Natural ligands of the B cell adhesion molecule CD22 beta can be masked by 9-O-acetylation of sialic acids

CD22 beta is a B cell-restricted phosphoprotein expressed on the surface of mature resting B cells. It mediates interactions with other cells partly or exclusively via recognition of alpha 2-6-linked sialic acids on glycoconjugates. The sialylated N-linked oligosaccharides recognized best by CD22 beta are common to many glycoproteins, suggesting that additional regulatory mechanisms may exist. Since the exocyclic side chain of sialic acid is required for recognition, we explored the effects of a naturally occurring modification of the side chain, 9-O-acetylation. Semisynthetic N-linked oligosaccharides terminating with 9-O-acetylated, alpha 2-6-linked sialic acids showed markedly reduced binding to CD22 beta relative to their non-O-acetylated counterparts. Murine lymphoid cells were probed for natural CD22 beta ligands that might be O-acetylated using recombinant soluble forms of CD22 beta (CD22 beta Rg) and influenza C esterase (CHE-Fc, which specifically removes 9-O-acetyl esters from sialic acids). By flow cytometry analysis, CD22 beta Rg binding to splenic B cells and a subset of T cells was increased by pretreatment with CHE-Fc, indicating that some potential CD22 beta ligands are naturally "masked" by 9-O-acetylation. Unmasking of these CD22 beta ligands by removal of 9-O-acetyl esters from intact splenocytes substantially increases their CD22 beta-dependent adhesion in an in vitro adhesion assay. Probing of murine lymphoid tissue sections by CD22 beta Rg and CHE-Fc treatment demonstrates regionally restricted and differentially expressed patterns of distribution between masked and unmasked ligands. For example, lymph node-associated follicular B cells express high levels of CD22 beta ligands, none of which are masked by 9-O-acetylation. In contrast, the ligands on lymph node-associated dendritic cells are almost completely masked by 9-O-acetylation, suggesting that masking may regulate interactions between CD22 beta-positive B cells and dendritic cells. In the thymus, only medullary cells express CD22 beta ligands, and a significant portion of these are masked by 9-O-acetylation, particularly at the cortical-medullary junction. Thus, 9-O-acetylation of sialic acids on immune cells is in a position to negatively regulate CD22 beta adhesion events in a manner depending on both cell type and tissue localization.     

Molecular mechanisms of serum resistance of human influenza H3N2 virus and their involvement in virus adaptation in a new host

H3N2 human influenza viruses that are resistant to horse, pig, or rabbit serum possess unique amino acid mutations in their hemagglutinin (HA) protein. To determine the molecular mechanisms of this resistance, we characterized the receptor-binding properties of these mutants by measuring their affinity for total serum protein inhibitors and for soluble receptor analogs. Pig serum-resistant variants displayed a markedly decreased affinity for total pig serum sialylglycoproteins (which contain predominantly 2-6 linkage between sialic acid and galactose residues) and for the sialyloligosaccharide 6'-sialyl(N-acetyllactosamine). These properties correlated with the substitution 186S-->I in HA1. The major inhibitory activity in rabbit serum was found to be a beta inhibitor with characteristics of mannose-binding lectins. Rabbit serum-resistant variants exhibited decreased sensitivity to this inhibitor due to the loss of a glycosylation sequon at positions 246 to 248 of the HA. In addition to a somewhat reduced affinity for 6'-sialyl(N-acetyllactosamine)-containing receptors, horse serum-resistant variants lost the ability to bind the viral neuraminidase-resistant 4-O-acetylated sialic acid moieties of equine alpha2-macroglobulin because of the mutation 145N-->K/D in their HA1. These results indicate that influenza viruses become resistant to serum inhibitors because their affinity for these inhibitors is reduced. To determine whether natural inhibitors play a role in viral evolution during interspecies transmission, we compared the receptor-binding properties of H3N8 avian and equine viruses, including two strains isolated during the 1989 to 1990 equine influenza outbreak, which was caused by an avian virus in China. Avian strains bound 4-O-acetylated sialic acid residues of equine alpha2-macroglobulin, whereas equine strains did not. The earliest avian-like isolate from a horse influenza outbreak bound to this sialic acid with an affinity similar to that of avian viruses; a later isolate, however, displayed binding properties more similar to those of classical equine strains. These data suggest that the neuraminidase-resistant sialylglycoconjugates present in horses exert selective pressure on the receptor-binding properties of avian virus HA after its introduction into this host.     

