Carbohydrate on human factor VIII/von Willebrand factor. Impairment of function by removal of specific galactose residues.

Human factor VIII/von Willebrand factor protein containing 120 +/- 12 nmol of sialic acid and 135 +/- 13 nmol of galactose/mg of protein was digested with neuraminidase. The affinity of native factor VIII/von Willebrand factor and its asialo form for the hepatic lectin that specifically binds asialoglycoproteins was assessed from in vitro binding experiments. Native factor VIII/von Willebrand factor exhibited negligible affinity while binding of the asialo derivative was comparable to that observed for asialo-alpha1-acid glycoprotein. Incubation of asialo-factor VIII/von Willebrand factor with Streptococcus pneumoniae beta-galactosidase removed only 62% of the galactose but abolished binding to the purified hepatic lectin. When the asialo derivative was incubated with purified beta-D-galactoside alpha2 leads to 6 sialyltransferase and CMP-[14C]NeuAc, only 61% of the galactose incorporated [14C]NeuAc. From the known specificites of these enzymes, it is concluded that galactose residues important in lectin binding are present in a terminal Gal/beta1 leads to 4GlcNAc sequence on asialo-factor VIII/von Willebrand factor. The relative ristocetin-induced platelet aggregating activity of native, asialo-, and agalacto-factor VIII/von Willebrand factor was 100:38:12, respectively, while procoagulant activity was 100:100:103.     

Some biochemical properties of human breast tumor sialyltransferase

Sialyltransferase activity in normal human breast tissue and tumors was investigated with lactose, desialylated fetuin, and bovine submaxillary mucin as the acceptors. While microsomal preparations from the normal tissue showed little or no sialyltransferase activity toward these acceptors, tumors showed elevated enzymic activities. Tween-20 at 0.5% concentrations stimulated sialic acid transfer to all three acceptors. Another nonionic detergent, Triton X-100, stimulated asialo fetuin sialyltransferase activity while inhibiting activity toward asialo BSM and lactose. Interestingly, lysolecithin, a normal cellular constituent which possesses detergent properties also had an effect similar to that of Triton X-100. Thermal denaturation curves of enzymic activity toward asialo BSM, however, resembled those seen with asialo fetuin as the acceptor. Kinetic studies showed that at acceptor concentrations of 500 micrograms each, sialyl transfers to asialo fetuin, asialo BSM, and lactose showed apparent Km values of 50, 60, and 300 microM, respectively. At CMP-sialic acid concentrations of 300 microM, the Km values for the above acceptors were 25, 15, and 5000 microM.     

An N-linked glycoprotein with alpha(2,3)-linked sialic acid is a receptor for BK virus

BK virus (BKV) is a common human polyomavirus infecting >80% of the population worldwide. Infection with BKV is asymptomatic, but reactivation in renal transplant recipients can lead to polyomavirus-associated nephropathy. In this report, we show that enzymatic removal of alpha(2,3)-linked sialic acid from cells inhibited BKV infection. Reconstitution of asialo cells with alpha(2,3)-specific sialyltransferase restored susceptibility to infection. Inhibition of N-linked glycosylation with tunicamycin reduced infection, but inhibition of O-linked glycosylation did not. An O-linked-specific alpha(2,3)-sialyltransferase was unable to restore infection in asialo cells. Taken together, these data indicate that an N-linked glycoprotein containing alpha(2,3)-linked sialic acid is a critical component of the cellular receptor for BKV.     

The function of the human factor V carbohydrate moiety in blood coagulation

Human factor V was subjected to desialation and deglycosylation to investigate the function of the molecular carbohydrate moiety. Removal of 90% of the sialic acid residues resulted in a 1.5-2-fold increase in clotting activity, and up to 70% deglycosylation in a concurrent decrease in clotting activity. Desialation had no effect on thrombin-induced activation, whereas deglycosylated factor V activation was impaired. Lectin-blot experiments with sialic-acid-specific Limax flavus agglutinin (LFA), galactose-specific Ricinus communis agglutinin (RCA-II) and mannose-specific concanavalin A on thrombin-induced factor V fragments revealed the presence of carbohydrate residues in fragments B, C1, D and F1F2. Interestingly, sialic acid was present in C1 whilst galactose was not detectable. Fragment F1F2 contained terminal galactose residues. LFA and RCA-II inhibited the procoagulant activity of native factor V and of desialated factor V respectively. These investigations distinctly indicate the important role of the human factor V carbohydrate moiety in the process of blood coagulation.     

