Sialic Acid Acceptor Research Articles

Nuclear and cytoplasmic lectin receptor sites in rat Py1a osteoblasts.

The intracellular distribution of lectin receptor sites was studied in the rat Pyla osteoblasts using immunofluorescence at the confocal microscopy level. This immortalized cell line was found to represent a satisfactory model to study the occurrence and distribution of sugar moieties. Our data showed distinct affinity patterns of lectins recognizing different terminal or internal sugar residues. For some lectins, the binding patterns appeared to be cell cycle-independent, whereas for PNA the cell cycle greatly influenced the nuclear binding. By combining lectin affinity with sialidase degradation and alcoholic saponification the sialic acid acceptor sugars and derivatives were also visualized. In particular, glycoconjugates with sialic acids linked to beta-galactose, and mainly C4 acetylated, were located in the cytoplasm, while glycoconjugates characterized by sialic acids linked to alpha-N-acetylgalactosamine, and devoid of acetyl groups at C4, were almost exclusively found in the nucleus. The comparison of lectin affinities, with and without prior glycosidase digestions, allowed us to gain further insight into the chemical composition of glycoconjugates that act as the lectin receptor sites that appeared to belong to O- and N-linked glycoconjugates. The use of additional enzymatic treatments were useful to better establish the localization of nuclear receptor sites and results were compared with previous studies about endogenous and exogenous lectins in an attempt to reconcile the association of lectins and sugars within the nucleus and their possible involvement in modulation of cell proliferation and/or response to chemical signals. The above digestions also provided information about the cytoplasmic binding patterns.

Trypanosoma cruzi Surface Mucins with Exposed Variant Epitopes

The protozoan parasite Trypanosoma cruzi, the agent of Chagas disease, has a large number of mucin molecules on its surface, whose expression is regulated during the life cycle. These mucins are the main acceptors of sialic acid, a monosaccharide that is required by the parasite to infect and survive in the mammalian host. A large mucin-like gene family named TcMUC containing about 500 members has been identified previously in T. cruzi. TcMUC can be divided into two subfamilies according to the presence or absence of tandem repeats in the central region of the genes. In this work, T. cruzi parasites were transfected with one tagged member of each subfamily. Only the product from the gene with repeats was highly O-glycosylated in vivo. The O-linked oligosaccharides consisted mainly of beta-d-Galp(1-->4)GlcNAc and beta-d-Galp(1-->4)[beta-d-Galp(1-->6)]-d-GlcNAc. The same glycosyl moieties were found in endogenous mucins. The mature product was anchored by glycosylphosphatidylinositol to the plasma membrane and exposed to the medium. Sera from infected mice recognized the recombinant product of one repeats-containing gene thus showing that they are expressed during the infection. TcMUC genes encode a hypervariable region at the N terminus. We now show that the hypervariable region is indeed present in the exposed mature N termini of the mucins because sera from infected hosts recognized peptides having sequences from this region. The results are discussed in comparison with the mucins from the insect stages of the parasite (Di Noia, J. M., D'Orso, I., Sánchez, D. O., and Frasch, A. C. C. (2000) J. Biol. Chem. 275, 10218-10227) which do not have variable regions.

Development of spectrophotometric method for monitoring trans-sialidase reaction and its application

A spectrophotometric method that can monitor the trans-sialidase catalyzed reaction was devised by using a chromogenic substrate, o-nitrophenyl-β-d-galactopyranoside (ONPG), as a sialic acid acceptor. This method yielded results that were consistent with those from HPLC, and could be very conveniently used in the screening of sialic acid donor or inhibitor of trans-sialidase.

Characterisation of the enzymatic 4-O-acetylation of sialic acids in microsomes from equine submandibular glands.