Sialylation of the sialic acid binding lectin sialoadhesin regulates its ability to mediate cell adhesion

The macrophage-specific cell surface receptor sialoadhesin, which is a member of the newly recognized family of sialic acid binding lectins called siglecs, binds glycoprotein and glycolipid ligands containing a2-3-linked sialic acid on the surface of several leukocyte subsets. Recently, the sialic acid binding activity of the siglec CD22 has been demonstrated to be regulated by sialylation of the CD22 receptor molecule. In the present work, we show that desialylation of in vivo macrophage sialylconjugates enhances sialoadhesin-mediated lectin activity. Herein, we show that receptor sialylation of soluble sialoadhesin inhibits its binding to Jurkat cell ligands, and that charge-dependent repulsion alone cannot explain this inhibition. Furthermore, we show that the inhibitory effect of sialic acid is partially dependent on the presence of an intact exocyclic side chain. These results, in conjunction with previous findings, suggest that sialylation of siglecs by specific glycosyltransferases may be a common mechanism by which siglec-mediated adhesion is regulated.     

Monomeric, monovalent derivative of Maackia amurensis leukoagglutinin. Preparation and application to the study of cell surface glycoconjugates by flow cytometry

A stable subunit of Maackia amurensis leukoagglutinin (MMAL) was prepared by the selective reduction of disulfide bridges between the subunits followed by alkylation with 4-vinylpyridine. MMAL failed to precipitate fetuin, whereas it retained its ability to bind to the same glycoprotein coated on a plastic plate, indicating the monovalency of this derivative. This binding to immobilized fetuin was inhibited by a haptenic sugar, Neu5Ac alpha 2-3lactose, with the same inhibitory potency as against the native M. amurensis leukoagglutinin. Microscopic observation as well as flow cytometric analyses showed that Chinese hamster ovary cells were clearly stained with fluorescein isothiocyanate-labeled MMAL without any detectable agglutination. This staining was inhibited by the addition of fetuin or by the sugar chains of fetuin. Differences in the types of sialylated glycoconjugates on the cell surface of several cell lines were detected by the combined use of fluorescein isothiocyanate-labeled MMAL and the monomeric derivative of elderberry bark lectin (specific for the Neu5Ac alpha 2-6Gal/GalNAc sequence) by flow cytometry. These results demonstrate the usefulness of these monovalent derivatives of sialylated oligosaccharide-specific lectins as probes for the analysis of cell surface glycoconjugates containing sialic acid by the technique of flow cytometry.     

Glycan specificity of myelin-associated glycoprotein and sialoadhesin deduced from interactions with synthetic oligosaccharides

Myelin-associated glycoprotein (MAG) and sialoadhesin (Sn) bind to sialylated glycans on cell surfaces and are thought to be involved in cell-cell interactions. In order to investigate how the interactions of these proteins are influenced by the glycan structure, we compared the inhibitory potencies of different synthetic monovalent oligosaccharides and polyvalent polyacrylamide derivatives. Using oligosaccharides with modifications in the sialic acid, galactose or N-acetylglucosamine moieties, we could demonstrate that both MAG and Sn bind with high preference to alpha2,3-linked sialic acid and interact at least with the three terminal monosaccharide units. For MAG, contacts with even more distant monosaccharides are likely, since pentasaccharides are bound better than trisaccharides. Also, an additional sialic acid at position six of the third-terminal monosaccharide unit enhances binding to MAG, whereas it does not influence binding to Sn significantly. Modifications of the sialic acid glycerol side chain demonstrated that the hydroxy groups at positions 8 and 9 are required for binding to both proteins. Surprisingly, MAG binds 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid significantly better than N-acetylneuraminic acid, whereas Sn prefers the latter structure. These results indicate that the interactions of MAG and Sn are mainly with sialic acid and that additional contacts with the subterminal galactose and N-acetylglucosamine residues also contribute to the binding strength, although to a lesser degree.     

Lack of volatilization and escape of orally administered trichloroethylene from the gastrointestinal tract of rats

The sugar residues in glycoconjugates present in the parotid and mandibular glands of the adult fallow-deer were detected and characterized by using a battery of eight different lectin-horseradish peroxidase conjugates. In some cases a treatment with sialidase preceded the lectin staining. Parotid secretory cells produced glycoconjugates with N-acetylgalactosamine, N-acetylglucosamine and mannose residues. Mucous acinar cells were the most reactive sites of the mandibular gland and contained conspicuous quantities of oligosaccharides with terminal sialic acid radicals. Galactosil-(beta 1-->3)N-acetylgalactosamine was the most abundant penultimate sugar linked to N-acetylneuraminic acid. Mandibular mucous cells also presented N-acetylglucosamine and sialylated components with the terminal dimer sialic acid-N-acetylgalactosamine. Demilunar cells contained glycoconjugates with fucose and mannose residues. The apical surface of duct cells was stained by all the lectins.     

