia coli, all of which are translated in the same frame as AngB. The results of site-directed mutagenesis and in vivo phosphorylation experiments suggest that the carboxy-terminal end of AngB and the AngG polypeptide(s) function as aryl carrier proteins involved in the assembly of the anguibactin molecule. Our results also show that the regulatory functions of the TAF are encoded in a region, TAFr, which is distinct from and independent of the angB and angG genes.

scholarly journals N-glycosylation requirements for the AT1a angiotensin II receptor delivery to the plasma membrane

1999 ◽  
Vol 339 (2) ◽  
pp. 397-405 ◽  
Author(s):  
Benoit DESLAURIERS ◽  
Cecilia PONCE ◽  
Colette LOMBARD ◽  
Renée LARGUIER ◽  
Jean-Claude BONNAFOUS ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Cellular Localization ◽  
Inositol Phosphate ◽  
Glycosylation Site ◽  
Receptor Expression ◽  
Site Directed Mutagenesis ◽  
Pharmacological Properties ◽  
Peptide Epitope ◽  
Glycosylation Sites

The purpose of this work was to investigate the role of N-glycosylation in the expression and pharmacological properties of the the rat AT1a angiotensin II (AII) receptor. Glycosylation-site suppression was carried out by site-directed mutagenesis (Asn → Gln) of Asn176 and Asn188 (located on the second extracellular loop) and by the removal of Asn4 at the N-terminal end combined with the replacement of the first four amino acids by a 10 amino acid peptide epitope (c-Myc). We generated seven possible N-glycosylation-site-defective mutants, all tagged at their C-terminal ends with the c-Myc epitope. This double-tagging strategy, associated with photoaffinity labelling, allowed evaluation of the molecular masses and immunocytochemical cellular localization of the various receptors transiently expressed in COS-7 cells. We showed that: (i) each of the three N-glycosylation sites are utilized in COS-7 cells; (ii) the mutant with three defective N-glycosylation sites was not (or was very inefficiently) expressed at the plasma membrane and accumulated inside the cell at the perinuclear zone; (iii) the preservation of two sites allowed normal receptor delivery to the plasma membrane, the presence of only Asn176 ensuring a behaviour similar to that of the wild-type receptor; and (iv) all expressed receptors displayed unchanged pharmacological properties (Kd for 125I-sarcosine1-AII; sarcosine1-AII-induced inositol phosphate production). These results demonstrate that N-glycosylation is required for the AT1 receptor expression. They are discussed in the light of current knowledge of membrane-protein maturation and future prospects of receptor overexpression for structural studies.

scholarly journals Synthetic Fragments of Vibrio cholerae O1 Inaba O-Specific Polysaccharide Bound to a Protein Carrier Are Immunogenic in Mice but Do Not Induce Protective Antibodies

2004 ◽  
Vol 72 (7) ◽  
pp. 4090-4101 ◽  
Author(s):  
Michael D. Meeks ◽  
Rina Saksena ◽  
Xingquan Ma ◽  
Terri K. Wade ◽  
Ronald K. Taylor ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Cell Epitope ◽  
Immunoglobulin M ◽  
Hydroxyl Group ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protective Antibody ◽  
Infant Mouse ◽  
Molar Ratios ◽  
Specific Polysaccharide

ABSTRACT Development of Vibrio cholerae lipopolysaccharide (LPS) as a cholera vaccine immunogen is justified by the correlation of vibriocidal anti-LPS response with immunity. Two V. cholerae O1 LPS serotypes, Inaba and Ogawa, are associated with endemic and pandemic cholera. Both serotypes induce protective antibody following infection or vaccination. Structurally, the LPSs that define the serotypes are identical except for the terminal perosamine moiety, which has a methoxyl group at position 2 in Ogawa but a hydroxyl group in Inaba. The terminal sugar of the Ogawa LPS is a protective B-cell epitope. We chemically synthesized the terminal hexasaccharides of V. cholerae serotype Ogawa, which comprises in part the O-specific polysaccharide component of the native LPS, and coupled the oligosaccharide at different molar ratios to bovine serum albumin (BSA). Our initial studies with Ogawa immunogens showed that the conjugates induced protective antibody. We hypothesized that antibodies specific for the terminal sugar of Inaba LPS would also be protective. Neoglycoconjugates were prepared from synthetic Inaba oligosaccharides (disaccharide, tetrasaccharide, and hexasaccharide) and BSA at different levels of substitution. BALB/c mice responded to the Inaba carbohydrate (CHO)-BSA conjugates with levels of serum antibodies of comparable magnitude to those of mice immunized with Ogawa CHO-BSA conjugates, but the Inaba-specific antibodies (immunoglobulin M [IgM] and IgG1) were neither vibriocidal nor protective in the infant mouse cholera model. We hypothesize that the anti-Inaba antibodies induced by the Inaba CHO-BSA conjugates have enough affinity to be screened via enzyme-linked immunosorbent assay but not enough to be protective in vivo.

