Effect of genetic background on glycosylation heterogeneity in human antithrombin produced in the mammary gland of transgenic goats

Glycosylation is involved in the correct folding, targeting, bioactivity and clearance of therapeutic glycoproteins. With the development of transgenic animals as expression systems it is important to understand the impact of different genetic backgrounds and lactations on glycosylation. We have evaluated the glycosylation of recombinant antithrombin produced in several transgenic goat lines, from cloned animals and from different types of lactation including induced lactations. Our results show glycosylation patterns from the protein expressed in animals, derived from the same founder goat, are mostly comparable. Furthermore, the protein expressed in two cloned goats had highly consistent oligosaccharide profiles and similar carbohydrate composition. However, there were significantly different oligosaccharide profiles from the proteins derived from different founder goats. Artificial induction of lactation did not have significant effects on overall carbohydrate structures when compared to natural lactation. The only major difference was that recombinant antithrombin from induced lactations contained a slightly higher ratio of N-acetylneuraminic acid to N-glycolylneuraminic acid and less amount of oligosaccharides containing N-glycolylneuraminic acid. The oligosaccharides from all animals were a mixture of high mannose-, hybrid- and complex-type oligosaccharides. Sialic acid was present as alpha-2,6-linkage and no alpha-1,3-linked galactose was observed. These results indicate that transgenic animals with closely related genetic backgrounds express recombinant protein with comparable glycosylation.     

CTLA4Ig融合蛋白在CHO细胞中的表达

CTLA4Ig是人CTLA4胞外区与人免疫球蛋白铰链区、CH2区、CH3区组成的融合蛋白,可以与B7结合,通过阻断B7与CD28的结合,从而阻断B7介导的T细胞活化必需的共刺激信号,可作为免疫抑制剂用于器官移植。将CTLA4Ig融合分子克隆到真核表达载体pCI-dhfr,并用脂质体方法转染到COS7和CHO-dhfr-细胞中,用氨甲喋呤筛选转染的CHO-dhfr-细胞。用RT-PCR、ELISA、细胞免疫荧光染色和Western-blot鉴定重组蛋白的表达。采用A蛋白纯化重组蛋白。     

Identification of two binding sites for wheat-germ agglutinin on polylactosamine-type oligosaccharides.

The carbohydrate-binding properties of wheat-germ agglutinin (WGA) have been studied by using glycopeptides isolated from the cell surfaces of a cultured murine myeloid cell line (416B). The glycopeptides were passed through affinity columns of lentil lectin (LCA), concanavalin A (Con A) and WGA arranged in series so that material reaching the WGA column had failed to bind to LCA or Con A. WGA-binding glycopeptides were step-eluted with 0.01 M, 0.1 M and 0.5 M-N-acetylglucosamine (GlcNAc), to yield weak (WGA-W), intermediate (WGA-I) and strong (WGA-S) affinity fractions. WGA-W and WGA-I contained 'N'- and 'O'-linked oligosaccharides bound to separate polypeptides. WGA-S consisted almost entirely of N-linked components. Our analytical work was concentrated mainly on the N-linked fractions. In these carbohydrates WGA affinity was directly proportional to molecular size but inversely related to N-acetylneuraminic acid content. The binding of the weak-affinity fraction was dependent on N-acetylneuraminic acid, but the intermediate- and strong-binding species interacted with the lectin by N-acetylneuraminic acid-independent mechanisms. N-linked glycopeptides in each WGA-binding class were almost totally degraded to monosaccharides by the concerted action of the exoglycosidases neuraminidase, beta-galactosidase and beta-N-acetylglucosaminidase. Treatment with endo-beta-galactosidase caused partial depolymerization, yielding some disaccharides but also a heterogeneous population of partially degraded components. These findings suggest that WGA binds with high affinity to internal GlcNAc residues in large oligosaccharides containing repeat sequences of Gal beta(1----4)GlcNAc beta(1----3) (i.e. polylactosamine-type glycans). N-Acetylneuraminic acid is involved only in low-affinity interactions with WGA. WGA therefore displays an intricate pattern of saccharide specificities that can be profitably utilized for structural analysis of complex carbohydrates.     

