scholarly journals Sites of Vulnerability on Ricin B Chain Revealed through Epitope Mapping of Toxin-Neutralizing Monoclonal Antibodies

2020 ◽  
Author(s):  
David J. Vance ◽  
Amanda Y. Poon ◽  
Nicholas J. Mantis
Keyword(s):  
Monoclonal Antibodies ◽  
Mammalian Cells ◽  
Type I ◽  
Type Ii ◽  
Single Chain ◽  
B Subunit ◽  
Carbohydrate Recognition Domains ◽  
Efficient Uptake ◽  
And Function ◽  
Golgi Network

AbstractRicin toxin’s B subunit (RTB) is a multifunctional galactose (Gal)-/N-acetylgalactosamine (GalNac)-specific lectin that promotes efficient uptake and intracellular trafficking of ricin’s ribosome-inactivating subunit (RTA) into mammalian cells. Structurally, RTB consists of two globular domains (RTB-D1, RTB-D2), each divided into three homologous sub-domains (α, β, γ). The two carbohydrate recognition domains (CRDs) are situated on opposite sides of RTB (sub-domains 1α and 2γ) and function non-cooperatively. Previous studies have revealed two distinct classes of toxin-neutralizing, anti-RTB monoclonal antibodies (mAbs). Type I mAbs, exemplified by SylH3, inhibit (∼90%) toxin attachment to cell surfaces, while type II mAbs, epitomized by 24B11, interfere with intracellular toxin transport between the plasma membrane and the trans-Golgi network (TGN). Localizing the epitopes recognized by these two classes of mAbs has proven difficult, in part because of RTB’s duplicative structure. To circumvent this problem, full-length RTB or the two individual domains, RTB-D1 and RTB-D2, were expressed as pIII fusion proteins on the surface of filamentous phage M13 and subsequently used as “bait” in mAb capture assays. The results indicated that SylH3 captured RTB-D1, while 24B11 captured RTB-D2. Analysis of additional toxin-neutralizing and non-neutralizing mAbs along with single chain antibodies (VHHs) known to compete with SylH3 or 24B11 confirmed these domain assignments. These results not only indicate that so-called type I and type II mAbs segregate on the basis of domain specificity, but suggest that RTB’s two domains may contribute to distinct steps in the intoxication pathway.

scholarly journals Sites of vulnerability on ricin B chain revealed through epitope mapping of toxin-neutralizing monoclonal antibodies

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0236538
Author(s):  
David J. Vance ◽  
Amanda Y. Poon ◽  
Nicholas J. Mantis
Keyword(s):  
Monoclonal Antibodies ◽  
Mammalian Cells ◽  
Type I ◽  
Dependent Manner ◽  
Type Ii ◽  
Single Chain ◽  
Carbohydrate Recognition Domains ◽  
And Function ◽  
Golgi Network ◽  
Dose Dependent

Ricin toxin’s B subunit (RTB) is a multifunctional galactose (Gal)-/N-acetylgalactosamine (GalNac)-specific lectin that promotes uptake and intracellular trafficking of ricin’s ribosome-inactivating subunit (RTA) into mammalian cells. Structurally, RTB consists of two globular domains (RTB-D1, RTB-D2), each divided into three homologous sub-domains (α, β, γ). The two carbohydrate recognition domains (CRDs) are situated on opposite sides of RTB (sub-domains 1α and 2γ) and function non-cooperatively. Previous studies have revealed two distinct classes of toxin-neutralizing, anti-RTB monoclonal antibodies (mAbs). Type I mAbs, exemplified by SylH3, inhibit (~90%) toxin attachment to cell surfaces, while type II mAbs, epitomized by 24B11, interfere with intracellular toxin transport between the plasma membrane and the trans-Golgi network (TGN). Localizing the epitopes recognized by these two classes of mAbs has proven difficult, in part because of RTB’s duplicative structure. To circumvent this problem, RTB-D1 and RTB-D2 were expressed as pIII fusion proteins on the surface of filamentous phage M13 and subsequently used as “bait” in mAb capture assays. We found that SylH3 captured RTB-D1 (but not RTB-D2) in a dose-dependent manner, while 24B11 captured RTB-D2 (but not RTB-D1) in a dose-dependent manner. We confirmed these domain assignments by competition studies with an additional 8 RTB-specific mAbs along with a dozen a single chain antibodies (VHHs). Collectively, these results demonstrate that type I and type II mAbs segregate on the basis of domain specificity and suggest that RTB’s two domains may contribute to distinct steps in the intoxication pathway.