Microsomes prepared from equine submandibular glands and incubated with tritium-labelled AcCoA incorporated acid-insoluble radioactivity in a manner dependent on time, protein, membrane integrity and AcCoA concentration, with incorporation being optimal at 37 degrees C and pH 6.6. Under the experimental conditions used a K(M) of 32.1 microM for AcCoA and a V(max) of 1.2 pmol/mg protein x min was obtained. The incorporation of acid-insoluble radioactivity was also inhibited by CoA in a competitive manner (K(i)=240 microM), as well as by para-chloromercuribenzoate, 3'-dephospho-CoA, 5'-IDP, 5'-ADP, beta-NAD and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate. We demonstrate here that this incorporation of radioactivity into endogenous sialic acid is due to the action of an AcCoA:sialate-4-O-acetyltransferase [EC 2.3.1.44]. Radio thin-layer chromatography analyses of propionic acid-released sialic acids showed that the incorporation of radioactivity correlated with the formation of a radiolabelled species that co-migrated with authentic Neu4,5Ac2. Saponification experiments using NaOH, mouse hepatitis virus strain S and Influenza C/JJ/50 virus also showed that the transfer of [3H]acetyl groups from [3H]AcCoA to endogenous sialic acid acceptors was occurring exclusively at carbon 4 of the pyranose ring.

Relevant glycoconjugates on the surface of Trypanosoma cruzi.

Although Chagas disease has been discovered90 years ago, only after 1970 substantial progresshas been made in the knowledge of molecular as-pects of the host-parasite interaction. The amountof information gathered in the past 30 years wasconsiderable, making it virtually impossible tocover all aspects of the biochemistry and molecu-lar biology of the Trypanosoma cruzi , the etiologicagent of the disease. T. cruzi colonizes differenthabitats, from the intestinal tract of an insect to thecytoplasm of a mammalian cell, with a brief pas-sage by the blood of the vertebrate host. An adap-tation of the parasite to these different environmentsshould occur and, consequently, differences in thecomposition of the plasma membrane should beexpected among the parasite stages. In fact, in eachparasite stage, molecules belonging to the surfacemembrane are the ones most probably involved ininteraction with the hosts. In the vertebrate side ofthe infective cycle, the parasite has to leave theblood by crossing the capillary walls, the basallaminae and the extracellular matrix before encoun-tering receptors located on the host cell plasmamembrane. In the invertebrate phase of the cycle,epimastigotes must attach to the perimicrovillarmembrane of the reduviid intestine in order to getprepared to differentiate to metacyclic trypo-mastigotes.This review will cover mainly the evolution ofknowledge which occurred with the parasite mac-romolecular structures residing in the plasma mem-brane and originally described by our laboratory.In fact, by extracting epimastigote forms with phe-nol it was possible to separate in polyacrylamidegels four bands developed by the Schiff reagentthat were called bands A, B, C, and D (Alves &Colli 1975). Band D was baptized as lipopeptidophosphoglycan (LPPG) (Lederkremer et al. 1976),and more recently T. cruzi Type-1 GIPL (Ferguson1997), and bands A, B, C have been shown to becompounds which are the main sialic acid accep-tors on T. cruzi membranes, including the now wellstudied mucins (Acosta Serrano et al. 1995). Inaddition, trypomastigotes have on their surface afamily of glycoproteins (Abuin et al. 1989) prob-ably involved in parasite interiorization (Alves etal. 1986) and adhesion (Giordano et al. 1994, 1999,Alves 1996) which have been collectively calledTc-85 (Katzin & Colli 1983).Excellent reviews on related issues were re-cently published on surface receptors and trans-porters of trypanosomatids, including the glucosetransporter (Tetaud et al. 1997, Borst & Fairlamb1998) and trans-sialidase (Cross & Takle 1993,Colli 1993, Schenkman et al. 1994).

Sex-related expression of sialic acid acceptor sugars in the mouse submandibular gland. Simultaneous visualization by confocal laser scanning microscopy.

