Showing papers on "Sialic acid published in 1987"

Inhibitors of the Biosynthesis and Processing of N-Linked Oligosaccharide Chains

Abstract: A number of glycoproteins have oligosaccharides linked to protein in a GlcNAc----asparagine bond. These oligosaccharides may be either of the complex, the high-mannose or the hybrid structure. Each type of oligosaccharides is initially biosynthesized via lipid-linked oligosaccharides to form a Glc3Man9GlcNAc2-pyrophosphoryl-dolichol and transfer of this oligosaccharide to protein. The oligosaccharide portion is then processed, first of all by removal of all three glucose residues to give a Man9GlcNAc2-protein. This structure may be the immediate precursor to the high-mannose structure or it may be further processed by the removal of a number of mannose residues. Initially four alpha 1,2-linked mannoses are removed to give a Man5 - GlcNAc2 -protein which is then lengthened by the addition of a GlcNAc residue. This new structure, the GlcNAc- Man5 - GlcNAc2 -protein, is the substrate for mannosidase II which removes the alpha 1,3- and alpha 1,6-linked mannoses . Then the other sugars, GlcNAc, galactose, and sialic acid, are added sequentially to give the complex types of glycoproteins. A number of inhibitors have been identified that interfere with glycoprotein biosynthesis, processing, or transport. Some of these inhibitors have been valuable tools to study the reaction pathways while others have been extremely useful for examining the role of carbohydrate in glycoprotein function. For example, tunicamycin and its analogs prevent protein glycosylation by inhibiting the first step in the lipid-linked pathway, i.e., the formation of Glc NAc-pyrophosphoryl-dolichol. These antibiotics have been widely used in a number of functional studies. Another antibiotic that inhibits the lipid-linked saccharide pathway is amphomycin, which blocks the formation of dolichyl-phosphoryl-mannose. In vitro, this antibiotic gives rise to a Man5GlcNAc2 -pyrophosphoryl-dolichol from GDP-[14C]mannose, indicating that the first five mannose residues come directly from GDP-mannose rather than from dolichyl-phosphoryl-mannose. Other antibodies that have been shown to act at the lipid-level are diumycin , tsushimycin , tridecaptin, and flavomycin. In addition to these types of compounds, a number of sugar analogs such as 2-deoxyglucose, fluoroglucose , glucosamine, etc. have been utilized in some interesting experiments. Several compounds have been shown to inhibit glycoprotein processing. One of these, the alkaloid swainsonine , inhibits mannosidase II that removes alpha-1,3 and alpha-1,6 mannose residues from the GlcNAc- Man5GlcNAc2 -peptide. Thus, in cultured cells or in enveloped viruses, swainsonine causes the formation of a hybrid structure.(ABSTRACT TRUNCATED AT 400 WORDS)

An IgG monoclonal antibody to group B meningococci cross-reacts with developmentally regulated polysialic acid units of glycoproteins in neural and extraneural tissues.

Abstract: The structurally similar polysialic acid capsules of group B meningococci and Escherichia coli K1 are poor immunogens, and attempts are currently being made to improve their immunogenicity by chemical modifications. An IgG monoclonal antibody to these polysialic acid capsules was used for the study of the presence of structurally similar components in tissue glycoproteins to investigate the reasons for the poor immunogenicity and to evaluate potential dangers in active or passive immunization. By immunoblotting polysialic acid was detected outside the brain in newborn rat kidney, heart, and muscle. It appeared in immunoblots as one component and with similar mobility to the neural cell adhesion molecule N-CAM. Specificity studies of the antibody and endosialidase treatment showed that the polysialic acid glycans detected were composed of chains as long as eight sialic acid residues or more. The polysialic acid was not detected in the corresponding tissues of the adult animal. These results indicate that polysialic acid units are developmentally regulated components of both neural and extraneural tissues, and are bound to components with properties similar to a known cell-adhesion molecule. This together with the presence of low amounts of polysialic acid even in the adult brain, suggests potential hazards in vaccination trials and suggested immunotherapy of meningitis caused by group B meningococci or E. coli K1, which should be carefully assessed.

Tyrosine sulfation is a trans-Golgi-specific protein modification.