Investigations on the oligosaccharide units of an A myeloma globulin

The carbohydrate content of an A myeloma globulin was investigated. The carbohydrate content was found to be unchanged when the protein was isolated from the patient over a period of 18 months. The various polymeric forms of the protein contained similar proportions of carbohydrate. The A myeloma globulin contained approx. 2 residues of 6-deoxy-l-galactose (l-fucose), 14-15 of d-mannose, 12-13 of d-galactose, 12-13 of 2-acetamido-2-deoxy-d-glucose (N-acetyl-d-glucosamine), 6 of 2-acetamido-2-deoxy-d-galactose (N-acetyl-d-galactosamine) and 5 of N-acetylneuraminic acid (sialic acid), and these were distributed between six oligosaccharide units all of which were present on the heavy polypeptide chains. The oligosaccharide units showed two kinds of heterogeneity, which have been termed central and peripheral. Central heterogeneity was shown by the presence of three completely different core units, which had the following compositions: (1) 3 residues of d-galactose and 3 of 2-acetamido-2-deoxy-d-galactose, joined to protein by an O-glycosidic linkage between acetamidohexose and serine; (2) 3 residues of d-mannose, 2 of d-galactose and 3 of 2-acetamido-2-deoxy-d-glucose, joined to protein by an N-glycosidic linkage between acetamidohexose and aspartic acid; (3) 4 residues of d-mannose and 3 of 2-acetamido-2-deoxy-d-glucose with a linkage similar to that in (2). The core oligosaccharide units showed peripheral heterogeneity in the attachment of 6-deoxy-l-galactose, 2-acetamido-2-deoxy-d-glucose and N-acetylneuraminic acid. Tentative structures are proposed for these various types of oligosaccharide unit. Glycopeptides were isolated in which the sialic acid content exceeded that of d-galactose. Explanations are given for the electrophoretic mobility and staining characteristics of the various glycopeptides.     

A study of the carbohydrate present in three type K macroglobulins

For a monomeric molecular weight of 180000 three type K macroglobulins (IgM) contained 6-deoxygalactose, mannose, galactose, 2-acetamido-2-deoxyglucose and N-acetylneuraminic acid in the molar proportions 5:38:11:27:7 for Row IgM, 5:31:9:21:7 for Sha IgM, and 5:29:11:26:8 for Tya IgM. The first two proteins were euglobulins whereas Tya IgM was a pseudoglobulin, and therefore the total content of carbohydrate does not appear to be related to the physicochemical properties of the proteins. The three proteins appeared to contain different numbers of oligosaccharide units, Row IgM having about ten units/monomer, and Sha IgM and Tya IgM about eight each. All three proteins had two types of oligosaccharide unit, which by analogy with an immunoglobulin A myeloma globulin were called Type 2 and Type 3 respectively. The Type 2 units had molecular weights equal to or greater than 2000 and contained 1 residue of 6-deoxygalactose, 3-4 of mannose, 1-2 of galactose, 3-4 of 2-acetamido-2-deoxyglucose and 0-2 of N-acetylneuraminic acid. The Type 3 units had molecular weights of less than 2000 and contained 0-1 residue of 6-deoxygalactose, 3-6 of mannose, 0-1 of galactose, 1-3 of 2-acetamido-2-deoxyglucose and no N-acetylneuraminic acid. Glycopeptides corresponding to the two types of unit varied in their aspartic acid content in that most of the Type 3 glycopeptides possessed only 1 residue of aspartic acid whereas most of the Type 2 glycopeptides had an average content greater than 1 residue.     

Studies on the glycopeptides isolated from the urinary protein in heavy-chain disease

The urinary protein excreted in heavy-chain disease was separated by ion-exchange chromatography into two broad fractions designated Cra-1 and Cra-2. For a dimeric molecular weight of approx. 51000, Cra-1 contained 3-4 residues of 6-deoxy-l-galactose (l-fucose), 10 of d-mannose, 5-6 of d-galactose, 12 of 2-acetamido-2-deoxy-d-glucose (N-acetyl-d-glucosamine) and 4-5 of N-acetylneuraminic acid (sialic acid), whereas the corresponding values for Cra-2 were 2, 10, 7, 12 and 7. Cra-2 contained in addition 1 residue of 2-acetamido-2-deoxy-d-galactose (N-acetyl-d-galactosamine). Cra-1 contained an average of four oligosaccharide units, two of which contained 1 residue of 6-deoxy-l-galactose, 3 of d-mannose, 1 of d-galactose and 3 of 2-acetamido-2-deoxy-d-glucose, whereas the other two units contained the same proportions of 6-deoxy-l-galactose, d-mannose and 2-acetamido-2-deoxy-d-glucose but 2 residues of d-galactose and 2 of N-acetylneuraminic acid. Cra-2 also contained an average of four oligosaccharide units, but the range of glycopeptides was much wider, containing 0-1 residue of 6-deoxy-l-galactose, 2-3 of d-mannose, 2-3 of d-galactose, 2-3 of 2-acetamido-2-deoxy-d-glucose and 1-3 of N-acetylneuraminic acid. Possible reasons for this heterogeneity are discussed. Glycopeptides were also isolated from Cra-2 that contained 1 residue of d-mannose, 2 of d-galactose, 1 of 2-acetamido-2-deoxy-d-galactose and 0-3 of N-acetylneuraminic acid.     

