The lectin-like interaction between recombinant tumor necrosis factor and uromodulin

The polypeptide of uromodulin, an immunosuppressive glycoprotein isolated from human urine, has been shown to be identical to that of Tamm-Horsfall glycoprotein and is synthesized exclusively in the kidney (Hession, C., Decker, J. M., Sherblom, A. P., Kumar, S. (1987) Science 237, 1479-1484). Uromodulin binds recombinant murine interleukin 1 alpha with high affinity, and this binding can be inhibited by addition of specific saccharides (Muchmore, A. V., and Decker, J. M. (1987) J. Immunol. 138, 2541-2546). We now report that uromodulin binds recombinant human tumor necrosis factor (rTNF) with high affinity. Both diacetylchitobiose and Man(alpha 1-6)(Man(alpha 1-3]-Man-O-ethyl are effective inhibitors of the binding, whereas a wide variety of other saccharides are not inhibitory. Although Tamm-Horsfall glycoprotein contains predominantly tetraantennary N-linked chains, the binding to rTNF is unaffected by removal of terminal sialic acid, galactose, and N-acetylhexosamine residues. Fractionation of a Pronase digest of uromodulin by gel filtration yields material that inhibits the binding of uromodulin to rTNF but is of lower molecular weight than the major oligosaccharide. Uromodulin does not inhibit the cytotoxic activity of rTNF as monitored by lysis of tumor cell targets but effectively protects mice from lethal challenge with lipopolysaccharide, an event that may involve lymphokine toxicity. We have previously shown that rTNF binds to sections of human kidney and is localized in the same region as uromodulin. Thus, rTNF interacts with uromodulin via carbohydrate chains that are less processed than the major tetraantennary chain, and this interaction may be critical in promoting clearance and/or reducing toxicity of TNF and other lymphokines.     

Crystal structures of the HIV-1 inhibitory cyanobacterial protein MVL free and bound to Man3GlcNAc2: structural basis for specificity and high-affinity binding to the core pentasaccharide from n-linked oligomannoside

The cyanobacterial protein MVL inhibits HIV-1 envelope-mediated cell fusion at nanomolar concentrations by binding to high mannose N-linked carbohydrate on the surface of the envelope glycoprotein gp120. Although a number of other carbohydrate-binding proteins have been shown to inhibit HIV-1 envelope-mediated cell fusion, the specificity of MVL is unique in that its minimal target comprises the Man(alpha)(1-->6)Man(beta)(1-->4)GlcNAc(beta)(1-->4)GlcNAc tetrasaccharide core of oligomannosides. We have solved the crystal structures of MVL free and bound to the pentasaccharide Man3GlcNAc2 at 1.9- and 1.8-A resolution, respectively. MVL is a homodimer stabilized by an extensive intermolecular interface between monomers. Each monomer contains two structurally homologous domains with high sequence similarity connected by a short five-amino acid residue linker. Intriguingly, a water-filled channel is observed between the two monomers. Residual dipolar coupling measurements indicate that the structure of the MVL dimer in solution is identical to that in the crystal. Man3GlcNAc2 binds to a preformed cleft at the distal end of each domain such that a total of four independent carbohydrate molecules associate with each homodimer. The binding cleft provides shape complementarity, including the presence of a deep hydrophobic hole that accommodates the N-acetyl methyl at the reducing end of the carbohydrate, and specificity arises from 7-8 intermolecular hydrogen bonds. The structures of MVL and the MVL-Man3GlcNAc2 complex further our understanding of the molecular basis of high affinity and specificity in protein-carbohydrate recognition.     

Mutation of Arg(273) to Leu alters the specificity of the yeast N-glycan processing class I alpha1,2-mannosidase

Class I alpha1,2-mannosidases (glycosyl hydrolase family 47) involved in the processing of N-glycans during glycoprotein maturation have different specificities. Enzymes in the endoplasmic reticulum of yeast and mammalian cells remove a single mannose from Man(9)GlcNAc(2) to form Man(8)GlcNAc(2) isomer B (lacking the alpha1, 2-mannose residue of the middle alpha1, 3-arm), whereas other alpha1,2-mannosidases, including Golgi alpha1,2-mannosidases IA and IB, can convert Man(9)GlcNAc(2) to Man(5)GlcNAc(2). In the present work, it is demonstrated that with a single mutation in its catalytic domain (Arg(273) --> Leu) the yeast endoplasmic reticulum alpha1,2-mannosidase acquires the ability to transform Man(9)GlcNAc to Man(5)GlcNAc. High resolution proton nuclear magnetic resonance analysis of the products shows that the order of removal of mannose from Man(9)GlcNAc is different from that of other alpha1, 2-mannosidases that remove four mannose from Man(9)GlcNAc. These results demonstrate that Arg(273) is in part responsible for the specificity of the endoplasmic reticulum alpha1,2-mannosidase and that small differences in non-conserved amino acids interacting with the oligosaccharide substrate in the active site of class I alpha1, 2-mannosidases are responsible for the different specificities of these enzymes.     