Abstract: The trans-Golgi has been recognized as having a key role in terminal glycosylation and sorting of proteins. Here we show that tyrosine sulfation, a frequent modification of secretory proteins, occurs specifically in the trans-Golgi. The heavy chain of immunoglobulin M (IgM) produced by hybridoma cells was found to contain tyrosine sulfate. This finding allowed the comparison of the state of sulfation of the heavy chain with the state of processing of its N-linked oligosaccharides. First, the pre-trans-Golgi forms of the IgM heavy chain, which lacked galactose and sialic acid, were unsulfated, whereas the trans-Golgi form, identified by the presence of galactose and sialic acid, and the secreted form of the IgM heavy chain were sulfated. Second, the earliest form of the heavy chain detectable by sulfate labeling, as well as the heavy chain sulfated in a cell-free system in the absence of vesicle transport, already contained galactose and sialic acid. Third, sulfate-labeled IgM moved to the cell surface with kinetics identical to those of galactose-labeled IgM. Lastly, IgM labeled with sulfate at 20 degrees C was not transported to the cell surface at 20 degrees C but reached the cell surface at 37 degrees C. The data suggest that within the trans-Golgi, tyrosine sulfation of IgM occurred at least in part after terminal glycosylation and therefore appeared to be the last modification of this constitutively secreted protein before its exit from this compartment. Furthermore, the results establish the covalent modification of amino acid side chains as a novel function of the trans-Golgi.

Sialic acid of group B Neisseria meningitidis regulates alternative complement pathway activation.

Abstract: The effect of meningococcal cell-associated sialic acid on activation of the human alternative complement pathway was examined by using a quantitative fluorescence immunoassay to assess alternative pathway-mediated C3 binding to a group B strain of Neisseria meningitidis from which graded amounts of sialic acid had been removed with neuraminidase. Using human serum absorbed with strain B16B6 (B:2a:L2,3) and chelated with 10 mM MgCl2 and 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, we found an increase in the amount of C3 bound by enzymatically desialylated B16B6 organisms over the amount bound by fully sialylated organisms. This increase was proportional to the amount of sialic acid cleaved from the bacteria. Enhanced C3 binding was accompanied by an increase in factor B deposition. A sialic acid-deficient mutant of strain B16B6, designated 2T4-1, bound C3 via the alternative pathway at a level equivalent to that bound by wild-type meningococci from which 88% of the sialic acid had been removed. Strain B16B6 was resistant to the alternative pathway-mediated bactericidal activity of both absorbed and hypogammaglobulinemic human sera, whereas noncapsular variant 2T4-1 was sensitive to these sera. The addition of purified immune immunoglobulin M (IgM) and IgG significantly increased the alternative pathway-mediated killing of strain B16B6 organisms. IgM mediated increased bactericidal activity without an increase in C3 or factor B deposition. In contrast, the IgG-mediated killing was associated with increased binding of C3 and factor B to the organisms. Absorption studies showed that the IgM bound to the sialic acid capsule, whereas the IgG bound to noncapsular surface antigens. We conclude from these results that the group B meningococcal sialic acid capsule inhibits activation of the alternative pathway in the nonimmune host and that both IgM and IgG, although specific for different surface antigens, are capable of augmenting the alternative pathway-mediated killing of group B meningococci.

Purification and biochemical characterization of three major acidic proteins (BSP-A1, BSP-A2 and BSP-A3) from bovine seminal plasma.