The receptor destroying enzyme of influenza viruses. The role of the receptor destroying enzyme on the budding and the release of influenza viruses from the host cells

Host-range of influenza viruses are established by many factors in nature such as the quantitative, qualitative aspects of the viral receptor, the permissive and non permissive states of host-cell condition, and also the antibody and inhibitor pressure present in the host. Influenza virus hemagglutinin recognizes specific sialyl-sugar chains of the host cell membranes. In this paper, the structure and function of sialyl-sugar chains as influenza virus receptors are described in relation to the variation of influenza viruses and the molecular evolution of influenza virus hemagglutinin.     

Variation of influenza viruses and their recognition of the receptor sialo-sugar chains

Influenza A, B viruses contain 2 viral specific, membrane associated glycoprotein antigens, hemagglutinin and sialidase. Hemagglutinin is essential for the initial binding of the virus to the cell membrane receptors that contain sialic acid such as gangliosides and sialo-glycoproteins. Hemagglutinin is also important for the intracellular viral uncoating by the low pH fusion processes. The evolution of the influenza viruses and host range variation come from the mutation of hemagglutinin and sialidase genes and change of their sialo-sugar chain recognition together with alteration in the antigenic epitopes. In this report, the molecular mechanism of the relationship between the evolutional change of the viral glycoproteins, especially hemagglutinin molecules and the change of the receptor binding specificity is reported, and also the strategy for the development of a new universal vaccine which generates the antibody whose supervariable region mimics the common receptor sialo-sugar chains for all the subtypes of influenza viruses is also described.     

Structure-activity relationship studies of novel carbocyclic influenza neuraminidase inhibitors

A series of influenza neuraminidase inhibitors with the cyclohexene scaffold containing lipophilic side chains have been synthesized and evaluated for influenza A and B neuraminidase inhibitory activity. The size and geometry of side chains have been modified systematically in order to investigate structure-activity relationships of this class of compounds. The X-ray crystal structures of several analogues complexed with neuraminidase revealed that the lipophilic side chains bound to the hydrophobic pocket consisted of Glu276, Ala246, Arg224, and Ile222 of the enzyme active site. The structure-activity relationship studies of this series have also demonstrated remarkably different inhibitory potency between influenza A and B neuraminidase. This indicated that the lipophilic side chains had quite different hydrophobic interactions with influenza A and B neuraminidase despite their complete homology in the active site. Influenza B neuraminidase appeared to be much more sensitive toward the increased steric bulkiness of inhibitors compared to influenza A neuraminidase. From the extensive structure-activity relationship investigation reported in this article, GS 4071 emerged as one of the most potent influenza neuraminidase inhibitors against both influenza A and B strains.     

Influenza A virus hemagglutinin is a B cell-superstimulatory lectin

Influenza A viruses display T cell-independent polyclonal B cell-activating properties which are mediated by the B cell-superstimulatory envelope glycoprotein hemagglutinin (HA). In this report, the receptor-binding requirements for B cell activation by influenza viruses were expected. Neuraminidase treatment of resting mature B cells from BALB/c mice abrogated late (proliferation/immunoglobulin synthesis), early (up-regulation of cell surface markers, including CD25, B220, and B7-1) and very-early events (homotypic adhesion) in virus-responding B lymphocytes. Similarly, pretreatment of murine responder cells with different inhibitors of N-glycosylation (tunicamycin, deoxymannojirimycin) significantly suppressed subsequent B lymphocyte activation by HA, but not control responses to lipopolysaccharide or anti-mu. Assays with chimeric HA transfectants, expressing the loop region of epitope B (amino acids 155-160) of the globular head of H2 (high B cell-stimulatory subtype) or H3 (medium-stimulatory subtype) on the protein backbone of a low-stimulatory subtype (H1) failed to alter the B cell-stimulatory activity of the virus, suggesting that the hypervariable loop region is not crucial in determining the B cell-activating properties of the protein. Collectively, our results imply that the B cell-superstimulatory function of influenza virus HA is not mediated by a direct protein/protein interaction, but via binding of HA to terminal sialic acid residues on cell surface receptor glycoproteins. These findings identify the influenza virus HA glycoprotein as the first viral lectin with lymphocyte-activating properties.     