The novel combination of sialidase digestion with simultaneous PNA and DBA binding yielded marked differences on sialoglycoconjugate occurrence and distribution in the mouse submandibular gland acinar cells of the two sexes. Striking differences in the structure of terminal disaccharides within stored secretory sialoglycoconjugates were also found. High content of sialic acid, characterized by the terminal sequence sialic acid-alpha-N-acetylgalactosamine, was established to only occur in the male acini where secretory cells appeared to be differently stained; indeed, some cells exhibited codistribution of sialic acid-alpha-N-acetylgalactosamine and sialic acid-beta-galactose terminal disaccharides, whereas other ones exclusively contained one of the two kinds of terminal sequences. In the female acinar cells, the secretory products were found to be almost exclusively composed by glycoconjugates having sialic acid subtended to beta-galactose without appreciable differences between acinar cells. Our finding of such extensive differences in the acinar cells of male and female mice adds new insights into the submandibular gland sexual dimorphism, commonly attributed to the androgen responsiveness of the granular convoluted tubule portion of the gland.

Two distinct groups of mucin-like genes are differentially expressed in the developmental stages of Trypanosoma cruzi

Sialic acid acceptors of Trypanosoma cruzi are abundant mucin-like glycoproteins linked to the parasite membrane by a glycosylphosphatidyl inositol (GPI) anchor. They are heterogeneous and variable in different parasite stages. The protein portion of these mucins contains many threonine residues, and is thought to be encoded by a heterogeneous gene family. To investigate whether the high degree of heterogeneity in the mucin gene family is responsible for the diversity of mucins expressed on the parasite surface, we have studied the expression of mucin genes in several developmental stages of T. cruzi. We have found that mucins are expressed in all parasite stages. By using conserved sequences at 3′ end of translated sequences of the gene family and the splice leader sequence, we have isolated 120 mucin-like cDNAs by RT-PCR from epimastigote and trypomastigote mRNAs. All transcribed genes contain conserved 5′ and 3′ regions, which code for the signal peptide, the sequence for GPI anchor addition, and a conserved domain rich in threonine residues. The internal portions of these genes are highly variable in size and sequence, and can be grouped in two major categories. One group contains KP (1-2)T (6-8) repeats, a motif found in mammalian mucins in the central region. This group is expressed preferentially in the trypomastigote forms ready to be released from the infected mammalian cell. The other has highly variable sequences in the central portion, and is expressed in all parasite stages. Because the number of synonymous substitutions is equivalent to the non-synonymous substitutions in the second group, they are probably evolving neutrally. On the other hand, the KP (1-2)T (6-8) containing genes have more synonymous substitutions and are most likely under a strong selective pressure. We propose that the group of KP (1-2)T (6-8) motif corresponds to the highly glycosylated mucins of the trypomastigote stages. In the other group proteolysis may remove the central domain yielding small mucins, such as the mucins found in insect derived stages of T. cruzi.

Altered development of nerve fibers in the hippocampus of rats with methylazoxymethanol-induced microcephaly

The chapter discusses the investigations according to which the specificity of sialyltransferases from different animal species and tissues varies appreciably. However, as a result of the lack of well-defined macromolecular sialic-acid acceptors, the study was difficult. The influence of several parameters of the acceptor substrate on enzyme activity has been studied: the nature of the accepting sugar, the distance of the acceptor sugar from the polypeptide chain, and the presence of hydrophobic arms between the sugar and the protein. In a study described in the chapter, lysozyme was chosen as protein. The various synthetic substrates were tested for N-acetylneuraminic acid acceptor activity. The sialyltransferase studies were carried out with particulate (microsome) enzyme preparations obtained from frog (Rana esculenta) and cow liver, and from pig and cow submandibular glands. As a result of examinations, it was deduced that in liver and porcine submandibular gland glycoproteins, sialic acids are found to be linked to galactose residues, whereas in bovine submandibular gland glycoproteins, sialic acids are mainly linked to N-acetylgalactosamine.

Sialylation of Neisseria meningitidis lipooligosaccharide inhibits serum bactericidal activity by masking lacto-N-neotetraose.