Biological and physicochemical characterization of recombinant human erythropoietins fractionated by Mono Q column chromatography and their modification with sialytransferase

Ten erythropoietin (EPO) fractions differing in sialic acid content, ranging from 9.5 to 13.8 mol mol^–1 of EPO, were obtained from baby hamster kidney cell-derived recombinant human EPO by Mono Q column chromatography. The mean pI values of the EPO fractions determined by IEF-gel electrophoresis systematically shifted from 4.11 to 3.31, coinciding with the sialic acid content, without a change in the constitution of asialo N-linked oligosaccharides of each fraction. Although a linear relationship between thein vivo bioactivity and the sialic acid content of the fractionated, samples was observed until 12.1 mol mol^–1 of EPO, there was no further increase in their activity over 12.4 mol mol^–1 of EPO. On the other hand, an inverse relationship between thein vitro bioactivity and sialic acid content of EPO was observed. Also, we showed that thein vivo bioactivity of some fractions with low sialic acid contents was increased after treatment with 2,6-sialyltransferase, but thein vivo bioactivity of the other fractions with high sialic acid contents was either decreased or not affected.Abbreviations EPO erythropoietin - rHuEPO recombinant human erythropoietin - hCG human chorionic gonadotropin - BHK baby hamster kidney - CHO Chinese hamster ovary - NeuAc N-acetyl neuraminic acid - Gal galactose - HRCs hemolyser-resistant cells - WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Na - IEF isoelectric focusing - pI isoelectric point     

The effect of neuraminidase on genetic variants of alpha anitrypsin.

Sera of Pi types M, F, S, Z, IM, FM, MS, and MZ were incubated with neuraminidase and the reaction products followed by electrophoresis. The alpha1 antitrypsin components showed a series of changes in mobility as sialic residues were removed. Removal of sialic acid was confirmed by chemical assay. Results of studies with two different electrophoretic systems suggested that the Z type alpha1 antitrypsin has less sialic acid than the M, F, and S types. There was no evidence that other genetic variants have a reduced sialic acid content. The two major bands of alpha1 antitrypsin seen in certain electrophoretic systems may reflect a difference of one sialic acid residue. It is proposed that the Z protein lacks a carbohydrate chain with two terminal sialic acid residues. This carbohydrate deficiency results in lack of secretion of type Z alpha1 antitrypsin from the endoplasmic reticulum, perhaps because of binding to sites specific for the incomplete glycoprotein or because of aggregation of the Z asialo protein. A carbohydrate chain could be prevented from attaching to the Z type either because of a conformational change or because of the replacement of a carbohydrate-binding asparagine residue in the Z protein.     

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.     

Membrane receptors for Vicia graminea anti-N lectin and its binding to native and neuraminidase-treated human erythrocytes