Abstract: Three major acidic proteins of bovine seminal plasma, BSP-A1, BSP-A2 and BSP-A3, were purified to homogeneity, by employing fast protein liquid chromatography, gel filtration and h.p.l.c. The proteins were purified on the basis of their stimulatory effect on the basal release of gonadotropins by rat anterior-pituitary cells in culture. All three proteins migrated as distinct single bands in the presence or absence of 2-mercaptoethanol in SDS/polyacrylamide-gel electrophoresis. Their Mr values were estimated to be between 15,000 and 16,500 by SDS/polyacrylamide-gel electrophoresis. Similar Mr estimates were obtained when they were subjected to gel filtration on a calibrated column of Sephadex G-75 equilibrated in 0.05 M-acetic acid, pH 3.0. However, BSP-A1 and BSP-A2 were eluted as aggregated molecules (Mr 60,000-120,000) during gel filtration on Sephadex G-200 equilibrated in 0.05 M-NH4HCO3, pH 8.5, or phosphate buffer, pH 7.0, containing 0.15 M-NaCl. In the presence of 8 M-urea both BSP-A1 and BSP-A2 were eluted at positions corresponding to Mr values of 17,000-20,000. BSP-A1 and BSP-A2 had an identical amino acid composition, which differed largely from that of BSP-A3. All three proteins contained aspartic acid as the N-terminal residue, and cysteine was identified as the C-terminal residue. BSP-A1 and BSP-A2 are glycoproteins containing galactosamine, sialic acid and neutral sugars, but BSP-A3 did not contain any covalently attached sugars. Whereas BSP-A2 and BSP-A3 were eluted unadsorbed, BSP-A1 bound to wheat-germ lectin-Sepharose 6MB and could be eluted by the competing sugar N-acetyl-D-glucosamine. Treatment of BSP-A1 and BSP-A2 with trypsin resulted in complete loss of gonadotropin-release activity, but BSP-A3 retained full activity. Antibody raised against BSP-A1 did not cross-react with BSP-A3, or vice versa. All these properties indicated marked structural differences between BSP-A3 and BSP-A1 (or BSP-A2). On the basis of amino acid composition it was concluded that BSP-A1, BSP-A2 and BSP-A3 are the same as the gonadostatins [Esch, Ling, Bohlen, Ying & Guillemin (1983) Biochem. Biophys. Res. Commun. 113, 861-867].

Plasminogen activator-specific inhibitors produced by human monocytes/macrophages.

Abstract: Human monocytes/macrophages produce plasminogen activator-specific inhibitors (PAIs) that form covalent complexes with urokinase-type plasminogen activator (uPA). We have characterized two functionally and antigenically related forms of PAIs produced by resting and phorbol myristate acetate (PMA)-treated U 937 cells: an Mr 40,000 form, presumably nonglycosylated, with a pI of 5.2, that is constitutively synthetized by these cells and that remains predominantly intracellular; a PMA-induced form of heterogeneous Mr (50,000-65,000) with a pI of 4.7, that is preferentially secreted; this PAI is glycosylated with terminal sialic acid residue(s). Biosynthetic labeling experiments demonstrated that both PAIs are synthetized by U 937 cells. They are inactivated upon treatment with propanol, heat, and acid; the covalent and equimolar complexes formed between these PAIs and 125I-uPA are dissociated by ammonium hydroxide, suggesting that the PAIs are linked to uPA via an ester bond. Human peripheral blood monocytes/macrophages also produce the two forms of PAI. These PAIs are clearly different from the main plasma protease inhibitors and they are both antigenically related to the PAI-2 characterized in human placenta.

Distribution and epitope analysis of the cell membrane glycoprotein (HPCA-1) associated with human hemopoietic progenitor cells.

Abstract: Monoclonal antibodies My10, BI.3C5, 12.8, and ICH3 identify a monomeric cell surface glycoprotein (HPCA-1) of 100-120 kD, which is selectively expressed on human hemopoietic progenitor cells. Other tissues are nonreactive with the exception of capillary endothelia and basement membrane in some sites. In addition, the antigen can be detected on cell lines that exhibit characteristics associated with early T cell precursors. HPCA-1 is therefore associated with myeloid, B, and T lineage precursors. Sequential immunoprecipitation and Western blotting studies demonstrate that BI.3C5, ICH3, My10, and an antibody directed against endothelial cells, 188.27, all react with the same glycoprotein species, although the epitopes involved may be distinct. The epitope recognized by BI.3C5 is sialic acid dependent, whereas that recognized by ICH3 is not. The My10 epitope has partial sensitivity to neuraminidase. Competitive/additive binding experiments suggest that these epitopes, although probably distinct, may be closely associated.