Studies on the structure of the oligosaccharide chains of alpha 1-acid glycoprotein associated with rough membranes from rat liver

The high mannose form of rat alpha 1-acid glycoprotein was isolated from rough membranes of rat liver using methods described previously. The high mannose glycopeptides were prepared by Pronase digestion, and oligosaccharides were isolated following digestion with endohexosaminidase-H. The structure of the carbohydrate chains of the high mannose glycopeptide and the oligosaccharides was examined by 300 MHz nuclear magnetic resonance spectroscopy. The glycopeptide contained a mixture of about equal amounts of AsnGlcNAc2Man9 and AsnGlcNAc2Man8. Analysis of the oligosaccharide fraction showed that it consisted of about equal amounts of GlcNAc Man9 and GlcNAc Man8; the GlcNAc Man8 fraction contained 85% of the "A" isomer (which was missing the terminal mannose from the middle antenna). The results suggested that mannose processing of alpha 1-acid glycoprotein in rough membranes of rat liver in vivo occurred only as far as the Man8 structure and that the "A" isomer was the main isomer formed.     

Importance of glycosidases in mammalian glycoprotein biosynthesis

Processing glycosidases play an important role in N-glycan biosynthesis in mammalian cells by trimming Glc(3)Man(9)GlcNAc(2) and thus providing the substrates for the formation of complex and hybrid structures by Golgi glycosyltransferases. Processing glycosidases also play a role in the folding of newly formed glycoproteins and in endoplasmic reticulum quality control. The properties and molecular nature of mammalian processing glycosidases are described in this review. Membrane-bound alpha-glucosidase I and soluble alpha-glucosidase II of the endoplasmic reticulum remove the alpha1,2-glucose and alpha1,3-glucose residues, respectively, beginning immediately following transfer of Glc(3)Man(9)GlcNAc(2) to nascent polypeptides. The alpha-glucosidases participate in glycoprotein folding mediated by calnexin and calreticulin by forming the monoglucosylated high mannose oligosaccharides required for the interaction with the chaperones. In some mammalian cells, Golgi endo alpha-mannosidase provides an alternative pathway for removal of glucose residues. Removal of alpha1,2-linked mannose residues begins in the endoplasmic reticulum where trimming of mannose residues in the endoplasmic reticulum has been implicated in the targeting of malfolded glycoproteins for degradation. Removal of mannose residues continues in the Golgi with the action of alpha1, 2-mannosidases IA and IB that can form Man(5)GlcNAc(2) and of alpha-mannosidase II that removes the alpha1,3- and alpha1,6-linked mannose from GlcNAcMan(5)GlcNAc(2) to form GlcNAcMan(3)GlcNAc(2). These membrane-bound Golgi enzymes have been cloned and shown to have very distinct patterns of tissue-specific expression. There are also broad specificity alpha-mannosidases that can trim Man(4-9)GlcNAc(2) to Man(3)GlcNAc(2), and provide an alternative pathway toward complex oligosaccharide formation. Cloning of the remaining alpha-mannosidases will be required to evaluate their specific functions in glycoprotein maturation.     

The crystal structures of Man(alpha1-3)Man(alpha1-O)Me and Man(alpha1-6)Man(alpha1-O)Me in complex with concanavalin A.

