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

2000 ◽  
Vol 74 (18) ◽  
pp. 8472-8479 ◽  
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.

scholarly journals Sialic acid-Dependent Binding and Viral Entry of SARS-CoV-2

2021 ◽  
Author(s):  
Linh Nguyen ◽  
Kelli McCord ◽  
Duong Bui ◽  
Kim Bouwman ◽  
Elena Kitova ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Receptor Binding ◽  
Viral Entry ◽  
Receptor Binding Domain ◽  
Artificial Membrane ◽  
Neuraminidase Treatment ◽  
S Protein ◽  
Knock Out

Abstract Emerging evidence suggests that host glycans influence infection by SARS-CoV-2. Here, we reveal that the receptor-binding domain (RBD) of the spike (S)-protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (SA), with preference for the oligosaccharide of monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind the RBD. The monomeric affinities (Kd = 100-200 μM) of gangliosides for the RBD are similar to heparan sulfate, another negatively charged glycan ligand of the RBD proposed as a viral co-receptor. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to ACE2-expressing cells is decreased upon depleting cell surface SA level using three approaches: sialyltransferase inhibition, genetic knock-out of SA biosynthesis, or neuraminidase treatment. These effects on RBD binding and pseudotyped viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2.

Partial resialylation of human asialotransferrin types 1 and 2 in the rat

1984 ◽  
Vol 62 (9) ◽  
pp. 853-858 ◽  
Author(s):  
Erwin Regoeczi ◽  
Paul A. Chindemi ◽  
Maria T. Debanne
Keyword(s):  
Sialic Acid ◽  
Electrophoretic Mobility ◽  
Acid Content ◽  
Sialic Acid Content ◽  
Small Doses ◽  
Type 3 ◽  
Time Type

125I-labeled asialotransferrin types 1 and 2 were administered in small doses to rats. The protein still in the plasma after 1–12 h was partially repurified and electrophoresed at pH 8.1, together with a transferrin standard that is composed of all six forms of the protein with respect to sialic acid content. The electrophoretic mobility of both asialotransferrins increased with time, type 2 being affected sooner than type 1. The changed mobility was due to increased electronegativity that was fully reversible by treatment of the samples with neuraminidase, thus identifying the underlying cause as partial resialylation. Asialotransferrin incubated in vitro with serum, plasma, or whole blood for 16 h exhibited no change in electrophoretic mobility. In conjunction with an earlier study on asialotransferrin type 3, it was found that the apparent speeds of resialylation of the three asialotransferrins were in the same order as their affinities for the asialoglycoprotein-binding hepatic lectin. This suggests the involvement of an endo- rather than of an ecto-transferase. Transfer of 59Fe from asialotransferrins to the liver was used to monitor the frequency of hepatocyte–asialotransferrin interactions. Iron deposition in the liver took place much more rapidly than the appearance of detectable quantities of partially resialylated asialotransferrin molecules in the circulation. It is concluded that each asialotransferrin molecule probably undergoes several passages through the hepatocyte before its glycans become modified.

scholarly journals Glanzmann thrombasthenia due to a two amino acid deletion in the fourth calcium-binding domain of alpha IIb: demonstration of the importance of calcium-binding domains in the conformation of alpha IIb beta 3

Blood ◽  
1996 ◽  
Vol 88 (1) ◽  
pp. 167-173 ◽  
Author(s):  
RB Basani ◽  
G Vilaire ◽  
SJ Shattil ◽  
MA Kolodziej ◽  
JS Bennett ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Calcium Binding ◽  
Binding Domain ◽  
In Vitro Mutagenesis ◽  
Amino Acid Deletion ◽  
Glanzmann Thrombasthenia ◽  
Binding Domains ◽  
Intracellular Processing ◽  
Calcium Binding Domain

The integrin alpha IIb beta 3, a calcium-dependent heterodimer, plays a critical role in platelet aggregation. The alpha IIb subunit of the heterodimer contains four highly conserved putative calcium-binding domains in its extracellular portion. During studies of the molecular basis of Glanzmann thrombasthenia in a child of mixed Caucasian background whose platelets expressed little alpha IIb beta 3 on their surface, we found the patient heterozygous for a two amino acid deletion in the fourth alpha IIb calcium-binding domain. When this alpha IIb mutant was expressed in COS-1 cells, we found that the deletion did not interfere with the assembly of alpha IIb beta 3 heterodimers, but altered their conformation such that they were neither recognized by the heterodimer-specific antibody A2A9 nor able to undergo further intracellular processing or transport to the cell surface. These results suggest that the calcium-binding domains in alpha IIb play an important role maintaining the overall conformation of alpha IIb beta 3. To confirm this suggestion, we deleted each of the four 12 amino acid calcium-binding domains in alpha IIb by in vitro mutagenesis and expressed the mutants along with beta 3 in COS-1 cells. Each construct formed a heterodimer with beta 3, but none of the heterodimers interacted with A2A9 or underwent further intracellular processing. These data indicate that the calcium-binding domains in alpha IIb are not involved in alpha IIb beta 3 heterodimer formation, but their presence is required for the intracellular transport of alpha IIb beta 3 to the cell surface.