Membrane receptors for Vicia graminea (Vg) lectin on human red cells were analyzed using deoxycholate lysates obtained from ¹²⁵I-erythrocyte membranes incubated with a purified lectin immobilized on Sepharose 4B. The glycoproteins (GP) specifically bound to the gel were eluted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Using native erythrocytes the results obtained demonstrate that N red cells have exposed Vg receptors located on GPα (synonym glycophorin A) and GPδ (synonym glycophorin B) whereas on M erythrocytes the Vg receptors are restricted to GPδ. The presence of Vg receptors was also found on the hybrid glycoprotein (made of the N-ter of GPδ and C-ter of GPα) carried by St(a+) erythrocytes. A similar amount of radioactivity was bound to Vg-Sepharose incubated with neuraminidase-treated N or M membranes. The material eluted was tentatively identified as asialo GPα and asialo GPδ, suggesting that numerous receptors have been uncovered mainly on asialo GPα species from M erythrocytes. No glycoprotein component could be identified from the material eluted from Vg Sepharose incubated with native or neuraminidase-treated membrane from a Tn(+) individual. Scatchard plot analysis obtained from binding experiments at equilibrium with M, N, and St(a+) cells revealed the existence of at least two classes of receptors both on native and neuraminidase-treated erythrocytes. Desialylation of the M, N, and St(a+) erythrocytes resulted in an increase in the number of low- and high-affinity binding sites but had no significant effect on the association constants. However, high-affinity binding constants were about six times higher with N (7.07 × 10⁷ and 6.61 × 10⁷m^?1 for native and neuraminidase-treated N cells, respectively) as compared to M erythrocytes (1.13 × 10⁷ and 1.17 × 10⁷m^?1 for native and neuraminidase-treated M cells, respectively) whereas the low-affinity binding constants were similar for all types of cells (in the range of 0.1 to 0.3 × 10⁷m^?1). The number of Vg binding sites increases from 0.085 × 10⁵ to 0.8 × 10⁵ (high affinity) and from 2.10 × 10⁵ to 6.25 × 10⁵ (low affinity) per native and neuraminidase-treated N cell, respectively. On native and neuraminidase-treated M cells the number of Vg receptors increases from 0.011 × 10⁵ to 0.51 × 10⁵ (high affinity) and 0.13 × 10⁵ (low affinity), respectively. The large increase in the number of Vg receptors on neuraminidase-treated M cells is correlated with a large increase in agglutinability. Under similar treatment St(a+) cells behave like N erythrocytes whereas only 0.16 × 10⁵ Vg receptors of low affinity could be detected on neuraminidase-treated Tn erythrocytes. The results demonstrate that sialic acid is not required for binding and favor the view that the binding site of V. graminea lectin accommodates with two types of erythrocyte membrane receptors, one including both a contribution of polypeptide and oligosaccharide chains and a second which involves a simple interaction with sugar sequence Galβ1–3GalNAc available only when sialic acids are removed. The latter disaccharide is recognized by the Arachis hypogea lectin which therefore inhibits further binding of the V. graminea to neuraminidase-treated erythrocytes.     

Functional characterization of vesicular excitatory amino acid transport by human sialin

Sialin, the protein coded by SLC17A5, is responsible for membrane potential (Δψ)-driven aspartate and glutamate transport into synaptic vesicles in addition to H+/sialic acid co-transport in lysosomes. Rodent sialin mutants harboring the mutations associated with Salla disease in humans did not transport aspartate and glutamate whereas H+/sialic acid co-transport activity was about one-third of the wild-type protein. In this study, we investigate the effects of various mutations on the transport activities of human sialin. Proteoliposomes containing purified heterologously expressed human sialin exhibited both Δψ-driven aspartate and glutamate transport activity and H+/sialic acid co-transport activity. Aspartate and glutamate transport was not detected in the R39C and K136E mutant forms of SLC17A5 protein associated with Salla disease, whereas H+/sialic acid co-transport activity corresponded to 30-50% of the recombinant wild-type protein. In contrast, SLC17A5 protein harboring the mutations associated with infantile sialic acid storage disease, H183R and Δ268SSLRN272 still showed normal levels of Δψ-driven aspartate and glutamate transport even though H+/sialic acid co-transport activity was absent. Human sialin carrying the G328E mutation that causes both phenotypes, and P334R and G378V mutations that cause infantile sialic acid storage disease showed no transport activity. These results support the idea that people suffering from Salla disease have been defective in aspartergic and glutamatergic neurotransmissions.     

NMR spectroscopic and molecular modeling investigations of the trans-sialidase from Trypanosoma cruzi

Nuclear magnetic resonance (NMR) spectroscopy was used to investigate the transfer of sialic acid from a range of sialic acid donor compounds to acceptor molecules, catalyzed by Trypanosoma cruzi trans-sialidase (TcTS). We demonstrate here that NMR spectroscopy is a powerful tool to monitor the trans-sialidase enzyme reaction for a variety of donor and acceptor molecules. The hydrolysis or transfer reactions that are catalyzed by TcTS were also investigated using a range of N-acetylneuraminosyl-based donor substrates and asialo acceptor molecules. These studies showed that the synthetic N-acetylneuraminosyl donor 4-methylumbelliferyl alpha-d-N-acetylneuraminide (MUN) is hydrolyzed by the enzyme approximately 3-5 times faster than either the disaccharide Neu5Acalpha(2,3)Galbeta1Me or the trisaccharide Neu5Acalpha(2,3)Lacbeta1Me. In the transfer reaction, we show that Neu5Acalpha(2,3)Lacbeta1Me is the most favorable substrate for TcTS and is a better substrate than the naturally-occurring N-acetylneuraminosyl donor alpha1-acid glycoprotein. In the case of MUN as the donor molecule, the transfer of Neu5Ac to different acceptors is significantly slower than when other N-acetylneuraminosyl donors are used. We hypothesize that when MUN is bound by the enzyme, the orientation and steric bulk of the umbelliferyl aglycon moiety may restrict the access for the correct positioning of an acceptor molecule. AutoDock studies support our hypothesis and show that the umbelliferyl aglycon moiety undergoes a strong pi-stacking interaction with Trp-312. The binding properties of TcTS towards acceptor (lactose) and donor substrate (Neu5Ac) molecules have also been investigated using saturation transfer difference (STD) NMR experiments. These experiments, taken together with other published data, have clearly demonstrated that lactose in the absence of other coligands does not bind to the TcTS active site or other binding domains. However, in the presence of the sialic acid donor, lactose (an asialo acceptor) was observed by NMR spectroscopy to interact with the enzyme's active site. The association of the asialo acceptor with the active site is an absolute requirement for the transfer reaction to proceed.     