Activation of protein kinase C induces cortical granule exocytosis in a Ca(2+)-independent manner, but not the resumption of cell cycle in porcine eggs

N-Acetylneuraminic acid (1) is a common sugar in many biological recognition processes. Neuraminidase enzymes recognize and cleave terminal sialic acids from cell surfaces. Viral entry into host cells requires neuraminidase activity, thus inhibition of neuraminidase is a useful strategy for development of drugs for viral infections. A recent crystal structure for influenza viral neuraminidase with sialic acid bound shows that the sialic acid is in a boat conformation [Prot Struct Funct Genet 14: 327 (1992)]. Our studies seek to determine if structural pre-organization can be achieved through the use of sialyllactones. Determination of whether siallylactones are pre-organized in a binding conformation requires conformational analysis. Our inability to find a systematic study comparing the results obtained by various computational methods for carbohydrate modeling led us to compare two different conformational analysis techniques, four different force fields, and three different solvent models. The computational models were compared based on their ability to reproduce experimental coupling constants for sialic acid, sialyl-1,4-lactone, and sialyl-1,7-lactone derivatives. This study has shown that the MM3 forcefield using the implicit solvent model for water implemented in Macromodel best reproduces the experimental coupling constants. The low-energy conformations generated by this combination of computational methods are pre-organized toward conformations which fit well into the active site of neuraminidase.     

Infection of glial cells by the human polyomavirus JC is mediated by an N-linked glycoprotein containing terminal alpha(2-6)-linked sialic acids

The human JC polyomavirus (JCV) is the etiologic agent of the fatal central nervous system (CNS) demyelinating disease progressive multifocal leukoencephalopathy (PML). PML typically occurs in immunosuppressed patients and is the direct result of JCV infection of oligodendrocytes. The initial event in infection of cells by JCV is attachment of the virus to receptors present on the surface of a susceptible cell. Our laboratory has been studying this critical event in the life cycle of JCV, and we have found that JCV binds to a limited number of cell surface receptors on human glial cells that are not shared by the related polyomavirus simian virus 40 (C. K. Liu, A. P. Hope, and W. J. Atwood, J. Neurovirol. 4:49-58, 1998). To further characterize specific JCV receptors on human glial cells, we tested specific neuraminidases, proteases, and phospholipases for the ability to inhibit JCV binding to and infection of glial cells. Several of the enzymes tested were capable of inhibiting virus binding to cells, but only neuraminidase was capable of inhibiting infection. The ability of neuraminidase to inhibit infection correlated with its ability to remove both alpha(2-3)- and alpha(2-6)-linked sialic acids from glial cells. A recombinant neuraminidase that specifically removes the alpha(2-3) linkage of sialic acid had no effect on virus binding or infection. A competition assay between virus and sialic acid-specific lectins that recognize either the alpha(2-3) or the alpha(2-6) linkage revealed that JCV preferentially interacts with alpha(2-6)-linked sialic acids on glial cells. Treatment of glial cells with tunicamycin, but not with benzyl N-acetyl-alpha-D-galactosaminide, inhibited infection by JCV, indicating that the sialylated JCV receptor is an N-linked glycoprotein. As sialic acid containing glycoproteins play a fundamental role in mediating many virus-cell and cell-cell recognition processes, it will be of interest to determine what role these receptors play in the pathogenesis of PML.     

Matrix-assisted laser desorption/ionization mass spectrometry of acidic glycoconjugates facilitated by the use of spermine as a co-matrix

Negative-ion matrix-assisted laser desorption/ionization mass spectra of sialyated glycoconjugates were acquired employing 2,5-dihydroxybenzoic acid (DHB) in conjunction with spermine as a co-matrix. The addition of spermine to DHB permitted an improved crystal formation as well as a higher analyte solubility. Moreover, DHB/spermine appears to minimize alkali adduct formation, thus allowing the sample analysis without desalting. The combined matrix permitted the analysis of complex sialylated and sialylated/fucosylated structures down to the femotomole range. The ability to use such a matrix also facilitates determination of the sialic acid linkages (in combination with a specific enzyme cleavage). The matrix also appears suitable for studies on gangliosides.     