Structures of the asparagine-linked sugar chains of human chorionic gonadotropin.

The asparagine-linked sugar chains of human chorionic gonadotropin were released from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. More than 90% of the released radioactive oligosaccharides contained N-acetylneuraminic acid residues. After removal of N-acetylneuraminic acid residues by sialidase treatment, two neutral oligosaccharide fractions were obtained by paper chromatography. Sequential exoglycosidase digestion revealed that one of them was a mixture of two neutral oligosaccharides. The complete structures of the three oligosaccharides were elucidated by methylation analysis. It was confirmed that all the N-acetylneuraminic acid residues of the asparagine-linked sugar chains of human chorionic gonadotropin occur as NeuAc alpha 2 leads to 3Gal groupings by comparing the methylation analysis data for the acidic oligosaccharide mixture before and after sialidase treatment. Based on these results, the structures of the asparagine-linked sugar chains of human chorionic gonadotropin were confirmed to be +/- NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(+/- Fuc alpha 1 leads to 6)GlcNAc and Man alpha 1 leads to 6(NeuAc alpha 2 leads to 3 Gal beta 1 leads to 4 GlcNAc beta 1 leads to Man alpha 1 leads to 3)Man beta 1 leads to 4 GlcNAc beta 1 leads to 4GlcNAc.     

Structural characterization of the oligosaccharides of a human monoclonal anti-lipopolysaccharide immunoglobulin M

The oligosaccharide side chains of a human anti-lipopolysaccharide IgM produced by a human-human-mouse heterohybridoma were analyzed at each of its five conserved N-glycosylation sites. This antibody also has a potential sixth N-glycosylation site in the variable region of its heavy chain which is not glycosylated. The oligosaccharides were released by digestion with various endo- and exoglycosidases and analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and fluorophore-assisted carbohydrate electrophoresis. The antibody has various complex- and hybrid-type oligosaccharide structures at Asn 171, various sialylated complex-type oligosaccharides at Asn 332 and 395, and high-mannose-type oligosaccharides at Asn 402 and 563. Of note is the presence in this human IgM of oligosaccharides containing N-glycolylneuraminic acid and N-acetylneuraminic acid in the ratio of 98:2 as determined using anion- exchange chromatography. Furthermore, we observed oligosaccharide structures containing Gal alpha (1,3)Gal that have not been reported as components of human glycoproteins.      

Glycopeptides of immunoglobulins: Investigations on IgA myeloma globulins

The oligosaccharide units of a type K and a type L IgA immunoglobulin have been examined. The two proteins differed in their content of 6-deoxy-l-galactose and N-acetylneuraminic acid, and in the d-mannose/d-galactose ratio. With glycopeptides prepared from the type K protein, specific glycosidases liberated the N-acetylneuraminic acid and 7-8 residues of 2-acetamido-2-deoxy-d-glucose, and mild acid hydrolysis released most of the 6-deoxy-l-galactose. The type K immunoglobulin appeared to contain 3 oligosaccharide units, whereas the type L protein probably contained 3 or more units.     

Fractionation and Characterization of the Sulfated Oligosaccharide Chains of Ovomucin

The O-glycosidically linked carbohydrate units of ovomucin were released from serine and threonine in peptide as oligosaccharide chains by alkali treatment with and without borohydride. Two sulfated oligosaccharides were fractionated by using gel filtration and ion-exchange chromatography. The yield of sulfated oligosaccharides released by alkali treatment was higher in the presence of borohydride than in the absence of borohydride. The sulfated oligosaccharides released by alkali treatment with borohydride were as follows: an oligosaccharide composed of N-acetylgalactosaminitol, galactose, N-acetylneuraminic acid and sulfate in a molar ratio of about 1: 1: 1: 1 and another oligosaccharide in a molar ratio of about 1:1: 0.6: 0.5.     