Gangliosides of Human Melanoma

Abstract: Human melanoma synthesizes a large quantity of gangliosides, glycosphingolipids containing sialic acid. The authors previously have demonstrated that the ganglioside profile differs among individual melanomas and is widely heterogenous. In the current study, a retrospective study was performed to compare the relationship between the quantity of five major gangliosides of human melanoma (GM3, GM2, GD3, GD2, and alkali-labile ganglioside) and nine clinical factors (sex, age, site, stage, tumor size, pigmentation, histopathologic type of primary tumor, chemosensitivity, and prognosis). Melanoma specimens studied were obtained from patients of our clinic and included 52 biopsy specimens and 28 cultured cell lines. Analysis of melanoma biopsy specimens have shown a differential ganglioside expression among different sites of tumor, pigmentation, and histopathologic types. Results of cultured melanoma cell lines differed from those of biopsy specimens, but ganglioside expression also differed among the site of tumor, tumor size, histopathologic types, and chemosensitivity. GM3 positively correlated with a good prognosis in both biopsy and cultured melanomas. Cuncer 63:1166-1174, 1989. ANGLIOSIDES are membrane-bound glycosphingoG lipids containing sialic acids that are found in high concentrations on neural tissues. Malignant melanoma, a tumor of neural crest origin, also synthesizes large quantities of gangliosides. GM3, GM2, GD3, GD2, and alkali-labile gangliosides all are expressed on melanoma but their composition and total sialic acid content are widely heterogene~us.~ Gangliosides have been shown to be important as cell membrane receptors for viruses, hormones, toxin^,^ cellular interactions and adhe~ion,~ oncogene~is,~ and immunologic recognition.6 The biologic properties of melanomas, such as histopathologic features, growth rate, metastatic properties, and drug resistance are also widely heterogene~us.~-~ From these previous investigations, it appeared possible that differences in gangliosides of human melanoma might be related to certain clinical factors. Therefore, in this study we have investigated the relationship between various clinical factors and the ganglioside composition of human melanoma.

Tumor cell surface carbohydrate and the metastatic phenotype

Abstract: The synthesis and expression of cell surface carbohydrates is a developmentally regulated process that appears to affect a number of cell-cell interactions. To determine whether specific oligosaccharide structures present on highly malignant cells are required for expression of the metastatic phenotype, we have isolated lectin resistant tumor cell mutants with defects in the biosynthesis of oligosaccharides. The mutants selected from the highly aggressive lymphoreticular-like tumor line MDAY-D2 were grouped into genetic complementation classes, compared for metastatic ability and for changes in cell surface glycoconjugates. The Asn-linked oligosaccharides and glycolipids of class 1 mutants were deficient in both sialic acid and galactose and the cells showed a greatly attenuated metastatic phenotype compared to the parental cells. A revertant of the class 1 mutation selected in vitro regained the wild type glycoconjugate profile and the highly metastatic phenotype indicating a direct association between the mutation and the loss of metastatic potential. Class 2 mutants remained highly metastatic and had Asn-linked oligosaccharide structures very similar to those found in the wild type cells with N-glycolylneuraminic acid rather than the N-acetylneuraminic acid. Swainsonine, an inhibitor of golgi α-mannosidase II, blocks the synthesis of complex-type Asn-linked oligosaccharides resulting in the expression of hybrid-type oligosaccharides at the cell surface and the cells display a lectin resistant phenotype. Although swainsonine inhibited neither tumor cell growth in vitro nor solid tumor growth in situ, the drug dramatically reduced the incidence of lung colonies after i.v. inoculation of both MDAY-D2 and B16F10 melanoma cells. These results, taken together, indicate that certain sialylated Asn-linked oligosaccharides found on metastatic tumor cells are required for expression of the metastatic phenotype.

The Growth of Oral Bacteria on Saliva

Abstract: The present experiments were aimed at studying the degradation of salivary glycoproteins by the oral microflora. To this end, S. sanguis I strain Ny476 and S. sanguis II (S. mitior) strain Ny581 were grown continuously in human-whole saliva. Under these conditions, the strains produced a variety of cell-associated hydrolytic activities, including glycosidases, exo- and endopeptidases, and esterases. S. sanguis II generally exhibited higher levels of enzyme activity than did S. sanguis I, in particular of neuraminidase that was produced only by S. sanguis II. In accordance, S. sanguis II had a higher cell yield and consumed a higher proportion of the sugars and sialic acid in the glycoproteins than did S. sanguis I. Interestingly, S. sanguis I, which is devoid of neuraminidase, is known to have a lectin with specificity for sialic acid, whereas S. sanguis II has affinity for galactose residues in the glycoproteins. We propose that specific binding of glycoproteins by oral bacteria constitutes a mechanism to collect nutrients in the vicinity of the cell. The special ability of S. sanguis II to utilize saliva for growth was further exemplified by its selection in batch-wise enrichments of dental plaque on saliva. The microflora in these enrichment cultures always consisted of Peptostreptococcus micros, S. sanguis II, and Fusobacterium nucleatum as the dominant organisms. Further, S. mitis and Gemella haemolysans were generally found to be present. The enrichment cultures produced a wide variety of mainly cell-bound hydrolytic enzymes. This resulted in almost complete breakdown of salivary glycoproteins in the culture.

