The carbohydrate-binding domain on galectin-1 is more extensive for a complex glycan than for simple saccharides: implications for galectin–glycan interactions at the cell surface

gal-1 (galectin-1) mediates cell–cell and cell–extracellular matrix adhesion, essentially by interacting with β-galactoside-containing glycans of cell-surface glycoconjugates. Although most structural studies with gal-1 have investigated its binding to simple carbohydrates, in particular lactose and N-acetyl-lactosamine, this view is limited, because gal-1 functions at the cell surface by interacting with more complex glycans that are heterogeneous in size and composition. In the present study we used NMR spectroscopy to investigate the interaction of human gal-1 with a large (120 kDa) complex glycan, GRG (galactorhamnogalacturonate glycan), that contains non-randomly distributed mostly terminal β(1→4)-linked galactose side chains. We used 15N–1H-HSQC (heteronuclear single quantum coherence) NMR experiments with 15N-enriched gal-1 to identify the GRG-binding region on gal-1 and found that this region covers a large surface area on gal-1 that includes the quintessential lactose-binding site and runs from that site through a broad valley or cleft towards the dimer interface. HSQC and pulsed-field-gradient NMR diffusion experiments also show that gal-1 binds GRG with a gal-1:GRG stoichiometry of about 5:1 (or 6:1) and with average macroscopic and microscopic equilibrium dissociation constants (Kd) of 8×10−6 M and 40×10−6 M (or 48×10−6 M) respectively, indicating stronger binding than to lactose (Kd=520×10−6 M). Although gal-1 may bind GRG in various ways, the glycan can be competed for by lactose, suggesting that there is one major mode of interaction. Furthermore, even though terminal motifs on GRG are Gal-β(1→4)-Gal rather than the traditional Gal-β(1→4)-Glc/GlcNAc (where GlcNAc is N-acetylglucosamine), we show that the disaccharide Gal-β(1→4)-Gal can bind gal-1 at the lactose-binding domain. In addition, gal-1 binding to GRG disrupts inter-glycan interactions and decreases glycan-mediated solution viscosity, a glycan decongestion effect that may help explain why gal-1 promotes membrane fluidity and lateral diffusion of glycoconjugates within cell membranes. Overall, our results provide an insight into the function of galectin in situ and have potential significant biological consequences.

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Identification of Carbohydrate-Binding Domains in the Attachment Proteins of Type 1 and Type 3 Reoviruses

Journal of Virology ◽

10.1128/jvi.74.18.8472-8479.2000 ◽

2000 ◽

Vol 74 (18) ◽

pp. 8472-8479 ◽

Cited By ~ 93

Author(s):

James D. Chappell ◽

Joy L. Duong ◽

Benjamin W. Wright ◽

Terence S. Dermody

Keyword(s):

Sialic Acid ◽

Cell Surface ◽

Receptor Binding ◽

Binding Domain ◽

Carbohydrate Binding ◽

Tail Domain ◽

Amino Terminal ◽

Binding Domains ◽

Type 3

ABSTRACT The reovirus attachment protein, ς1, is responsible for strain-specific patterns of viral tropism in the murine central nervous system and receptor binding on cultured cells. The ς1 protein consists of a fibrous tail domain proximal to the virion surface and a virion-distal globular head domain. To better understand mechanisms of reovirus attachment to cells, we conducted studies to identify the region of ς1 that binds cell surface carbohydrate. Chimeric and truncated ς1 proteins derived from prototype reovirus strains type 1 Lang (T1L) and type 3 Dearing (T3D) were expressed in insect cells by using a baculovirus vector. Assessment of expressed protein susceptibility to proteolytic cleavage, binding to anti-ς1 antibodies, and oligomerization indicates that the chimeric and truncated ς1 proteins are properly folded. To assess carbohydrate binding, recombinant ς1 proteins were tested for the capacity to agglutinate mammalian erythrocytes and to bind sialic acid presented on glycophorin, the cell surface molecule bound by type 3 reovirus on human erythrocytes. Using a panel of two wild-type and ten chimeric and truncated ς1 proteins, the sialic acid-binding domain of type 3 ς1 was mapped to a region of sequence proposed to form the more amino terminal of two predicted β-sheet structures in the tail. This unit corresponds to morphologic region T(iii) observed in computer-processed electron micrographs of ς1 protein purified from virions. In contrast, the homologous region of T1L ς1 sequence was not implicated in carbohydrate binding; rather, sequences in the distal portion of the tail known as the neck were required. Results of these studies demonstrate that a functional receptor-binding domain, which uses sialic acid as its ligand, is contained within morphologic region T(iii) of the type 3 ς1 tail. Furthermore, our findings indicate that T1L and T3D ς1 proteins contain different arrangements of receptor-binding domains.