Involvement of sialic acid in the regulation of γ--aminobutyric acid uptake activity of γ-aminobutyric acid transporter 1

The γ-aminobutyric acid (GABA) transporters (GATs) have long been recognized for their key role in the uptake of neurotransmitters. The GAT1 belongs to the family of Na(+)- and Cl(-)-coupled transport proteins, which possess 12 putative transmembrane (TM) domains and three N-glycosylation sites on the extracellular loop between TM domains 3 and 4. Previously, we demonstrated that terminal trimming of N-glycans is important for the GABA uptake activity of GAT1. In this work, we examined the effect of deficiency, removal or oxidation of surface sialic acid residues on GABA uptake activity to investigate their role in the GABA uptake of GAT1. We found that the reduced concentration of sialic acid on N-glycans was paralleled by a decreased GABA uptake activity of GAT1 in Chinese hamster ovary (CHO) Lec3 cells (mutant defective in sialic acid biosynthesis) in comparison to CHO cells. Likewise, either enzymatic removal or chemical oxidation of terminal sialic acids using sialidase or sodium periodate, respectively, resulted in a strong reduction in GAT1 activity. Kinetic analysis revealed that deficiency, removal or oxidation of terminal sialic acids did not affect the K(m) GABA values. However, deficiency and removal of terminal sialic acids of GAT1 reduced the V(max) GABA values with a reduced apparent affinity for extracellular Na(+). Oxidation of cell surface sialic acids also strongly reduced V(max) without affecting both affinities of GAT1 for GABA and Na(+), respectively. These results demonstrated for the first time that the terminal sialic acid of N-linked oligosaccharides of GAT1 plays a crucial role in the GABA transport process.     

Mucin biosynthesis. Characterization of UDP-galactose: alpha-N-acetylgalactosaminide beta 3 galactosyltransferase from human tracheal epithelium

We have characterized the UDP-galactose: alpha-N-acetylgalactosaminide beta 3 galactosyltransferase in human tracheal epithelium using asialo ovine submaxillary mucin as the acceptor. Maximal enzyme activity was obtained at pH 6.0-7.5 and at 20-25 mM MnCl2 and at 2% Triton X-100. Cd2+ could substitute for Mn2+ as the divalent ion cofactor. Spermine, spermidine, putrecine, cadaverine, and poly-L-lysine stimulated the enzyme activity at low (2.5 mM) MnCl2 concentration. The apparent Michaelis constants for N-acetylgalactosamine, asialo ovine submaxillary mucin, and UDP-galactose were 15.5, 1.14, and 1.36 mM, respectively. The enzyme activity was not affected by alpha-lactalbumin. The alpha-N-acetygalactosaminide beta 3 galactosyltransferase was shown to be different from the N-acetylglucosamine galactosyltransferase by acceptor competition studies. The product of galactosyltransferase was identified as Gal beta 1 leads to 3GalNAc alpha Ser (Thr) by (a) isolation of [14C]Gal-GalNAc-H2 after alkaline borohydride treatment of the 14C-labeled product, (b) establishment of the beta-configuration of the newly synthesized glycosidic bond by its complete cleavage by bovine testicular beta-galactosidase, and (c) assignment of the 1 leads to 3 linkage by identification of threosaminitol obtained from the oxidation of the disaccharide with periodic acid followed by reduction with sodium borohydride, hydrolysis in 4 N HCl, and analysis on an amino acid analyzer. The 1 leads to 3 linkage was confirmed by its resistance to jack bean beta-galactosidase and by the presence of a m/e 307 ion fragment and the absence of a m/e 276 ion by gas-liquid chromatography-mass spectrometry analysis. When acid and beta-galactosidase-treated human tracheobronchial mucin was used as the acceptor, 3.3% of the product was found as [14C]Gal-GalNAc-H2. The remainder of the [14C]Gal was found in longer oligosaccharides formed by a different beta-galactosyltransferase. This galactosyltransferase is slightly inhibited by alpha-lactalbumin and stimulated by spermine.     