Structural Studies of the Carbohydrate Moieties of Carcinoembryonic Antigens

Abstract: Three samples of carcinoembryonic antigens were purified from liver metastases of primary colon cancer. The asparagine-linked sugar chains of carcinoembryonic antigens (CEA) were released as oligosaccharides by hydrazinolysis and the structures of oligosaccharides, thus obtained, was studied in combination with methylation analysis and several limited exoglycosidase digestions. All three CEAs contain approximately 25 asparagine-linked sugar chains in one molecule and about 10% of them was high mannose type. However, structural features of the outer chain moieties of the remaining complex-type sugar chains were different by CEA samples. The complex-type sugar chains were mono-, bi-, tri-, and tetraantennary with Man alpha 1----6(+/- GlcNAc beta 1----4)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc as their cores, half of which were bisected; 86% of their proximal N-acetylglucosamine was fucosylated. The major outer chains in two samples were N-acetyllactosamine and Gal beta 1----4(Fuc alpha 1----3)GlcNAc (X-antigenic determinant) and the remaining one sample contained Fuc alpha 1----2Gal beta 1----4(Fuc alpha 1----3)GlcNAc (Y-antigenic determinant) as an additional major outer chain. Furthermore, small amounts of type 1 chain and Lea antigenic determinant were found in some samples. Acidic oligosaccharides consisted of sialic acid containing fractions and sialidase-resistant fractions, and their contents seemed to be in a reciprocal relationship. Sialic acid was linked at the C-3 and C-6 positions of the nonreducing terminal galactose residues of the outer chains.

Sialic acid glycoproteins inhibit in vitro and in vivo replication of rotaviruses.

Abstract: We investigated the interactions of rotaviruses with glycoproteins and cells that support rotaviral replication. We found that a wide range of naturally occurring glycoproteins, including ovalbumins and ovomucoids from chicken and turkey eggs, and mucin derived from bovine submaxillary glands, inhibit the replication of rotaviruses in MA-104 cells. Our studies further indicated that the glycoproteins bind directly to rotaviruses and that virus-glycoprotein binding is dependent largely upon interactions with sialic acid oligosaccharides. We found that accessible sialic acid oligosaccharides are required for efficient rotavirus infection of MA-104 cells, thus demonstrating that sialic acid oligosaccharides play an important role in the interactions of rotaviruses with both glycoproteins and cells that support rotaviral replication. Bovine submaxillary mucin and chicken ovoinhibitor can also prevent the shedding of rotavirus antigen and the development of rotavirus gastroenteritis in a mouse model of rotavirus infection. Our findings document that a range of glycoproteins inhibit the in vivo and in vitro replication of rotaviruses and suggest that the alteration in the quantity or chemical composition of intestinal glycoproteins is a potential means for the modulation of enteric infections.

Gangliosides induce selective modulation of CD4 from helper T lymphocytes.

Abstract: The cluster designation (CD)4 molecule is one of several nonpolymorphic T lymphocyte surface proteins that have been implicated in T cell-target cell interactions, and is thought to play an important role in regulating T helper cell function. Previously, we found that gangliosides inhibited the function of rat T helper cell lines, and simultaneously inhibited the expression of the rat CD4 molecule identified by the W3/25 antibody. We have now evaluated the generality and mechanism(s) of ganglioside-induced modulation of CD4 expressed by mouse, rat, and human T helper lymphocytes. Ganglioside pretreatment induced rapid and selective disappearance of the CD4 molecule from T helper cells of all three species. The ganglioside effect was temperature- and dose-dependent, reversible within 24 hr of ganglioside removal, azide-insensitive, and was neutralized completely by 10% serum. CD4 modulation appeared to be a general property of gangliosides since the effect could be induced similarly by highly purified individual gangliosides with varying amounts of sialic acid, or by mixed gangliosides. The activity of gangliosides appeared to require both the lipid and sialated oligosaccharide moieties. Gangliosides did not inactivate antibody function, but prevented binding at the cell surface by 12 different monoclonal antibodies specific for a variety of different CD4 epitopes. Preclearance of CD4 by antibody-mediated capping reduced binding of 3H-GM1 to T helper cells. Labeled GM1 bound to several detergent-extracted and transblotted lymphocyte-associated proteins, but apparently did not bind directly to the CD4 molecule under these conditions. These results indicate that gangliosides induce a profound change in the molecular orientation of CD4 within the T helper cell membrane which renders epitopes on the CD4 molecule inaccessible to antibody. This ganglioside effect represents a novel pathway which may contribute to the understanding of the role of CD4 as a regulatory molecule and as a specific receptor for the acquired immune deficiency syndrome virus.