scholarly journals Structural analysis of Pore-Forming CEL-III

2014 ◽  
Vol 70 (a1) ◽  
pp. C1679-C1679
Author(s):  
Hideaki Unno ◽  
Shuichiro Goda ◽  
Tomomitsu Hatakeyama
Keyword(s):  
Cell Surface ◽  
Structural Transition ◽  
Carbohydrate Binding ◽  
Binding Domains ◽  
Cell Surface Carbohydrate ◽  
Transmembrane Pores ◽  
Flat Ring ◽  
Domain 3 ◽  
Monomeric Structure ◽  
Carbohydrate Chains

CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1-2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1-2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore-formation mechanism of CEL-III, the crystal structure of the CEL-III oligomer was determined. The CEL-III oligomer has a heptameric structure with a long β-barrel as a transmembrane pore. This β-barrel is composed of 14 β-strands resulting from a large structural transition of α-helices accommodated in the interface between domains 1-2 and domain 3 in the monomeric structure, suggesting that the dissociation of these α-helices triggered their structural transition into a β-barrel. After heptamerization, domains 1-2 form a flat ring, in which all carbohydrate- binding sites remain bound to cell surface carbohydrate chains, stabilizing the transmembrane β-barrel in a position perpendicular to the plane of the lipid bilayer.

Gliolectin is a novel carbohydrate-binding protein expressed by a subset of glia in the embryonic Drosophila nervous system

Development ◽  
1996 ◽  
Vol 122 (3) ◽  
pp. 925-936
Author(s):  
M. Tiemeyer ◽  
C.S. Goodman
Keyword(s):  
Nervous System ◽  
Cell Surface ◽  
Neural Development ◽  
Small Subset ◽  
Carbohydrate Binding ◽  
Calculated Molecular Mass ◽  
Amino Terminal ◽  
Surface Carbohydrates ◽  
Drosophila Embryos ◽  
Cell Cell

Interactions between embryonic neural cells generate the specific patterns of connectivity observed in nervous systems. Cell surface carbohydrates have been proposed to function in cellular recognition events guiding such interactions. Carbohydrate-binding proteins (lectins) that recognize specific oligosaccharide ligands in embryonic neural tissue provide a molecular mechanism for carbohydrate-mediated cell-cell interactions in neural development. Therefore, we have screened an embryonic Drosophila melanogaster cDNA library, expressed in COS1 cells, for carbohydrate-binding activity. COS1 cells expressing putative Drosophila lectins were identified and recovered based on their adhesion to immobilized preparations of neutral and zwitterionic glycolipids extracted from Drosophila embryos. We have identified an endogenous lectin expressed during Drosophila embryogenesis. The cloned lectin, designated ‘gliolectin’, possesses a novel protein sequence with a calculated molecular mass of 24,993. When expressed in Drosophila S2 cells, the lectin mediates heterophilic cellular aggregation. In embryos, gliolectin is expressed by a subset of glial cells found at the midline of the developing nervous system. Expression is highest during the formation of the Drosophila embryonic axonal commissures, a process requiring midline glial cell funcion. Immunoprecipitation with a monoclonal antibody against gliolectin yields a protein of Mr=46,600 from Drosophila embryonic membranes, suggesting that post-translational modification of gliolectin is extensive. Epitope- tagged chimericproteins composed of the amino terminal one-half of gliolectin and the Fc region of human IgG bind a small subset of the total glycolipids extracted from Drosophila embryos, demonstrating that the lectin activity of gliolectin can discriminate between oligosaccharide structures. The presence of gliolectin in the developing Drosophila embryonic nervous system further supports a role for cell surface carbohydrates in cell-cell recognition and indicates that the molecular diversity of animal lectins is not yet completely defined.

scholarly journals Comprehensive Mutational Analysis of the Moloney Murine Leukemia Virus Envelope Protein

2001 ◽  
Vol 75 (23) ◽  
pp. 11851-11862 ◽  
Author(s):  
S. Michael Rothenberg ◽  
Mari N. Olsen ◽  
Louise Chang Laurent ◽  
Rachel Adams Crowley ◽  
Patrick O. Brown
Keyword(s):  
Cell Surface ◽  
Receptor Binding ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Receptor Binding Domain ◽  
Amino Terminal ◽  
Carboxy Terminal Region ◽  
Env Protein ◽  
Carboxy Terminal ◽  
Murine Leukemia

ABSTRACT The envelope (Env) protein of Moloney murine leukemia virus is the primary mediator of viral entry. We constructed a large pool of insertion mutations in the env gene and analyzed the fitness of each mutant in completing two critical steps in the virus life cycle: (i) the expression and delivery of the Env protein to the cell surface during virion assembly and (ii) the infectivity of virions displaying the mutant proteins. The majority of the mutants were poorly expressed at the producer cell surface, suggesting folding defects due to the presence of the inserted residues. The mutants with residual infectivity had insertions either in the amino-terminal signal sequence region, two disulfide-bonded loops in the receptor binding domain, discrete regions of the carboxy-terminal region of the surface subunit (SU), or the cytoplasmic tail. Insertions that allowed the mutants to reach the cell surface but not to mediate detectable infection were located within the amino-terminal sequence of the mature Env, within the SU carboxy-terminal region, near putative receptor binding residues, and throughout the fusion peptide. Independent analysis of select mutants in this group allowed more precise identification of the defect in Env function. Mapping of mutant phenotypes to a structural model of the receptor-binding domain provides insights into the protein's functional organization. The high-resolution functional map reported here will be valuable for the engineering of the Env protein for a variety of uses, including gene therapy.