Identification of intrinsic sialidase and sialoglycoprotein substrates in rat brain synaptic junctions

Sialidase activity associated with rat brain synaptic junctions (SJ) and synaptic membranes (SM) was determined. Both fractions released sialic acid from exogenous glycopeptides and gangliosides. SJ accounted for 5-10% of the total sialidase activity recovered from SM following extraction with Triton X-100, and the specific activity of SJ sialidase was 60% of that of the parent SM fraction. Intrinsic SJ sialidase hydrolysed 12-15% of the sialic acid associated with endogenous SJ glycoproteins. Sialic acid residues associated with SJ glycoproteins were labelled with sodium borotritide and SJ proteins fractionated by affinity chromatography on concanavalin A-agarose. SJ glycoproteins that reacted with concanavalin A (con A+ glycoproteins) accounted for 25% of the total SJ [3H]sialic acid. Intrinsic SJ sialidase hydrolysed 20% of the [3H]sialic acid associated with these glycoproteins. Each molecular weight class of con A+ glycoprotein previously shown to be a specific component of the postsynaptic apparatus contained sialic acid and was acted on by intrinsic SJ sialidase.     

Morphological and conformational studies of Tamm-Horsfall urinary glycoprotein

We have studied the circular dichroic properties of normal and cystic fibrotic Tamm-Horsfall urinary glycoproteins, and the asialo-derivatives (ca. 80% removal of sialic acid with neuraminidase). There was no evidence of α-helicity in Tamm-Horsfall urinary glycoprotein, but the results do indicate a significant amount of β-structure. The circular dichroic spectra of normal and cystic fibrotic Tamm-Horsfall urinary glycoproteins and the asialo-derivatives were identical, thus suggesting that there is no major difference in the ordered secondary structure of Tamm-Horsfall urinary glycoprotein in cystic fibrosis (relative to normal Tamm-Horsfall urinary glycoprotein), and that sialic acid exerts no major effect on the β-structure. Also, the circular dichroic spectrum of Tamm-Horsfall urinary glycoprotein was not affected by Ca²⁺ at concentrations just below that required for gel formation. Electron microscopic studies reveal the presence of a supramolecular helical structure arising from subunit interactions. This structure was characterized by a repeat of 120–130 Å and a minimal helix diameter of ca. 40 Å, although this value varied depending on the number of interacting helices. The helical structure was observed for normal, cystic fibrotic, and asialo derivatives of Tamm-Horsfall urinary glycoproteins, and was independent of added Ca²⁺. Guanidine hydrochloride treatment, followed by dialysis, irreversibly destroyed this supra-molecular helical structure, but the β-structure was partially restored, as indicated by the circular dichroic spectrum. The Ca²⁺-mediated gel formation was found to be inhibited in asialo-Tamm-Horsfall urinary glycoprotein.     

Blood clotting factor IX. Loss of activity after cleavage of sialic acid residues

Enzymatic cleavage of sialic acid from human blood clotting factor IX results in a loss of factor IX clotting activity. The loss of clotting activity and the rate of release of sialic acid follow the same time courses. Control experiments have ruled out several explanations for the loss of factor IX activity: proteolytic degradation, inhibitory effects of free sialic acid, and non-specific inhibition of the clotting assays. Furthermore, no inhibition was seen when similar enzymatic cleavage was carried out on factor X and factor VIII. Therefore, we suggest that the loss of factor IX activity is the direct result of cleavage of sialic acid from the protein. Most of the inhibition appeared to be an effect on the activity of factor IXa itself, and thus far, little or no effect has been shown on the activation of factor IX to IXa. The structural basis for this unusual effect of sialic acid on protein function currently is being investigated.     

Human fibrinogen and asialo-fibrinogen: a comparison of coagulation parameters.