New feature of angiotensin-converting enzyme: carbohydrate-recognizing domain

Self carbohydrate-mediated dimerization of glycoprotein angiotensin-converting enzyme (ACE) was demonstrated. The dimerization was studied in the reverse micelle experimental system as a model of biomembrane situation. Asialo-ACE or agalacto-ACE was able to form a dimer, whereas deglycosylated ACE and sequentially desialylated and degalactosylated ACE failed to dimerize. ACE-ACE interaction was competitively inhibited by Neu5Ac- or Gal-terminated saccharides. The results have allowed us to propose the existence of carbohydrate-recognizing domain (CRD) on ACE molecule. The structural requirements of this CRD were estimated based on the ability of saccharides to inhibit ACE dimerization. The most effective monosaccharides with equal inhibition potencies were shown to be galactose (as GalbetaOMe) and N-acetylneuraminic acid (as Neu5AcalphaOMe). Among oligosaccharides, the most effective ones were found to be 3'SiaLac and, especially, the whole pool of ACE oligosaccharide chains and biantennae complex oligosaccharide chains of other glycoproteins. Bovine and human ACEs were shown to be similar in terms of recognition of carbohydrates.     

Carbohydrate content of human erythrocyte membrane. Variations with ABO-blood group.

The study of the carbohydrates of human erythrocyte membranes has been mainly focused on their glycopeptidic and glycolipidic complexes. Modifications of these carbohydrates have been described in subjects with various pathological states. In order to characterize possible changes of the glycopeptides, or glycolipids obtained from erythrocyte membrane in various pathological situations, the determination of the carbohydrate content of the whole membrane appeared a necessary preliminary. This study concerns the determination of the normal values of the main carbohydrates of whole human erythrocyte membranes, with respect to their blood group. Erythrocyte membranes were prepared from donors of the four ABO blood groups. After acidic hydrolysis, the contents of fucose, mannose, galactose, glucose, glucosamine, galactosamine and N-acetylneuraminic acid in each blood group were determined and compared with one another. The galactosamine content of A, B and AB erythrocyte membranes is significantly higher than that of the O-erythrocyte. For galactose, the differences are significant for the following pairs: A/O; B/O; AB/O; A/B; A/AB. Significant differences in the mannose contents of O-erythrocytes and A, B and AB erythrocytes have also been found. This result suggests that a basic difference, in the core of the oligosaccharide chains, may exist between O and A, B, AB erythrocyte membranes.     

Expression of a soluble form of CTLA4 on macrophage and its biological activity.

Interaction between cytotoxic T lymphocyte-associated antigen-4(CTLA4,CD152) and B7 molecules (B7-1 and B7-2) is of importance in the cellular events of lymphocyte,including antigen-specific T-cell activation and induction of autoreactive T-cell.We describe haere the first introduction of a murine soluble CTLA4 gene,CTLA4Ig,to Mm1 cells,a macrophagic cell line.CTLA4Ig was successfully expressed on Mm1 cells and the expressed CTLA4Ig was found to be functionally active in their binding to B7 molecules by flow cytometry and immunofluorescence studies.The biological activity of CTLA4Ig from the transfected Mm1 cells was studied and showed inhibitory activity on mixed lymphocyte culture.A high CTLA4Ig producing macrophagic cell line was obtained.As Mm1 cells were regarded as difficult for gene transfection and there has so far been no report on expression of CTLA4Ig gene on Mm1 cells,these results suggested that the CELA4Ig expressing Mm1 cells could be useful for analysis of CTLA4 and B8 molecule interaction in both macrophage and T-cell.     

The specificity of viral and bacterial sialidases for alpha(2-3)- and alpha(2-6)-linked sialic acids in glycoproteins

The anomeric specificity of six sialidases (Vibrio cholerae, Arthrobacter ureafaciens, Clostridium perfringens, Newcastle disease virus, fowl plague virus and influenza A2 virus sialidases) was assessed with sialylated antifreeze glycoprotein, ovine submandibular gland glycoprotein and alpha 1-acid glycoprotein, resialylated specifically in alpha(2-3) or alpha(2-6) linkage with N-acetylneuraminic acid or N-glycolylneuraminic acid using highly purified sialyltransferases. The rate of release of sialic acid from these substrates was found to correlate well with the specificity observed earlier with the same sialidases using small oligosaccharide substrates, i.e., alpha(2-3) glycosidic linkages are hydrolyzed faster than alpha(2-6) linkages, with the exception of the enzyme from A. ureafaciens. Sialidase activity was higher with N-acetylneuraminic acid when compared with N-glycolylneuraminic acid. The studies also showed that the core oligosaccharide and protein structure in glycoproteins may influence the rate of release for different glycosidic linkages.     