Exogenous sialylation of gonococcal lipooligosaccharide causes resistance to serum bactericidal activity. The aim of this study was to determine how lipooligosaccharide sialylation affects the serum sensitivities of group C Neisseria meningitidis strains. The relationship between the degree of sialylation or expression of the lipooligosaccharide sialic acid acceptor, lacto-N-neotetraose (LNnT), of nine meningococcal strains and their sensitivities to a pool of normal human sera was assessed. All strains expressed LNnT that was variously endogenously sialylated. Susceptibility to serum bactericidal activity ranged from extremely sensitive to resistant in 50% serum. For endogenously sialylated strains, the amount of killing correlated with the amount of free LNnT above a threshold of expression; strains that expressed less than the threshold survived in 25% serum. All strains added more sialic acid when they were grown in medium that contained cytidine monophospho-N-acetylneuraminic acid. Exogenous sialylation reduced the expression of free LNnT and significantly increased serum resistance. Exogenous sialylation affected killing through both classical and alternative complement pathways. The killing of exogenously sialylated strains also correlated with the amount of free LNnT. The amounts of endogenous, exogenous, and total sialic acid bound to LNnT did not correlate with the resistance of strains to serum bactericidal activity; rather, the loss of free LNnT expression by sialylation was associated with resistance. In conclusion, the expression of free LNnT by group C meningococcal strains is directly associated with the amount of killing of organisms in pooled human sera. Both endogenous and exogenous lipooligosaccharide sialylation are associated with increased serum resistance by masking LNnT.

Passive transfer of a monoclonal antibody specific for a sialic acid-dependent epitope on the surface of Trypanosoma cruzi trypomastigotes reduces infection in mice.

Trypanosoma cruzi, the parasite that causes Chagas' disease, proliferates in the cytosol of mammalian cells. When the trypomastigote forms exit the infected cell, they become extensively sialylated because the parasite contains an enzyme called trans-sialidase. This enzyme efficiently catalyzes the transfer of bound sialic acid residues from host glycoconjugates to acceptors containing terminal beta-galactosyl residues on the parasite surface. The sialic acid acceptors are developmentally regulated mucin-like glycoproteins that are extremely abundant on the trypomastigote surface. In the present study, we determined whether passive transfer of monoclonal antibodies specific for sialic acid acceptors could reduce the acute infection induced by T. cruzi in a highly susceptible mouse strain. We found that passive transfer to naive mice of an immunoglobulin G1 monoclonal antibody directed to a sialylated epitope of these mucin-like glycoproteins significantly decreased parasitemia and the number of tissue parasites as measured by a DNA probe specific for T. cruzi. Upon challenge with trypomastigotes, mice which received this antibody also had a significant increase in survival. A statistically significant reduction in parasitemia could be accomplished with relatively small doses of immunoglobulin, and Fab fragments alone could not mediate protective immunity. The precise mechanism of parasite elimination is unknown; however, this monoclonal antibody does not lyse trypomastigotes in vitro in the presence of human complement or mouse spleen cells.

Mucin-like glycoprotein genes are closely linked to members of the trans-sialidase super-family at multiple sites in the Trypanosoma cruzi genome

In Trypanosoma cruzi a cell surface enzyme with trans-sialidase (TS) activity has been implicated as an important factor in establishing infection. The enzyme is encoded by genes belonging to a large super-family which on the basis of sequence has been subdivided into 4 groups. TS mediates the transfer of sialic acid residues from host glycoconjugates to acceptor molecules on the parasite surface. To study the organisation of the TS genes we isolated several distinct cosmids from a library constructed with DNA from the T. cruzi X10.6 clone. In these cosmids, the TS genes (group I) were present either as single copies or as a direct tandem repeat. A common feature of the cosmids was the presence of a related group III gene located 10–12kb downstream of the TS gene(s) and arranged in the same orientation. In several of the cosmids we also identified a mucin-like glycoprotein gene located between the group I and group III genes. The mucin-like genes are part of a large polymorphic family and contour clamped homogeneous electric field electrophoresis (CHEFE) analysis showed that they were linked to members of the TS super-family at multiple sites in the X10.6 genome. Screening of a second cosmid library made with DNA from the CL-Brener clone confirmed this multiple linkage suggesting that it is a common feature of the species. This genetic organisation may have important functional significance since the mucin-like glycoproteins are the major cell surface acceptors of sialic acid.