The crystal structures of concanavalin A in complex with Man(alpha1-6)Man(alpha1-O)Me and Man(alpha1-3)Man(alpha1-O)Me were determined at resolutions of 2.0 and 2.8 A, respectively. In both structures, the O-1-linked mannose binds in the conserved monosaccharide-binding site. The O-3-linked mannose of Man(alpha1-3)Man(alpha1-O)Me binds in the hydrophobic subsite formed by Tyr-12, Tyr-100, and Leu-99. The shielding of a hydrophobic surface is consistent with the associated large heat capacity change. The O-6-linked mannose of Man(alpha1-6)Man(alpha1-O)Me binds in the same subsite formed by Tyr-12 and Asp-16 as the reducing mannose of the highly specific trimannose Man(alpha1-3)[Man(alpha1-6)]Man(alpha1-O)Me. However, it is much less tightly bound. Its O-2 hydroxyl makes no hydrogen bond with the conserved water 1. Water 1 is present in all the sugar-containing concanavalin A structures and increases the complementarity between the protein-binding surface and the sugar, but is not necessarily a hydrogen-bonding partner. A water analysis of the carbohydrate-binding site revealed a conserved water molecule replacing O-4 on the alpha1-3-linked arm of the trimannose. No such water is found for the reducing or O-6-linked mannose. Our data indicate that the central mannose of Man(alpha1-3)[Man(alpha1-6)]Man(alpha1-O)Me primarily functions as a hinge between the two outer subsites.     

Phosphorylation of the oligosaccharide of uteroferrin by UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferases from rat liver, Acanthamoeba castellani, and Dictyostelium discoideum requires alpha 1,2-linked mannose residues

We have investigated the oligosaccharide requirements of the UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferases from rat liver, Acanthamoeba castellani, and Dictyostelium discoideum. Uteroferrin, an acid hydrolase, was phosphorylated by the three N-acetylglucosaminylphosphotransferases, and the phosphorylated oligosaccharides were isolated and analyzed by ion suppression high performance liquid chromatography. In all three cases, the phosphorylated species contained 6 or more mannose residues. Phosphorylation of the Man5GlcNAc2 oligosaccharide could not be detected even though this was the major species on the native uteroferrin. The Man5GlcNAc2 oligosaccharides lack alpha 1,2-linked mannose residues, whereas the larger oligosaccharides contain 1 or more mannose residues in this linkage. Treatment of intact uteroferrin with an alpha 1,2-specific mannosidase-generated molecules whose oligosaccharides consisted almost entirely of species with 5 mannose residues. The N-acetylglucosaminylphosphotransferases could no longer phosphorylate such molecules. These data indicate that at least 1 alpha 1,2-linked mannose residue must be present on uteroferrin's oligosaccharide for phosphorylation to occur.     

Variation of high mannose chains of Tamm-Horsfall glycoprotein confers differential binding to type 1-fimbriated Escherichia coli

Tamm-Horsfall glycoprotein (THP), the most abundant protein in mammalian urine, has been implicated in defending the urinary tract against infections by type 1-fimbriated Escherichia coli. Recent experimental evidence indicates that the defensive capability of THP relies on its single high mannose chain, which binds to E. coli FimH lectin and competes with mannosylated uroplakin receptors on the bladder surface. Here we describe several major differences, on both structural and functional levels, between human THP (hTHP) and pig THP (pTHP). pTHP contains a much higher proportion (47%) of Man5GlcNAc2 than does hTHP (8%). FimH-expressing E. coli adhere to monomeric pTHP at an approximately 3-fold higher level than to monomeric hTHP. This suggests that the shorter high mannose chain (Man5GlcNAc2) is a much better binder for FimH than the longer chains (Man6-7GlcNAc2) and that pTHP is a more potent urinary defense factor than hTHP. In addition, unlike hTHP whose polyantennary glycans are exclusively capped by sialic acid and sulfate groups, those of pTHP are also terminated by Galalpha1,3Gal epitope. This is consistent with the fact that the outer medulla of pig kidney expresses the alpha1,3-galactosyltransferase, which is completely absent in human kidney. Finally, pTHP is more resistant to leukocyte elastase hydrolysis than hTHP, thus explaining why pTHP is much less prone to urinary degradation than hTHP. These results demonstrate for the first time that the species variations of the glycomoiety of THP can lead to the differential binding of THP to type 1-fimbriated E. coli and that the differences in high mannose processing may reflect species-specific adaptation of urinary defenses against E. coli infections.     