Expression of a soluble form of CTLA4 on macrophage and its biological activity

INTRODUCTI0NItisrecentlyreportedthatB7andCD28/CTLA4pathwayplaysacriticalroleintheactivationofT-ce1l[1-3],aswellasintheonsetofautoimmunitybothinhumanandmurinem0delofaut0immunity[4-7].B7moleculesareexpressedonavarietyofcelltypes,includingdentriticcells,Bcells,T-cellsandmacrophages[8-1l].CTLA4Ig,asolubleform0fCTLA4,cou1db10ckB7andCD28/CTLA4pathwayandresultsintheinhibitationofT-cellactivationandautoimmuneresponse[12-19].ThemacrophagicMm1ce1llinewasregardedasag0odmodelforstudyingmacr…     

One- and two-dimensional 90.5-MHz 13C-NMR spectroscopy of the N-linked triantennary oligosaccharide units of calf fetuin

Complete assignments of all anomeric resonances in the proton and carbon spectra of the N-linked oligosaccharide units of fetuin were made using one- and two-dimensional NMR spectroscopy. We are able to confirm the presence of microheterogeneity in the N-acetylneuraminic acid linkages to the galactose residues and the presence of a unique triantennary structure which carries a side chain: NeuAc alpha(1-3)Gal beta(1-3)GlcNac beta(1-4)-. Anomeric carbon chemical shifts changes resulting from long-range conformational effects were observed.     

A micromethod for the estimation of oligosaccharides containing glycosidically linked sialic acid or hexoses, or both, in glycoproteins

The peeling reaction, the process by which oligosaccharides are degraded in alkali, was used as the basis for an assay to provide structural information about glycosidically linked oligosaccharides in glycoproteins. Glycoproteins were treated with 0.05 M NaOH at 50 degrees to induce release, and subsequent degradation ("peeling"), of glycosidically linked, but not of N-glycosydically linked, oligosaccharides. Among the degradation products generated from O-linked chains were three 3-deoxy sugar acids whose formation was correlated with certain structural features of the oligosaccharides. N-Acetylneuraminic acid was released from terminal positions in the oligosaccharides, and iso- and meta-saccharinic acids were derived from the degradation of 4-O- and 3-O-substituted hexoses, respectively. All of these sugar acids were detected colorimetrically by periodate oxidation and reaction of the product with 2-thiobarbituric acid. The ability of the method to generate 3-deoxy sugar acids was tested in 8 alkali-treated glycoproteins. 3-Deoxy sugar acids were detected only in those glycoproteins whose glycosidically linked carbohydrates contained N-acetylneuraminic acid, or 3-O- or 4-O-substituted hexoses, or both. As little as 0.12 microgram of 3-deoxy sugar acid produced from 5 micrograms of human chorionic gonadotropin was sufficient for detection. This method is novel in its ability to distinguish sialylation of glycosidically linked carbohydrates. Furthermore, it combines the specificity of beta-elimination with the sensitivity of the 2-thiobarbituric acid assay in targeting degradation products of the peeling reaction as candidates for an assay method.     

Modification of the intramolecular turnover of terminal carbohydrates of dipeptidylaminopeptidase IV isolated from rat-liver plasma membrane during liver regeneration

An intramolecular turnover of the terminal carbohydrates L-fucose, N-acetylneuraminic acid and D-galactose is a characteristic property of several liver plasma membrane glycoproteins, first demonstrated for dipeptidylaminopeptidase IV (EC 3.4.14.5., DPP IV). The core carbohydrates D-mannose and N-acetyl-D-glucosamine turn over like the polypeptide chain. The ratio of apparent half-lives of L-fucose and L-methionine of DPP IV is shifted from 0.17 in normal liver to 0.60 in regenerating liver. The ratio of half-lives of N-acetylneuraminic acid and L-methionine is only slightly changed from 0.43 in normal liver to 0.61 in regenerating liver. The ratio of apparent half-lives of D-mannose and L-methionine amounts to 0.80 in normal liver and 0.71 after partial hepatectomy. From this a drastic reduction of the intramolecular turnover of L-fucose on plasma membrane DPP IV in regenerating liver can be derived. The intramolecular N-acetylneuraminic acid turnover is affected to only a minor extent. D-Mannose turns over like the polypeptide in both normal and regenerating liver. The intramolecular L-fucose turnover may be involved in membrane glycoprotein recycling, which presumably is altered in regenerating liver. Additionally, L-fucose could regulate the rate of degradation of DPP IV, since core-fucosylated glycoproteins appear to be resistant to mammalian endo-N-acetylglucosaminidase.     