The Lipid Structure of the Glycosylphosphatidylinositol-anchored Mucin-like Sialic Acid Acceptors of Trypanosoma cruzi Changes during Parasite Differentiation from Epimastigotes to Infective Metacyclic Trypomastigote Forms

The major acceptors of sialic acid on the surface of metacyclic trypomastigotes, which are the infective forms of Trypanosoma cruzi found in the insect vector, are mucin-like glycoproteins linked to the parasite membrane via glycosylphosphatidylinositol anchors. Here we have compared the lipid and the carbohydrate structure of the glycosylphosphatidylinositol anchors and the O-linked oligosaccharides of the mucins isolated from metacyclic trypomastigotes and noninfective epimastigote forms obtained in culture. The single difference found was in the lipid structure. While the phosphatidylinositol moiety of the epimastigote mucins contains mainly 1-O-hexadecyl-2-O-hexadecanoylphosphatidylinositol, the phosphatidylinositol moiety of the metacyclic trypomastigote mucins contains mostly (approximately 70%) inositol phosphoceramides, consisting of a C18:0 sphinganine long chain base and mainly C24:0 and C16:0 fatty acids. The remaining 30% of the metacyclic phosphatidylinositol moieties are the same alkylacylphosphatidylinositol species found in epimastigotes. In contrast, the glycosylphosphatidylinositol glycan cores of both molecules are very similar, mainly Man alpha 1-2Man alpha 1-2Man alpha 1- 6Man alpha 1-4GlcN. The glycans are substituted at the GlcN residue and at the third alpha Man distal to the GlcN residue by ethanolamine phosphate or 2-aminoethylphosphonate groups. The structures of the desialylated O-linked oligosaccharides of the metacyclic trypomastigote mucin-like molecules, released by beta-elimination with concomitant reduction, are identical to the structures reported for the epimastigote mucins (Previato, J. O., Jones, C., Gonçalves, L. P. B., Wait, R., Travassos, L. R., and Mendoça-Previato, L. (1994) Biochem. J. 301, 151-159). In addition, a significant amount of nonsubstituted N-acetylglucosaminitol was released from the mucins of both forms of the parasite. Taken together, these results indicate that when epimastigotes transform into infective metacyclic trypomastigotes, the phosphatidylinositol moiety of the glycosylphosphatidylinositol anchor of the major acceptor of sialic acid is modified, while the glycosylphosphatidylinositol anchor and O-linked sugar chains remain essentially unchanged.

Lectin binding and identification of sialic acid acceptor sugars in rabbit oviduct under hormone administration.

Localization of individual glycosidic residues and sialic acid acceptor sugars was investigated by conjugated lectins in the rabbit oviduct under physiological hormonal conditions and human chorionic gonadotropin (HCG) administration. Ampulla and isthmus were found to exhibit lectin binding profiles typical of each hormonal stage. Two different sialylated glycomolecules were identified within the epithelial lining; in particular, sialoglycoconjugates characterized by the terminal sequence sialic acid-galactose were visualized in the secretory cells and the sialic acid-N-acetylgalactosamine terminal disaccharides were localized on both ciliated and secretory cells of the entire oviduct. Surface and cytoplasmic sialoglycoconjugates were also found to exhibit a differential behaviour inside the two oviduct tracts examined. Present findings further supported the idea that in ampulla and isthmus, the greatest modifications consequent to hormone treatment take place at different times.

Distribution of developmentally regulated trans-sialidases in the Kinetoplastida and characterization of a shed trans-sialidase activity from procyclic Trypanosoma congolense