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Cryo-EM structure of the full-length WzmWzt ABC transporter required for lipid-linked O antigen transport

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2016144118 ◽

2020 ◽

Vol 118 (1) ◽

pp. e2016144118

Author(s):

Christopher A. Caffalette ◽

Jochen Zimmer

Keyword(s):

Cell Surface ◽

Abc Transporter ◽

Bound State ◽

Binding Domain ◽

Full Length ◽

Carbohydrate Binding ◽

Aquifex Aeolicus ◽

O Antigen ◽

Lipid Environment ◽

O Antigens

O antigens are important cell surface polysaccharides in gram-negative bacteria where they extend core lipopolysaccharides in the extracellular leaflet of the outer membrane. O antigen structures are serotype specific and form extended cell surface barriers endowing many pathogens with survival benefits. In the ABC transporter-dependent biosynthesis pathway, O antigens are assembled on the cytosolic side of the inner membrane on a lipid anchor and reoriented to the periplasmic leaflet by the channel-forming WzmWzt ABC transporter for ligation to the core lipopolysaccharides. In many cases, this process depends on the chemical modification of the O antigen’s nonreducing terminus, sensed by WzmWzt via a carbohydrate-binding domain (CBD) that extends its nucleotide-binding domain (NBD). Here, we provide the cryo-electron microscopy structure of the full-length WzmWzt transporter fromAquifex aeolicusbound to adenosine triphosphate (ATP) and in a lipid environment, revealing a highly asymmetric transporter organization. The CBDs dimerize and associate with only one NBD. Conserved loops at the CBD dimer interface straddle a conserved peripheral NBD helix. The CBD dimer is oriented perpendicularly to the NBDs and its putative ligand-binding sites face the transporter to likely modulate ATPase activity upon O antigen binding. Further, our structure reveals a closed WzmWzt conformation in which an aromatic belt near the periplasmic channel exit seals the transporter in a resting, ATP-bound state. The sealed transmembrane channel is asymmetric, with one open and one closed cytosolic and periplasmic portal. The structure provides important insights into O antigen recruitment to and translocation by WzmWzt and related ABC transporters.

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The organization of cell surface membrane domains

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100155992 ◽

1989 ◽

Vol 47 ◽

pp. 804-805

Author(s):

Michael Edidin

Keyword(s):

Cell Surface ◽

Membrane Lipids ◽

Surface Membrane ◽

Lateral Diffusion ◽

Cell Types ◽

Membrane Domains ◽

Membrane Biology ◽

Morphological Polarity ◽

Discrete Domains ◽

Membrane Components

Cell surface membranes are based on a fluid lipid bilayer and models of the membranes' organization have emphasised the possibilities for lateral motion of membrane lipids and proteins within the bilayer. Two recent trends in cell and membrane biology make us consider ways in which membrane organization works against its inherent fluidity, localizing both lipids and proteins into discrete domains. There is evidence for such domains, even in cells without obvious morphological polarity and organization [Table 1]. Cells that are morphologically polarised, for example epithelial cells, raise the issue of membrane domains in an accute form.The technique of fluorescence photobleaching and recovery, FPR, was developed to measure lateral diffusion of membrane components. It has also proven to be a powerful tool for the analysis of constraints to lateral mobility. FPR resolves several sorts of membrane domains, all on the micrometer scale, in several different cell types.

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Molecular Characterization of Rotavirus Porcine OSU Carbohydrate- Binding Domain VP8*

Current Proteomics ◽

10.2174/1570164612666150226232936 ◽

2015 ◽

Vol 12 (1) ◽

pp. 69-72

Author(s):

Hao Li ◽

Wei Zhang ◽

Hong-hao Zhou ◽

Xiao-li Li

Keyword(s):

Molecular Characterization ◽

Binding Domain ◽

Carbohydrate Binding

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Cell surface anchorage and ligand-binding domains of the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin, a member of the immunoglobulin superfamily.

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2554 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2554-2563 ◽

Cited By ~ 82

Author(s):

D Wojciechowicz ◽

C F Lu ◽

J Kurjan ◽

P N Lipke

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Adhesion ◽

Amino Acid ◽

Cell Surface ◽

Consensus Sequence ◽

Binding Domain ◽

Immunoglobulin Superfamily ◽

Cell Contact ◽

Amino Acid Residues ◽

Adhesion Proteins

alpha-Agglutinin is a cell adhesion glycoprotein expressed on the cell wall of Saccharomyces cerevisiae alpha cells. Binding of alpha-agglutinin to its ligand a-agglutinin, expressed by a cells, mediates cell-cell contact during mating. Analysis of truncations of the 650-amino-acid alpha-agglutinin structural gene AG alpha 1 delineated functional domains of alpha-agglutinin. Removal of the C-terminal hydrophobic sequence allowed efficient secretion of the protein and loss of cell surface attachment. This cell surface anchorage domain was necessary for linkage to a glycosyl phosphatidylinositol anchor. A construct expressing the N-terminal 350 amino acid residues retained full a-agglutinin-binding activity, localizing the binding domain to the N-terminal portion of alpha-agglutinin. A 278-residue N-terminal peptide was inactive; therefore, the binding domain includes residues between 278 and 350. The segment of alpha-agglutinin between amino acid residues 217 and 308 showed significant structural and sequence similarity to a consensus sequence for immunoglobulin superfamily variable-type domains. The similarity of the alpha-agglutinin-binding domain to mammalian cell adhesion proteins suggests that this structure is a highly conserved feature of adhesion proteins in diverse eukaryotes.