scholarly journals Biochemical and structural characterization of the novel sialic acid-binding site of Escherichia coli heat-labile enterotoxin LT-IIb

2016 ◽  
Vol 473 (21) ◽  
pp. 3923-3936 ◽  
Author(s):  
Dani Zalem ◽  
João P. Ribeiro ◽  
Annabelle Varrot ◽  
Michael Lebens ◽  
Anne Imberty ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Cholera Toxin ◽  
Carbohydrate Binding ◽  
Type I ◽  
Type Ii ◽  
E Coli ◽  
B Subunit ◽  
Heat Labile ◽  
B Subunits

The structurally related AB5-type heat-labile enterotoxins of Escherichia coli and Vibrio cholerae are classified into two major types. The type I group includes cholera toxin (CT) and E. coli LT-I, whereas the type II subfamily comprises LT-IIa, LT-IIb and LT-IIc. The carbohydrate-binding specificities of LT-IIa, LT-IIb and LT-IIc are distinctive from those of cholera toxin and E. coli LT-I. Whereas CT and LT-I bind primarily to the GM1 ganglioside, LT-IIa binds to gangliosides GD1a, GD1b and GM1, LT-IIb binds to the GD1a and GT1b gangliosides, and LT-IIc binds to GM1, GM2, GM3 and GD1a. These previous studies of the binding properties of type II B-subunits have been focused on ganglio core chain gangliosides. To further define the carbohydrate binding specificity of LT-IIb B-subunits, we have investigated its binding to a collection of gangliosides and non-acid glycosphingolipids with different core chains. A high-affinity binding of LT-IIb B-subunits to gangliosides with a neolacto core chain, such as Neu5Gcα3- and Neu5Acα3-neolactohexaosylceramide, and Neu5Gcα3- and Neu5Acα3-neolactooctaosylceramide was detected. An LT-IIb-binding ganglioside was isolated from human small intestine and characterized as Neu5Acα3-neolactohexaosylceramide. The crystal structure of the B-subunit of LT-IIb with the pentasaccharide moiety of Neu5Acα3-neolactotetraosylceramide (Neu5Ac-nLT: Neu5Acα3Galβ4GlcNAcβ3Galβ4Glc) was determined providing the first information for a sialic-binding site in this subfamily, with clear differences from that of CT and LT-I.

scholarly journals Mutants of Type II Heat-Labile Enterotoxin LT-IIa with Altered Ganglioside-Binding Activities and Diminished Toxicity Are Potent Mucosal Adjuvants

2006 ◽  
Vol 75 (2) ◽  
pp. 621-633 ◽  
Author(s):  
Hesham F. Nawar ◽  
Sergio Arce ◽  
Michael W. Russell ◽  
Terry D. Connell
Keyword(s):  
Escherichia Coli ◽  
Immune Responses ◽  
Binding Activity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Type Ii ◽  
Heat Labile ◽  
Enhanced Expression ◽  
Adhesin Protein ◽  
And Function