The effect of desialylation of fibrinogen on its conversion to fibrin has been investigated with particular reference to the kinetics of clot formation and structure. Also examined was the role of sialic acid in fibrinogen (factor I) poor in factor XIII (fibrinstabilizing factor) and factor I containing F XIII. The removal of more than 90% of the sialic acid of fibrinogen does not alter the thrombin clotting time, the clot solubility in monochloroacetic acid, the extent of cross-linking in the fibrin polymer, or the firmness and elasticity of the evolved clot. The data indicate that the sialic acid residues of fibrinogen do not contribute significantly to its conversion to fibrin by thrombin.     

SIALIC ACID AND THE SOCIAL BEEIAVIOUR OF CELLS

1. It is suggested that specific carbohydrate side-chains of membrane glycoproteins are the sites for cell recognition or adhesion when the terminal sugar, sialic acid, is absent. 2. It is suggested that sialic acid plays a ‘protective’ or ‘blocking’ role in cell interactions so that addition of sialic acid to asialo side-chains converts them to forms inactive for recognition. This principle of ‘blocking’ by sialic acid has been observed in other situations as in covering tumour antigens and in protecting glycoproteins from uptake by the liver. It is here extended to cell-cell adhesions. 3. It is to be expected that specific ‘protective’ actions of sialic acid in membrane-bound glycoproteins will be difficult to detect. As a charged residue, sialic acid is likely to have a strong influence both on the glycoproteins on which it is borne and on their interactions with each other at the cell surface. Removal of sialic acid by enzymes could therefore perturb the structure of the cell surface in several ways and so obscure the ‘protective’ effects of sialic acid. Sialic acid is therefore suggested to have a structural role also. 4. Evidence is assembled in favour of a model in which sialysation of specific adhesive receptors affects the social behaviour of cells. This may be an effect associated with growing cells since the contact properties of mitotic cells (and populations rich in dividing cells) are decreased by the increased sialysation of receptors. One of the factors associated with malignant behaviour could be that adhesive receptors are permanently blocked by sialic acid. 5. A schematic representation of some of the points is given in Fig. 4.     

Mediator-induced macrophage activation, as shown by enhanced cytotoxicity for tumor, requires macrophage surface fucose and sialic acid

Macrophage-activating factor (MAF) activates macrophages so that their cytotoxic capacity is enhanced. This effect of MAF is inhibited by removing fucose from the macrophage cell surface by incubation with fucosidase, or by removing sialic acid by treatment with neuraminidase. After incubation with fucosidase or neuraminidase the average inhibition of cytotoxicity was 92 and 73%, respectively. β-Galactosidase had no effect. Addition of the specific products, fucose or sialic acid, to the incubation mixture of macrophages and enzyme blocked the effect of the enzymes. Taken together these observations indicate that macrophage surface fucose and sialic acid are essential for the interaction of MAF with macrophages which results in enhanced cytotoxicity for tumor cells.     

Enhanced sialic acid-dependent endocytosis explains the increased efficiency of infection of airway epithelia by a novel adeno-associated virus

We previously used directed evolution in human airway epithelia to create adeno-associated virus 2.5T (AAV2.5T), a highly infectious chimera of AAV2 and AAV5 with one point mutation (A581T). We hypothesized that the mechanism for its increased infection may be a higher binding affinity to the surface of airway epithelia than its parent AAV5. Here, we show that, like AAV5, AAV2.5T, uses 2,3N-linked sialic acid as its primary receptor; however, AAV2.5T binds to the apical surface of human airway epithelia at higher levels and has more receptors than AAV5. Furthermore, its binding affinity is similar to that of AAV5. An alternative hypothesis is that AAV2.5T interaction with 2,3N-linked sialic acid may instead be required for cellular internalization. Consistent with this, AAV2.5T binds but fails to be internalized by CHO cells that lack surface expression of sialic acid. Moreover, whereas AAV2.5T binds similarly to human (rich in 2,3N-linked sialic acid) and pig airway epithelia (2,6N-linked sialic acid), significantly more virus was internalized by human airway. Subsequent transduction correlated with the level of internalized rather than surface-bound virus. We also found that human airway epithelia internalized significantly more AAV2.5T than AAV5. These data suggest that AAV2.5T has evolved to utilize specific 2,3N-linked sialic acid residues on the surface of airway epithelia that mediate rapid internalization and subsequent infection. Thus, sialic acid serves as not just an attachment factor but is also required for AAV2.5T internalization, possibly representing an important rate-limiting step for other viruses that use sialic acids.