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Inhibition of Transfer to Secondary Receptors by Heparan Sulfate-Binding Drug or Antibody Induces Noninfectious Uptake of Human Papillomavirus

Journal of Virology ◽

10.1128/jvi.00998-07 ◽

2007 ◽

Vol 81 (20) ◽

pp. 10970-10980 ◽

Cited By ~ 111

Author(s):

Hans-Christoph Selinka ◽

Luise Florin ◽

Hetal D. Patel ◽

Kirsten Freitag ◽

Michaela Schmidtke ◽

...

Keyword(s):

Cell Surface ◽

Neutralizing Antibodies ◽

Structural Similarity ◽

Hpv Infection ◽

Human Papillomaviruses ◽

Laminin 5 ◽

Entry Pathway ◽

Successful Infection ◽

Major Mode ◽

Endocytic Vesicles

ABSTRACT Infection with various human papillomaviruses (HPVs) induces cervical cancers. Cell surface heparan sulfates (HS) have been shown to serve as primary attachment receptors, and molecules with structural similarity to cell surface HS, like heparin, function as competitive inhibitors of HPV infection. Here we demonstrate that the N,N′-bisheteryl derivative of dispirotripiperazine, DSTP27, efficiently blocks papillomavirus infection by binding to HS moieties, with 50% inhibitory doses of up to 0.4 μg/ml. In contrast to short-term inhibitory effects of heparin, pretreatment of cells with DSTP27 significantly reduced HPV infection for more than 30 h. Using DSTP27 and heparinase, we furthermore demonstrate that HS moieties, rather than laminin 5, present in the extracellular matrix (ECM) secreted by keratinocytes are essential for infectious transfer of ECM-bound virions to cells. Prior binding to ECM components, especially HS, partially alleviated the requirement for cell surface HS. DSTP27 blocks infection by cell-bound virions by feeding into a noninfectious entry pathway. Under these conditions, virus colocalized with HS moieties in endocytic vesicles. Similarly, postattachment treatment of cells with heparinase, cytochalasin D, or neutralizing antibodies resulted in uptake of virions without infection, indicating that deviation into a noninfectious entry pathway is a major mode of postattachment neutralization. In untreated cells, initial colocalization of virions with HS on the cell surface and in endocytic vesicles was lost with time. Our data suggest that initial attachment of HPV to HS proteoglycans (HSPGs) must be followed by secondary interaction with additional HS side chains and transfer to a non-HSPG receptor for successful infection.

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The projection domain of MAP2b regulates microtubule protrusion and process formation in Sf9 cells

Journal of Cell Science ◽

10.1242/jcs.115.7.1523 ◽

2002 ◽

Vol 115 (7) ◽

pp. 1523-1539 ◽

Cited By ~ 1

Author(s):

Dave Bélanger ◽

Carole Abi Farah ◽

Minh Dang Nguyen ◽

Michel Lauzon ◽

Sylvie Cornibert ◽

...

Keyword(s):

Cell Surface ◽

Binding Domain ◽

Intramolecular Interactions ◽

Microtubule Assembly ◽

Sf9 Cells ◽

Microtubule Binding ◽

Process Formation ◽

Microtubule Binding Domain ◽

Projection Domain

The expression of microtubule-associated protein 2 (MAP2), developmentally regulated by alternative splicing, coincides with neurite outgrowth. MAP2 proteins contain a microtubule-binding domain (C-terminal) that promotes microtubule assembly and a poorly characterized domain, the projection domain(N-terminal), extending at the surface of microtubules. MAP2b differs from MAP2c by an additional sequence of 1372 amino acids in the projection domain. In this study, we examined the role of the projection domain in the protrusion of microtubules from the cell surface and the subsequent process formation in Sf9 cells. In this system, MAP2b has a lower capacity to induce process formation than MAP2c. To investigate the role of the projection domain in this event, we expressed truncated forms of MAP2b and MAP2c that have partial or complete deletion of their projection domain in Sf9 cells. Our results indicate that process formation is induced by the microtubule-binding domain of these MAP2 proteins and is regulated by their projection domain. Furthermore, the microtubule-binding activity of MAP2b and MAP2c truncated forms as well as the structural properties of the microtubule bundles induced by them do not seem to be the only determinants that control the protrusion of microtubules from the cell surface in Sf9 cells. Rather, our data suggest that microtubule protrusion and process formation are regulated by intramolecular interactions between the projection domain and its microtubule-binding domain in MAP2b.

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