Processing and trafficking of cysteine proteases in Leishmania mexicana

2000 ◽  
Vol 113 (22) ◽  
pp. 4035-4041 ◽  
Author(s):  
D.R. Brooks ◽  
L. Tetley ◽  
G.H. Coombs ◽  
J.C. Mottram
Keyword(s):  
Cysteine Protease ◽  
Mammalian Cells ◽  
Protozoan Parasite ◽  
Cysteine Proteases ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Flagellar Pocket ◽  
Leishmania Mexicana

Removal of the pro-domain of a cysteine protease is essential for activation of the enzyme. We have engineered a cysteine protease (CPB2.8) of the protozoan parasite Leishmania mexicana by site-directed mutagenesis to remove the active site cysteine (to produce CPB(C25G)). When CPB(C25G) was expressed in a L. mexicana mutant lacking all CPB genes, the inactive pro-enzyme was processed to the mature protein and trafficked to the lysosome. These results show that auto-activation is not required for correct processing of CPB in vivo. When CPB(C25G) was expressed in a L. mexicana mutant lacking both CPA and CPB genes, the majority of the pro-enzyme remained unprocessed and accumulated in the flagellar pocket. These data reveal that CPA can directly or indirectly process CPB(C25G) and suggest that cysteine proteases are targeted to lysosomes via the flagellar pocket. Moreover, they show that another protease can process CPB in the absence of either CPA or CPB, albeit less efficiently. Abolition of the glycosylation site in the mature domain of CPB did not affect enzyme processing, targeting or in vitro activity towards gelatin. This indicates that glycosylation is not required for trafficking. Together these findings provide evidence that the major route of trafficking of Leishmania cysteine proteases to lysosomes is via the flagellar pocket and therefore differs significantly from cysteine protease trafficking in mammalian cells.

Carboxypeptidase-catalyzed hydrolysis of C-terminal lysine: mechanism for in vivo production of multiple forms of creatine kinase in plasma.

1984 ◽  
Vol 30 (5) ◽  
pp. 662-664 ◽  
Author(s):  
M B Perryman ◽  
J D Knell ◽  
R Roberts
Keyword(s):  
Creatine Kinase ◽  
Specific Substrate ◽  
Multiple Forms ◽  
Carboxypeptidase B ◽  
Creatine Kinase Isoenzyme ◽  
Terminal Amino ◽  
Carboxy Terminal ◽  
Hydrolysis Of ◽  
Positively Charged

Abstract Human myocardial creatine kinase isoenzyme MM is present as a single form in tissue, but upon its release into plasma two additional forms, with faster anodal migration, are apparent on polyacrylamide electrophoresis. We designate the three forms as MM3, MM2, and MM1 in increasing order of anodal mobility. When tissue creatine kinase isoenzyme MM (MM3) is incubated with either carboxypeptidase N or carboxypeptidase B it is converted into the two additional forms, MM2 and MM1. The carboxy terminal amino acid of human, canine, and rabbit tissue MM3 was determined to be lysine, a specific substrate for carboxypeptidases N and B. Evidently the mechanism for the production of multiple forms of creatine MM in human plasma is the hydrolysis of a positively charged C-terminal lysine residue from one M subunit (MM2), followed by hydrolysis of the C-terminal lysine from the other subunit (MM1).

Glycosylation decreases aggregation and immunogenicity of adalimumab fab secreted from Pichia pastoris

2020 ◽  
Author(s):  
Hitomi Nakamura ◽  
Masato Kiyoshi ◽  
Makoto Anraku ◽  
Noritaka Hashii ◽  
Naoko Oda-Ueda ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Half Life ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Induced Stress ◽  
Glycan Chain ◽  
Elimination Half ◽  
Pharmacokinetic Behavior

Abstract Glycoengineering of therapeutic proteins has been applied to improve the clinical efficacy of several therapeutics. Here, we examined the effect of glycosylation on the properties of the Fab of the therapeutic antibody, adalimumab. An N-glycosylation site was introduced at position 178 of the H-chain constant region of adalimumab Fab through site-directed mutagenesis (H: L178N Fab), and the H: L178N Fab was produced in Pichia pastoris. Expressed mutant Fab contained long and short glycan chains (L-glyco Fab and S-glyco Fab, respectively). Under the condition of aggregation of Fab upon pH shift-induced stress, both of L-glyco Fab and S-glyco Fab were less prone to aggregation, with L-glyco Fab suppressing aggregation more effectively than the S-glyco Fab. Moreover, the comparison of the antigenicity of glycosylated and wild-type Fabs in mice revealed that glycosylation resulted in the suppression of antigenicity. Analysis of the pharmacokinetic behavior of the Fab, L-glyco Fab, and S-glyco Fab indicated that the half-lives of glycosylated Fabs in the rats were shorter than that of wild-type Fab, with L-glyco Fab having a shorter half-life than S-glyco Fab. Thus, we demonstrated that the glycan chain influences Fab aggregation and immunogenicity, and glycosylation reduces the elimination half-life in vivo.

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