Sialic Acid Binding Activity Research Articles

Is the sialic acid binding activity of the S protein involved in the enteropathogenicity of transmissible gastroenteritis virus?

Transmissible gastroenteritis virus (TGEV) is able to recognize sialic acid on sialo-glycoconjugates. Analysis of mutants indicated that single point mutations in the S protein (around amino acids 145-155) of TGEV may result both in the loss of the sialic acid binding activity and in a drastic reduction of the enteropathogenicity. From this observation we conclude that the sialic acid binding activity is involved in the enteropathogenicity of TGEV. On the basis of our recent results we propose that binding of sialylated macromolecules to the virions surface may increase virus stability. This in turn would explain how TGEV as an enveloped virus can survive the gastrointestinal passage and cause intestinal infections.

Isolation of hemagglutination-defective mutants for the analysis of the sialic acid binding activity of transmissible gastroenteritis virus.

The surface protein S of transmissible gastroenteritis virus (TGEV) has a sialic acid binding activity that enables the virus to agglutinate erythrocytes. A protocol is described that has been successfully applied to the isolation of hemgglutination-defective mutants. The potential of these mutants for the characterization of the sialic acid-binding site and the function of the binding activity is discussed.

Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus.

Enteropathogenic transmissible gastroenteritis virus (TGEV), a porcine coronavirus, is able to agglutinate erythrocytes because of sialic acid binding activity. Competitive inhibitors that may mask the sialic acid binding activity can be inactivated by sialidase treatment of virions. Here, we show that TGEV virions with efficient hemagglutinating activity were also obtained when cells were treated with sialidase prior to infection. This method was used to analyze TGEV mutants for hemagglutinating activity. Recently, mutants with strongly reduced enteropathogenicity that have point mutations or a deletion of four amino acids within residues 145 to 155 of the S protein have been described. Here, we show that in addition to their reduced pathogenicity, these mutants also have lost hemagglutinating activity. These results connect sialic acid binding activity with the enteropathogenicity of TGEV.

The Plasma-Based Cytolytic System of the American Horseshoe Crab, Limulus polyphemus: Cooperative Interaction of the Sialic Acid-Binding Lectin Limulin and Thiol Ester-Reacted α2-Macroglobulin

The cytolytic destruction of invading pathogens by proteins of the plasma is an important immune defense strategy of higher animals. The principal protein effector of the plasmabased cytolytic system of Limulus is limulin, a sialic acid-binding lectin present in the plasma. In assays with sheep red cells as the model foreign cell, purified limulin is hemolytic at 4-6 nM, and removal of the limulin from plasma eliminates hemolytic activity (1). Hemolysis depends on the sialic acid-binding activity of limulin, because sialylated glycoconjugates, such as fetuin, and the sialic acids N-acetyl neuraminic acid and colominic acid inhibit hemolysis. The third most abundant protein in Limulus plasma (after hemocyanin and C-reactive protein) is Limulus az-macroglobulin (LAM), which functions as the principal mediator of protease clearance from the plasma (2). The reaction of LAM with proteases activates a unique internal @-cysteinyl-y-glutamyl thiol ester bond, generating a reactive glutamyl residue and a free cysteinyl thiol. Activation of the thiol ester initiates a substantial molecular compaction (3) exposing a previously cryptic domain that is recognized by cell surface receptors. Recognition results in the binding, internalization, and degradation of LAM with its cargo ofbound protease (2, lkawi et al., unpub. data). Small primary amines such as methylamine (MA) react with the thiol-esterified glutamine and induce a nearly identical conformational change in LAM, with a concomitant molecular compaction and activation of receptor recognizability. In this report, we identify a new function for LAM: thiol ester-reacted LAM binds to limulin and potentiates its hemolytic activity. Native, unreacted LAM has no effect on the hemolytic activity of limulin. Limulin (1) and LAM (4) were purified as described previously. Hemolysis was conducted as described in (5). The action of the proteaseand MA-reacted forms of LAM is complex: at high concentrations of limulin and LAM, we see a modest depression of hemolysis (6) but at subhemolytic concentrations of limulin, we see a marked potentiation. Potentiation requires a 20-40-fold molar excess of proteaseor MAreacted LAM. This approximates the ratio of these proteins in the plasma: Limulus plasma contains l-5 ~.LM LAM (7) and about 30-50 nM limulin. Native, unreacted LAM failed to potentiate limulin-mediated hemolysis. The selective action of reacted forms of LAM on the hemo-

Transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (N-glycolylneuraminic acid) binding activity

The hemagglutinating activity of transmissible gastroenteritis virus (TGEV), an enteric porcine coronavirus, was analyzed and found to be dependent on the presence of alpha-2,3-linked sialic acid on the erythrocyte surface. N-Glycolylneuraminic acid was recognized more efficiently by TGEV than was N-acetylneuraminic acid. For an efficient hemagglutination reaction the virions had to be treated with sialidase. This result suggests that the sialic acid binding site is blocked by virus-associated competitive inhibitors. Porcine respiratory coronavirus (PRCV), which is serologically related to TGEV but not enteropathogenic, was found to be unable to agglutinate erythrocytes. Incubation with sialidase did not induce a hemagglutinating activity of PRCV, indicating that the lack of this activity is an intrinsic property of the virus and not due to the presence of competitive inhibitors. Only monoclonal antibodies to an antigenic site that is absent from the S protein of PRCV were able to prevent TGEV from agglutinating erythrocytes. The epitope recognized by these antibodies is located within a stretch of 224 amino acids that is missing in the S protein of PRCV. Our results indicate that the sialic acid binding activity is also located in that portion of the S protein. The presence of a hemagglutinating activity in TGEV and its absence in PRCV open the possibility that the sialic acid binding activity contributes to the enterotropism of TGEV.

A Single N-linked Glycosylation Site Is Implicated in the Regulation of Ligand Recognition by the I-type Lectins CD22 and CD33

CD22 is an immunoglobulin superfamily B lymphocyte-specific adhesion receptor and a member of the recently identified I-type class of lectins. Recent work has shown that CD22 specifically recognizes sialic acid linked alpha2,6 to terminal N-linked oligosaccharides on selected cell surface glycoproteins. CD22-ligand interaction is regulated by the activity of a beta-galactoside alpha2, 6-sialyltransferase that can inactivate CD22-mediated binding by sialylating the CD22 receptor itself. These observations suggest that N-linked glycosylation sites on the CD22 molecule may play a role in the regulation of CD22-mediated adhesion. In this work we have performed site-specific mutagenesis of potential N-linked glycosylation sites on CD22 in an effort to determine whether they might be involved in ligand recognition. We show that mutation of a single potential N-linked glycosylation site in the first immunoglobulin domain of CD22 completely abrogates ligand recognition. Interestingly, this site is characterized by the sequence NCT, where the cysteine is thought to be involved in an intrachain disulfide bond. Site-directed mutagenesis of similar NC(T/S) motifs in the first or second Ig domains of the I-type lectins myelin-associated glycoprotein, and sialoadhesin did not disrupt their ability to mediate sialic acid binding. In contrast, mutation of a NCS motif in the first Ig domain of the I-type lectin CD33 unmasked its sialic acid binding activity. These observations suggest that a single N-linked glycosylation site located at a similar position in the CD22 and CD33 glycoproteins is critical for regulating ligand recognition by both receptors.