Construction, and in vitro and in vivo analyses of tetravalent immunoadhesins

Previous observations demonstrated that various immunosuppressive agents and their combination therapies can increase allograft survival rates. However, these treatments may have serious side effects and cannot substantially improve or prolong graft survival in acute graft-versus-host disease (GVHD). To improve the therapeutic potency of divalent immunoadhesins, we have constructed and produced several tetravalent forms of immunoadhesins comprising each of cytotoxic T-lymphocyte-associated antigen-4 (CTLA4), CD2, and lymphocyte activation gene-3 (LAG3). Flow cytometric and T cell proliferation analyses displayed that tetravalent immunoadhesins have a higher binding affinity and more potent efficacy than divalent immunoadhesins. Although all tetravalent immunoadhesins possess better efficacies, tetravalent forms of CTLA4-Ig and LAG3-Ig revealed higher inhibitory effects on T cell proliferation than tetravalent forms of TNFR2-Ig and CD2-Ig. In vitro mixed lymphocytes reaction (MLR) showed that combined treatment with tetravalent CTLA4-Ig and tetravalent LAG3-Ig was highly effective for inhibiting T cell proliferation in both human and murine allogeneic stimulation. In addition, both single tetravalent-form and combination treatments can prevent the lethality of murine acute GVHD. The results of this study demonstrated that co-blockade of the major histocompatibility complex class (MHC)II:T cell receptor (TCR) and CD28:B7 pathways by using tetravalent human LAG3-Ig and CTLA4-Ig synergistically prevented murine acute GVHD.     

Uptake of N-acetylneuraminic acid by Escherichia coli K-235. Biochemical characterization of the transport system.

Kinetic measurement of the uptake of N-acetyl[4,5,6,7,8,9-14C]neuraminic acid by Escherichia coli K-235 was carried out in vivo at 37 degrees C in 0.1 M-Tris/maleate buffer, pH 7.0. Under these conditions uptake was linear for at least 30 min and the Km calculated for sialic acid was 30 microM. The transport system was osmotic-shock-sensitive and was strongly inhibited by uncouplers of oxidative phosphorylation [2,4-dinitrophenol (100%); NaN3 (66%]) and by the metabolic inhibitors KCN (84%) and sodium arsenate (76%). The thiol-containing compounds mercaptoethanol, glutathione, cysteine, dithiothreitol and cysteine had no significant effect on the sialic acid-transport rate, whereas the thiol-modifying reagents N-ethylmaleimide, iodoacetate and p-chloromercuribenzoate almost completely blocked (greater than 94%) the uptake of this N-acetyl-sugar. N-Acetylglucosamine inhibited non-competitively the transport of N-acetylneuraminic acid, whereas other carbohydrates (hexoses, pentoses, hexitols, hexuronic acids, disaccharides, trisaccharides) and N-acetyl-sugars or amino acid derivatives (N-acetylmannosamine, N-acetylcysteine, N-acetylproline and N-acetylglutamic acid) did not have any effect. Surprisingly, L-methionine and its non-sulphur analogue L-norleucine partially blocked the transport of this sugar (50%), whereas D-methionine, D-norleucine, several L-methionine derivatives (L-methionine methyl ester, L-methionine ethyl ester, L-methionine sulphoxide) and other amino acids did not affect sialic acid uptake. The N-acetylneuraminic acid-transport system is induced by sialic acid and is strictly regulated by the carbon source used for E. coli growth, arabinose, lactose, glucose, fructose and glucosamine being the carbohydrates that cause the greatest repressions in this system. Addition of cyclic AMP to the culture broth reversed the glucose effect, indicating that the N-acetylneuraminic acid-uptake system is under catabolic regulation. Protein synthesis is not needed for sialic acid transport.