scholarly journals Glanzmann thrombasthenia due to a two amino acid deletion in the fourth calcium-binding domain of alpha IIb: demonstration of the importance of calcium-binding domains in the conformation of alpha IIb beta 3

Blood ◽  
1996 ◽  
Vol 88 (1) ◽  
pp. 167-173 ◽  
Author(s):  
RB Basani ◽  
G Vilaire ◽  
SJ Shattil ◽  
MA Kolodziej ◽  
JS Bennett ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Calcium Binding ◽  
Binding Domain ◽  
In Vitro Mutagenesis ◽  
Amino Acid Deletion ◽  
Glanzmann Thrombasthenia ◽  
Binding Domains ◽  
Intracellular Processing ◽  
Calcium Binding Domain

Abstract The integrin alpha IIb beta 3, a calcium-dependent heterodimer, plays a critical role in platelet aggregation. The alpha IIb subunit of the heterodimer contains four highly conserved putative calcium-binding domains in its extracellular portion. During studies of the molecular basis of Glanzmann thrombasthenia in a child of mixed Caucasian background whose platelets expressed little alpha IIb beta 3 on their surface, we found the patient heterozygous for a two amino acid deletion in the fourth alpha IIb calcium-binding domain. When this alpha IIb mutant was expressed in COS-1 cells, we found that the deletion did not interfere with the assembly of alpha IIb beta 3 heterodimers, but altered their conformation such that they were neither recognized by the heterodimer-specific antibody A2A9 nor able to undergo further intracellular processing or transport to the cell surface. These results suggest that the calcium-binding domains in alpha IIb play an important role maintaining the overall conformation of alpha IIb beta 3. To confirm this suggestion, we deleted each of the four 12 amino acid calcium-binding domains in alpha IIb by in vitro mutagenesis and expressed the mutants along with beta 3 in COS-1 cells. Each construct formed a heterodimer with beta 3, but none of the heterodimers interacted with A2A9 or underwent further intracellular processing. These data indicate that the calcium-binding domains in alpha IIb are not involved in alpha IIb beta 3 heterodimer formation, but their presence is required for the intracellular transport of alpha IIb beta 3 to the cell surface.

scholarly journals Role of Neuraminidase in Influenza A(H7N9) Virus Receptor Binding

2017 ◽  
Vol 91 (11) ◽  
Author(s):  
Donald J. Benton ◽  
Stephen A. Wharton ◽  
Stephen R. Martin ◽  
John W. McCauley
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Binding Site ◽  
Influenza A Virus ◽  
Receptor Binding ◽  
Influenza A ◽  
Health Concern ◽  
H7n9 Virus ◽  
Virus Receptor ◽  
Sialic Acid Receptors

ABSTRACT Influenza A(H7N9) viruses have caused a large number of zoonotic infections since their emergence in 2013. They remain a public health concern due to the repeated high levels of infection with these viruses and their perceived pandemic potential. A major factor that determines influenza A virus fitness and therefore transmissibility is the interaction of the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) with the cell surface receptor sialic acid. Typically, the HA is responsible for binding to the sialic acid to allow virus internalization and the NA is a sialidase responsible for cleaving sialic acid to aid virus spread and release. N9 NA has previously been shown to have receptor binding properties mediated by a sialic acid binding site, termed the hemadsorption (Hb) site, which is discrete from the enzymatically active sialidase site. This study investigated the N9 NA from a zoonotic H7N9 virus strain in order to determine its possible role in virus receptor binding. We demonstrate that this N9 NA has an active Hb site which binds to sialic acid, which enhances overall virus binding to sialic acid receptor analogues. We also show that the N9 NA can also contribute to receptor binding due to unusual kinetic characteristics of the sialidase site which specifically enhance binding to human-like α2,6-linked sialic acid receptors. IMPORTANCE The interaction of influenza A virus glycoproteins with cell surface receptors is a major determinant of infectivity and therefore transmissibility. Understanding these interactions is important for understanding which factors are necessary to determine pandemic potential. Influenza A viruses generally mediate binding to cell surface sialic acid receptors via the hemagglutinin (HA) glycoprotein, with the neuraminidase (NA) glycoprotein being responsible for cleaving the receptor to allow virus release. Previous studies showed that the NA proteins of the N9 subtype can bind sialic acid via a separate binding site distinct from the sialidase active site. This study demonstrates for purified protein and virus that the NA of the zoonotic H7N9 viruses has a binding capacity via both the secondary binding site and unusual kinetic properties of the sialidase site which promote receptor binding via this site and which enhance binding to human-like receptors. This could have implications for understanding human-to-human transmission of these viruses.

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