O-acetylation of a cell-surface carbohydrate creates discrete molecular patterns during neural development

Abstract: The cell-surface antigen detected by the monoclonal antibody JONES is expressed in the retina and a number of other central nervous system regions of the rat during the latter part of embryonic development and the early postnatal period. In addition to the expression on certain neuroblast populations it is found on some but not all axons and is also expressed at high levels on the end feet of radial glia in regions through which axons actively grow. In the perinatal rat retina, almost all the antigenic activity was carried on a ganglioside migrating between GM1 and GM2. The epitope recognized by antibody JONES was base labile and treatment with 0.1 M sodium carbonate or ammonia vapor converted the antigen into GD3. Resistance to oxidation by sodium periodate and reformation of the epitope by chemical acetylation of base-treated gangliosides with N-acetylimidazole identify the antigen as 9-O-acetyl GD3. The acetylation of GD3 seems to be regulated independently from GD3 expression itself since acetylated and nonacetylated GD3 do not have identical immunocytochemical distributions in the developing central nervous system. In addition, five independent human melanoma cell lines varied substantially in their expression of 9-O-acetyl GD3, even though they all expressed high levels of GD3. Acetylation of ganglioside-linked sialic acid provides a mechanism for generating unique patterns of surface carbohydrates, which may influence cell interactions in development.

Two integral membrane proteins located in the cis-middle and trans-part of the Golgi system acquire sialylated N-linked carbohydrates and display different turnovers and sensitivity to cAMP-dependent phosphorylation.

Abstract: The localization and chemical characteristics of two Golgi integral membrane proteins (GIMPs) have been studied using monoclonal antibodies. The two proteins are segregated in different parts of the Golgi system and whereas GIMPc(130 kD) is located in the cis and medial cisternae, GIMPt (100 kD) is confined in the trans-most cisterna and trans-tubular network. Both GIMPs are glycoproteins that contain N- and O-linked carbohydrates. The N-linked carbohydrates were exclusively of the complex type. Although excluded from the trans-side of the Golgi system, where sialylation is believed to occur, GIMPc acquires sialic acid in both its N- and O-linked carbohydrates. Sialic acid was also detected in the N-linked carbohydrates of GIMPt. GIMPc is apparently phosphorylated in the luminal domain in vivo. Phosphorylation occurred exclusively on serine and was stimulated by dibutyryl cyclic AMP. GIMPc and GIMPt displayed half-lives of 20 and 9 h, respectively.

Gangliosides and Modulation of Neuronal Functions

Abstract: Gangliosides (= amphiphilic sialic acid containing glycosphingolipids) are components of all plasma membranes and as such ubiquitous in vertebrate tissues. They are especially abundant and complex in neuronal membranes, where they are anchored in the outer leaflet of the membrane bilayer by their hydrophobic ceramide moiety, while exposing their negatively charged hydrophilic and spacious sialo-oligosaccharide residue into the extracellular space (Fig. 1). Together with neutral glycolipids, glycoproteins and glycosaminoglycans, gangliosides comprise the glycocalix of the cell surface.

Selective inactivation of influenza C esterase: a probe for detecting 9-O-acetylated sialic acids

Abstract: The influenza C virus (INF-C) hemagglutinin recognizes 9-O-acetyl-N-acetylneuraminic acid. The same protein contains the receptor-destroying enzyme (RDE), which is a 9-O-acetyl-esterase. The RDE was inactivated by the serine esterase inhibitor di-isopropyl fluorophosphate (DFP). [3H]DFP-labeling localized the active site to the heavy chain of the glycoprotein. DFP did not alter the hemagglutination or fusion properties of the protein, but markedly decreased infectivity of the virus, demonstrating that the RDE is important for primary infection. Finally, DFP-treated INF-C bound specifically and irreversibly to cells expressing 9-O-acetylated sialic acids. This provides a probe for a molecule that was hitherto very difficult to study.