The expression of developmentally regulated sialidase and trans-sialidase activities in kinetoplastid protozoa was investigated. The occurrence of these enzymes was found not to be a common feature among the Kinetoplastida, but to be restricted to distinct developmental life cycle stages of only a few species. While sialidases without trans-sialylating activities were demonstrated in Trypanosoma vivax and T. rangeli, trans-sialidase activity is expressed throughout the brucei-group and in T. congolense. Neither T. evansi, nor T. equiperdum express sialidases or trans-sialidases. Furthermore, the absence of both, sialidase and trans-sialidase activities was proven in the Leishmania, Crithidia, Herpetomonas, Leptomonas and Phytomonas, respectively. In all species tested, the occurrence of sialic acids coincides with the expression of trans-sialidase activity. Those parasites, which lack trans-sialidases or only display regular sialidases, also lack cell-bound sialic acids. The regular sialidase activity from bloodstream form T. vivax was characterized. The trans-sialidase from T. congolense is restricted to the procyclic culture forms and is shed into the culture medium. The enzyme has a pH-optimum at pH 7.0, displays sensitivity towards chlorides and is resistant against commonly used sialidase inhibitors. T. congolense trans-sialidase transfers preferentially α(2–3)-linked sialic acids onto terminal β-galactose residues. Also hydroxylated sialic acids (Neu5Gc) are transferred. The major glycoprotein GARP from procyclic T. congolense was identified as one potential natural sialic acid acceptor on the parasite's surface. In order to facilitate the characterization of trans-sialidases a novel, fluorimetric trans-sialidase assay was developed.

A sialidase activity in the midgut of the insect Triatoma infestans is responsible for the low levels of sialic acid in Trypanosoma cruzi growing in the insect vector.

Trypanosoma cruzi expresses a unique trans-sialidase that is responsible for the transfer of sialic acid from host glycoproteins and glycolipids to mucin-like glycoprotein acceptors on the parasite surface. The enzyme and the sialic acid acceptors are present in the mammalian forms of the parasite and in the parasite forms that grow in axenic cultures, which correspond to the developmental stages found in the insect vectors. Here we show that parasite forms growing in the vector Triatoma infestans express trans-sialidase in the hindgut portions of the insect. However, the sialic acid acceptors are poorly sialylated due to the low concentration of sialic acid donors in the gut lumen of T.infestans, which feeds exclusively on blood that is rich in sialic acid donors. These low levels of sialic acid donors are due to a novel sialidase activity present mainly in the anterior midgut with high specificity for alpha-2,3-sialyllactose, but not for alpha-2,6-sialyllactose. The activity is present in starved insects or insects fed with culture medium, indicating that it did not originate from the blood meal. Enzyme activity does not decrease in insects fed with antibiotics, is present in the salivary glands, and the few bacteria isolated from the gut and faeces of T.infestans did not display sialidase activity, indicating that the enzyme is not derived from a commensal organism. This novel activity could have a nutritional role in the gut of haematophagous insects and indicates that acquisition of sialic acid is not required for parasite development in the gut of T.infestans.

Structural and functional properties of Trypanosoma trans-sialidase.

Sialic acids and sialidases play important roles in cellular interactions and modulate the recognition of pathogenic microbes by mammalian host cells. Protozoan parasites of the genus Trypanosoma express a unique sialic acid-metabolizing enzyme. This enzyme, named trans-sialidase (TS), catalyzes the transfer of sialic acids from host glycoconjugates to acceptor molecules of the parasite plasma membrane. In African trypanosomes, the agents of sleeping sickness, TS is found only in forms developing within the insect vector, and the enzyme sialylates the major surface protein. In Trypanosoma cruzi, the causative agent of Chagas' disease in Central and South America, TS is expressed both in the insect and mammalian forms of the parasite. The T. cruzi enzyme has been biochemically characterized, and the gene encoding the enzyme has been cloned. The enzyme sialylates abundant mucin-like molecules present on the surface of the parasite. Several lines of evidence suggest that TS and sialic acid acceptors on the surface of T. cruzi participate in host-parasite interactions and mediate the initial stages of the trypanosomes' invasion of host cells.

Trypanosoma rangeli sialidase lacks trans-sialidase activity

Extracts and tissue culture supernatants of axenic forms of T. rangeli were assayed for the presence of sialidase and transsialidase activities. Using sialyl(α2–3)lactose, sialyl(a2–6)lactose, poly( α2−8) N-acetylneuraminic acid, fetuin and 4-methylumbelliferyl- N-acetylneuraminic acid as sialic acid donors, and lactose as a sialic acid acceptor, no trans-sialidase activity was detected. Nevertheless, T. rangeli lysates and culture supernatants contain a sialidase that hydrolyzes sialyl(α2–3)lactose, and much less efficiently sialyl(α2–6)lactose, but not poly( α2−8) N-acetylneuraminic acid. T. cruzi trans-sialidase hydrolyzed only sialyl(α2–3)lactose under the same conditions. The T. rangeli and the T. cruzi enzymes differ antigenically and in their pH optimum for hydrolase activity.