ABSTRACT The structure and function LT-IIa, a type II heat-labile enterotoxin of Escherichia coli, are closely related to the structures and functions of cholera toxin and LT-I, the type I heat-labile enterotoxins of Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. While LT-IIa is a potent systemic and mucosal adjuvant, recent studies demonstrated that mutant LT-IIa(T34I), which exhibits no detectable binding activity as determined by an enzyme-linked immunosorbent assay, with gangliosides GD1b, GD1a, and GM1 is a very poor adjuvant. To evaluate whether other mutant LT-IIa enterotoxins that also exhibit diminished ganglioside-binding activities have greater adjuvant activities, BALB/c mice were immunized by the intranasal route with the surface adhesin protein AgI/II of Streptococcus mutans alone or in combination with LT-IIa, LT-IIa(T14S), LT-IIa(T14I), or LT-IIa(T14D). All three mutant enterotoxins potentiated strong mucosal immune responses that were equivalent to the response promulgated by wt LT-IIa. All three mutant enterotoxins augmented the systemic immune responses that correlated with their ganglioside-binding activities. Only LT-IIa and LT-IIa(T14S), however, enhanced expression of major histocompatibility complex class II and the costimulatory molecules CD40, CD80, and CD86 on splenic dendritic cells. LT-IIa(T14I) and LT-IIa(T14D) had extremely diminished toxicities in a mouse Y1 adrenal cell bioassay and reduced abilities to induce the accumulation of intracellular cyclic AMP in a macrophage cell line.

scholarly journals Human type II receptor for bone morphogenic proteins (BMPs): extension of the two-kinase receptor model to the BMPs.

1995 ◽  
Vol 15 (7) ◽  
pp. 3479-3486 ◽  
Author(s):  
F Liu ◽  
F Ventura ◽  
J Doody ◽  
J Massagué
Keyword(s):  
Transforming Growth Factor Beta ◽  
Mammalian Cells ◽  
Transforming Growth Factor ◽  
Transcriptional Response ◽  
Family Type ◽  
Type I ◽  
Type Ii ◽  
Receptor System ◽  
Bone Morphogenic Proteins ◽  
Bmp Receptors

Bone morphogenic proteins (BMPs) are universal regulators of animal development. We report the identification and cloning of the BMP type II receptor (BMPR-II), a missing component of this receptor system in vertebrates. BMPR-II is a transmembrane serine/threonine kinase that binds BMP-2 and BMP-7 in association with multiple type I receptors, including BMPR-IA/Brk1, BMPR-IB, and ActR-I, which is also an activin type I receptor. Cloning of BMPR-II resulted from a strong interaction of its cytoplasmic domain with diverse transforming growth factor beta family type I receptor cytoplasmic domains in a yeast two-hybrid system. In mammalian cells, however, the interaction of BMPR-II is restricted to BMP type I receptors and is ligand dependent. BMPR-II binds BMP-2 and -7 on its own, but binding is enhanced by coexpression of type I BMP receptors. BMP-2 and BMP-7 can induce a transcriptional response when added to cells coexpressing ActR-I and BMPR-II but not to cells expressing either receptor alone. The kinase activity of both receptors is essential for signaling. Thus, despite their ability to bind to type I and II receptors receptors separately, BMPs appear to require the cooperation of these two receptors for optimal binding and for signal transduction. The combinatorial nature of these receptors and their capacity to crosstalk with the activin receptor system may underlie the multifunctional nature of their ligands.

scholarly journals Fish type I and type II interferons: composition, receptor usage, production and function

2019 ◽  
Vol 12 (2) ◽  
pp. 773-804 ◽  
Author(s):  
Zhen Gan ◽  
Shan Nan Chen ◽  
Bei Huang ◽  
Jun Zou ◽  
Pin Nie
Keyword(s):  
Type I ◽  
Type Ii ◽  
Receptor Usage ◽  
And Function

scholarly journals Biosynthesis of Polyketides in Streptomyces

2019 ◽  
Vol 7 (5) ◽  
pp. 124 ◽  
Author(s):  
Chandra Risdian ◽  
Tjandrawati Mozef ◽  
Joachim Wink
Keyword(s):  
Secondary Metabolites ◽  
Structure And Function ◽  
Polyketide Synthases ◽  
Type I ◽  
Type Ii ◽  
Filamentous Form ◽  
Gram Positive Bacteria ◽  
Wide Range ◽  
Different Types ◽  
And Function