Sialic acid determinations of serum and urine specimens in calcium-containing urinary stone formers

PURPOSE: The aim of the present study was to compare sialic acid concentrations of serum and urine specimens in both calcium (Ca)-containing urinary stone formers and non-stone formers. Moreover, we studied inhibitory activity of sialic acid upon the calcium oxalate (CaOx) crystal aggregation and growth. MATERIALS AND METHODS: Sialic acid determinations were done on fresh serum and urine samples of 35 Ca-containing urinary stone formers (stone formers group) and 20 non-stone formers (patient controls group). Inhibitory activity of sialic acid upon the CaOx crystal aggregation and growth was studied by using in vitro assay method of seed crystal system. RESULTS: Serum sialic acid concentrations were found to be similar in the two groups. Urinary sialic acid concentrations were significantly lower in the urine specimens of stone formers than in their patient controls. Sialic acid showed a dose dependent inhibitory activity upon the CaOx crystal aggregation and growth into seed crystal method. CONCLUSION: It is suggested that urinary sialic acid may play some role during the phase of stone formation from the results of the present study, because sialic acid shows marked inhibitory activity upon the CaOx crystal aggregation and growth at concentrations higher than 100 mg/dl.     

13C nuclear magnetic resonance study of molecular motions and conformational transitions in muscle calcium binding parvalbumins

13C nuclear magnetic resonance is used to detect the Ca2+ ion controlled conformational transition in muscle calcium binding parvalbumin and to study its intramolecular motions. Nuclear relaxation parameters are used to evaluate the reorientation rates of the protein and some of the amino acid side chains. While peripheral residues exhibit greater motional freedom than the protein interior, an interesting finding is that significant rapid internal motion is present in the phenylalanine rings comprising the hydrophobic core of the protein.     

Differential and cooperative polysialylation of the neural cell adhesion molecule by two polysialyltransferases, PST and STX

PST and STX are polysialyltransferases that form polysialic acid in the neural cell adhesion molecule (NCAM), and these two polysialyltransferases often exist together in the same tissues. To determine the individual and combined roles of PST and STX in polysialic acid synthesis, in the present study we asked if PST and STX differ in the acceptor requirement and if PST and STX act together in polysialylation of NCAM. We first examined whether PST and STX differ in the requirement of sialic acid and core structures of N-glycans attached to NCAM. Polysialic acid was formed well on Lec4 and Lec13 cells, which are defective in N-acetylglucosaminyltransferase V and GDP-fucose synthesis, respectively, demonstrating that a side chain elongating from GlcNAcbeta1-->6Manalpha1-->6R and alpha-1,6-linked fucose are not required. PST and STX were found to add polysialic acid on NCAM.Fc molecules sialylated by alpha-2,3- or alpha-2,6-linkage in vitro, but not on NCAM.Fc lacking either sialic acid. These results indicate that both PST and STX have relatively broad specificity on N-glycan core structures in NCAM and no remarkable difference exists between PST and STX for the requirement of core structures and sialic acid attached to the N-glycans of NCAM. We then, using various N-glycosylation site mutants of NCAM, discovered that PST strongly prefer the sixth N-glycosylation site, which is the closest to the transmembrane domain, over the fifth site. STX slightly prefer the sixth N-glycosylation site over the fifth N-glycosylation site. The results also demonstrated that polysialic acid synthesized by PST is larger than that synthesized by STX in vitro. Moreover, a mixture of PST and STX more efficiently synthesized polysialic acid on NCAM than PST or STX alone. These results suggest that polysialylation of NCAM is influenced by the difference between PST and STX in their preference for N-glycosylation sites on NCAM. The results also suggest that PST and STX form polysialylated NCAM in a synergistic manner.     

Purification of the lysosomal sialic acid transporter. Functional characteristics of a monocarboxylate transporter

Sialic acid and glucuronic acid are monocarboxylated monosaccharides, which are normally present in sugar side chains of glycoproteins, glycolipids, and glycosaminoglycans. After degradation of these compounds in lysosomes, the free monosaccharides are released from the lysosome by a specific membrane transport system. This transport system is deficient in the human hereditary lysosomal sialic acid storage diseases (Salla disease and infantile sialic acid storage disease, OMIM 269920). The lysosomal sialic acid transporter from rat liver has now been purified to apparent homogeneity in a reconstitutively active form by a combination of hydroxyapatite, lectin, and ion exchange chromatography. A 57-kDa protein correlated with transport activity. The transporter recognized structurally different types of acidic monosaccharides, like sialic acid, glucuronic acid, and iduronic acid. Transport of glucuronic acid was inhibited by a number of aliphatic monocarboxylates (i.e. lactate, pyruvate, and valproate), substituted monocarboxylates, and several dicarboxylates. cis-Inhibition, trans-stimulation, and competitive inhibition experiments with radiolabeled glucuronic acid as well as radiolabeled L-lactate demonstrated that L-lactate is transported by the lysosomal sialic acid transporter. L-Lactate transport was proton gradient-dependent, saturable with a Km of 0.4 mM, and mediated by a single mechanism. These data show striking biochemical and structural similarities of the lysosomal sialic acid transporter with the known monocarboxylate transporters of the plasma membrane (MCT1, MCT2, MCT3, and Mev).     