The developmentally regulated trans-sialidase from Trypanosoma brucei sialylates the procyclic acidic repetitive protein

A developmentally regulated trans-sialidase activity is present on the surface of procyclic Trypanosoma brucei. Bloodstream stages display no trans-sialidase activity. T. brucei trans-sialidase is capable of transferring sialic acids from a variety of glycoconjugates into new glycosidic linkages without requirement for CMP-Neu5Ac. The enzyme is linked to the plasma-membrane via a GPI-PLC-resistant GPI-anchor. The comparison of enzymic and structural features of sialidase and trans-sialidase suggests that the two activities may be catalyzed by the same protein, since highly enriched sialidase fractions display trans-sialidase activity. 2-Deoxy-2,3-didehydro- N-acetylneuraminic acid is only a poor inhibitor for the two enzymic activities. Sialic acids are transferred to α(2–3)-positions of terminal β-galactose residues of oligosaccharides and glycoconjugates at various rates. Neu5Ac-α(2–3)-lactose is the best trans-sialylation donor tested. Lewis x is a poor sialic acid acceptor. T. brucei trans-sialidase utilizes serum glycoconjugates, human and bovine erythrocytes as sialic acid donors, and resialylates sialidase-treated erythrocytes. The enzyme transfers sialic acids from the GPI-anchor of procyclic acidic repetitive protein (PARP) onto lactose and vice versa. Also structures within a variant surface glycoprotein (sVSG MITat. 1.7.) can be trans-sialylated.

Mucin-like glycoproteins linked to the membrane by glycosylphosphatidylinositol anchor are the major acceptors of sialic acid in a reaction catalyzed by trans-sialidase in metacyclic forms of Trypanosoma cruzi

We have previously shown that 35- and 50-kDa glycoconjugates of cultured metacyclic trypomastigotes participate in the attachment of parasites to mammalian cells. Here we show that when metacyclic trypomastigotes are incubated with [ 3H]sialyllactose, most of the sialic acid is transferred to these 35/50-kDa molecules in a reaction catalyzed by a parasite transsialidase. The sialic acid is incorporated in oligosaccharides of about 10 glucose units in size that are released from the glycoconjugate by mild alkaline hydrolysis. Compositional analysis reveals that the 35/50-kDa molecules are highly glycosylated proteins rich in threonine, galactose, N-acetyl-glucosamine and sialic acid. These glycoproteins can be labeled in vivo with [ 3H]palmitate, and the labeled fatty acid is released by glycosylphosphatidylinositol specific phospholipases C. This result, associated with the fact that they contain mannose, ethanolamine, myo-inositol, and lipid, indicate that these glycoproteins are anchored to the membrane by glycosylphosphatidylinositol. During cell invasion, these molecules appear to be capped and locally released by the parasite.

Variation in Phenotypic Expression of the Opa Outer Membrane Protein and Lipooligosaccharide of Neisseria meningitidis Serogroup C Causing Periorbital Cellulitis and Bacteremia

Expression of the Opa outer membrane protein varies widely among isolates of Neisseria meningitidis; the clinical significance of this variation is unclear. A sialic-acid acceptor is present in the lipooligosaccharide of some strains of Neisseria and has been shown to render Neisseria gonorrhoeae serum-resistant. We report the case of a patient who had an unusual clinical manifestation of infection due to N. meningitidis serogroup C, periorbital cellulitis with concomitant bacteremia. Clinical isolates from the blood and aspirate of the periorbital cellulitis were identical except for the phenotypic expression of the Opa outer membrane protein in the isolate from the periorbital cellulitis and in the lipooligosaccharide phenotype of the sialic-acid acceptor as defined by monoclonal antibodies. We discuss the laboratory and clinical implications of these findings.