Polyketides are a large group of secondary metabolites that have notable variety in their structure and function. Polyketides exhibit a wide range of bioactivities such as antibacterial, antifungal, anticancer, antiviral, immune-suppressing, anti-cholesterol, and anti-inflammatory activity. Naturally, they are found in bacteria, fungi, plants, protists, insects, mollusks, and sponges. Streptomyces is a genus of Gram-positive bacteria that has a filamentous form like fungi. This genus is best known as one of the polyketides producers. Some examples of polyketides produced by Streptomyces are rapamycin, oleandomycin, actinorhodin, daunorubicin, and caprazamycin. Biosynthesis of polyketides involves a group of enzyme activities called polyketide synthases (PKSs). There are three types of PKSs (type I, type II, and type III) in Streptomyces responsible for producing polyketides. This paper focuses on the biosynthesis of polyketides in Streptomyces with three structurally-different types of PKSs.

GGA proteins: new players in the sorting game

2001 ◽  
Vol 114 (19) ◽  
pp. 3413-3418 ◽  
Author(s):  
Annette L. Boman
Keyword(s):  
Membrane Trafficking ◽  
Sequence Homology ◽  
Mammalian Cells ◽  
Gene Knockout ◽  
Protein Domains ◽  
Vesicle Formation ◽  
Trans Golgi Network ◽  
And Function ◽  
Golgi Network ◽  
Cargo Sorting

The GGA proteins are a novel family of proteins that were discovered nearly simultaneously by several labs studying very different aspects of membrane trafficking. Since then, several studies have described the GGA proteins and their functions in yeast and mammalian cells. Four protein domains are present in all GGA proteins, as defined by sequence homology and function. These different domains interact directly with ARF proteins, cargo and clathrin. Alteration of the levels of GGA proteins by gene knockout or overexpression affects specific trafficking events between the trans-Golgi network and endosomes. These data suggest that GGAs function as ARF-dependent, monomeric clathrin adaptors to facilitate cargo sorting and vesicle formation at the trans-Golgi network.

Disruption of Golgi structure and function in mammalian cells expressing a mutant dynamin

2000 ◽  
Vol 113 (11) ◽  
pp. 1993-2002 ◽  
Author(s):  
H. Cao ◽  
H.M. Thompson ◽  
E.W. Krueger ◽  
M.A. McNiven
Keyword(s):  
Mammalian Cells ◽  
Living Cells ◽  
Dominant Negative ◽  
Coated Vesicle ◽  
Large Numbers ◽  
Golgi Structure ◽  
And Function ◽  
Golgi Network ◽  
Mutant Cells

The large GTPase dynamin is a mechanoenzyme that participates in the scission of nascent vesicles from the plasma membrane. Recently, dynamin has been demonstrated to associate with the Golgi apparatus in mammalian cells by morphological and biochemical methods. Additional studies using a well characterized, cell-free assay have supported these findings by demonstrating a requirement for dynamin function in the formation of clathrin-coated, and non-clathrin-coated vesicles from the trans-Golgi network (TGN). In this study, we tested if dynamin participates in Golgi function in living cells through the expression of a dominant negative dynamin construct (K44A). Cells co-transfected to express this mutant dynamin and a GFP-tagged Golgi resident protein (TGN38) exhibit Golgi structures that are either compacted, vesiculated, or tubulated. Electron microscopy of these mutant cells revealed large numbers of Golgi stacks comprised of highly tubulated cisternae and an extraordinary number of coated vesicle buds. Cells expressing mutant dynamin and GFP-tagged VSVG demonstrated a marked retention (8- to 11-fold) of the nascent viral G-protein in the Golgi compared to control cells. These observations in living cells are consistent with previous morphological and in vitro studies demonstrating a role for dynamin in the formation of secretory vesicles from the TGN.

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