A H1 hemagglutinin of a human influenza A virus with a carbohydrate-modulated receptor binding site and an unusual cleavage site

Two receptor binding variants of the influenza virus A/Tübingen/12/85 (H1N1) were separated by their different plaque formation in MDCK cells. Hemagglutination of variant I was restricted to red blood cells of guinea pigs, whereas variant II also hemagglutinated chicken cells. The variants differed also in their ability to bind to alpha 2,6-linked sialic acid. Evidence is presented that this difference is determined by a complex carbohydrate side chain at asparagine131 near the receptor binding site which is absent in variant II. With both variants, the arginine found at the cleavage site of all other human isolates analyzed so far was replaced by lysine.     

Analysis of viral and cellular parameters which affect the fusion process of influenza viruses

In the present investigation we studied the fusogenic process developed by influenza A, B and C viruses on cell surfaces and different factors associated with virus and cell membrane structures. The biological activity of purified virus strains was evaluated in hemagglutination, sialidase and fusion assays. Hemolysis by influenza A, B and C viruses ranging from 77.4 to 97.2%, from 20.0 to 65.0% from 0.2 to 93.7% and from 9.0 to 76.1% was observed when human, chicken, rabbit and monkey erythrocytes, respectively, were tested at pH 5.5. At this pH, low hemolysis indexes for influenza A, B and C viruses were observed if horse erythrocytes were used as target cells for the fusion process, which could be explained by an inefficient receptor binding activity of influenza on N-glycolyl sialic acids. Differences in hemagglutinin receptor binding activity due to its specificity to N-acetyl or N-glycolyl cell surface oligosaccharides, density of these cellular receptors and level of negative charges on the cell surface may possibly explain these results, showing influence on the sialidase activity and the fusogenic process. Comparative analysis showed a lack of dependence between the sialidase and fusion activities developed by influenza B viruses. Influenza A viruses at low sialidase titers (< 2) also exhibited clearly low hemolysis at pH 5.5 (15.8%), while influenza B viruses with similarly low sialidase titers showed highly variable hemolysis indexes (0.2 to 78.0%). These results support the idea that different virus and cell-associated factors such as those presented above have a significant effect on the multifactorial fusion process.     

Unsteady flow in a rigid 3-D model of the carotid artery bifurcation

Colominic acid (CA), an alpha-(2-->8) N-acetylneuraminic acid (sialic acid) polymer (average molecular weight of 10 kDa) was activated by periodate oxidation of carbon 7 at the non-reducing end of the saccharide. The oxidized CA was then coupled to catalase by reductive amination in the presence of sodium cyanoborohydride. The extent of sialylation of catalase, estimated by ammonium sulfate precipitation as 3.8+/-0.4 (mean+/-S.D.) moles of CA per mole of catalase, did not improve significantly when depolymerized CA was used in the coupling reaction. At the end of the coupling reaction, sialylated catalase exhibited a two-fold (70%) retention of initial activity compared to enzyme controls (29-35%) subjected to the same conditions. Formation of sialylated catalase was confirmed by ammonium sulfate or trichloroacetic acid precipitation, molecular sieve chromatography and SDS-PAGE electrophoresis. Enzyme kinetics studies revealed an increase in the apparent Km of the enzyme from 70.0 (native) to 122.9 mmol l-1 H2O2 (sialylated catalase) indicating a reduction of enzyme affinity for the substrate (hydrogen peroxide) on sialylation. Compared to native enzyme, sialylated catalase was much more stable in the presence of specific proteinases, completely resisting degradation by chymotrypsin and losing only some of its activity in the presence of trypsin. The increased stability conferred to catalase by sialylation agrees with similar observations on enzymes modified by other hydrophilic molecules (e.g., monomethoxypoly(ethyleneglycol)) and suggests that steric stabilization with the biodegradable polysialic acid may prove an alternative means to render therapeutic proteins more effective in vivo.