Endoplasmic reticulum (ER) mannosidase I is compartmentalized and required for N-glycan trimming to Man5-6GlcNAc2 in glycoprotein ER-associated degradation

We had previously shown that endoplasmic reticulum (ER)-associated degradation (ERAD) of glycoproteins in mammalian cells involves trimming of three to four mannose residues from the N-linked oligosaccharide Man(9)GlcNAc(2). A possible candidate for this activity, ER mannosidase I (ERManI), accelerates the degradation of ERAD substrates when overexpressed. Although in vitro, at low concentrations, ERManI removes only one specific mannose residue, at very high concentrations it can excise up to four alpha1,2-linked mannose residues. Using small interfering RNA knockdown of ERManI, we show that this enzyme is required for trimming to Man(5-6)GlcNAc(2) and for ERAD in cells in vivo, leading to the accumulation of Man(9)GlcNAc(2) and Glc(1)Man(9)GlcNAc(2) on a model substrate. Thus, trimming by ERManI to the smaller oligosaccharides would remove the glycoprotein from reglucosylation and calnexin binding cycles. ERManI is strikingly concentrated together with the ERAD substrate in the pericentriolar ER-derived quality control compartment (ERQC) that we had described previously. ERManI knockdown prevents substrate accumulation in the ERQC. We suggest that the ERQC provides a high local concentration of ERManI, and passage through this compartment would allow timing of ERAD, possibly through a cycling mechanism. When newly made glycoproteins cannot fold properly, transport through the ERQC leads to trimming of a critical number of mannose residues, triggering a signal for degradation.     

Saccharomyces cerevisiae alpha1,6-mannosyltransferase has a catalytic potential to transfer a second mannose molecule

In yeast, the N-linked oligosaccharide modification in the Golgi apparatus is initiated by alpha1,6-mannosyltransferase (encoded by the OCH1 gene) with the addition of mannose to the Man(8)GlcNAc(2) or Man(9)GlcNAc(2) endoplasmic reticulum intermediates. In order to characterize its enzymatic properties, the soluble form of the recombinant Och1p was expressed in the methylotrophic yeast Pichia pastoris as a secreted protein, after truncation of its transmembrane region and fusion with myc and histidine tags at the C-terminus, and purified using a metal chelating column. The enzymatic reaction was performed using various kinds of pyridylaminated (PA) sugar chains as acceptor, and the products were separated by high performance liquid chromatography. The recombinant Och1p efficiently transferred a mannose to Man(8)GlcNAc(2)-PA and Man(9)GlcNAc(2)-PA acceptors, while Man(5)GlcNAc(2)-PA, which completely lacks alpha1,2-linked mannose residues, was not used as an acceptor. At high enzyme concentrations, a novel product was detected by HPLC. Analysis of the product revealed that a second mannose was attached at the 6-O-position of alpha1,3-linked mannose branching from the alpha1,6-linked mannose that is attached to beta1,4-linked mannose of Man(10)GlcNAc(2)-PA produced by the original activity of Och1p. Our results indicate that Och1p has the potential to transfer two mannoses from GDP-mannose, and strictly recognizes the overall structure of high mannose type oligosaccharide.     

Structural basis for the recognition of complex-type biantennary oligosaccharides by Pterocarpus angolensis lectin

The crystal structure of Pterocarpus angolensis lectin is determined in its ligand-free state, in complex with the fucosylated biantennary complex type decasaccharide NA2F, and in complex with a series of smaller oligosaccharide constituents of NA2F. These results together with thermodynamic binding data indicate that the complete oligosaccharide binding site of the lectin consists of five subsites allowing the specific recognition of the pentasaccharide GlcNAc beta(1-2)Man alpha(1-3)[GlcNAc beta(1-2)Man alpha(1-6)]Man. The mannose on the 1-6 arm occupies the monosaccharide binding site while the GlcNAc residue on this arm occupies a subsite that is almost identical to that of concanavalin A (con A). The core mannose and the GlcNAc beta(1-2)Man moiety on the 1-3 arm on the other hand occupy a series of subsites distinct from those of con A.     

Inhibitors of the biosynthesis and processing of N-linked oligosaccharides

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

Structural basis of oligomannose recognition by the Pterocarpus angolensis seed lectin

The crystal structure of a Man/Glc-specific lectin from the seeds of the bloodwood tree (Pterocarpus angolensis), a leguminous plant from central Africa, has been determined in complex with mannose and five manno-oligosaccharides. The lectin contains a classical mannose-specificity loop, but its metal-binding loop resembles that of lectins of unrelated specificity from Ulex europaeus and Maackia amurensis. As a consequence, the interactions with mannose in the primary binding site are conserved, but details of carbohydrate-binding outside the primary binding site differ from those seen in the equivalent carbohydrate complexes of concanavalin A. These observations explain the differences in their respective fine specificity profiles for oligomannoses. While Man(alpha1-3)Man and Man(alpha1-3)[Man(alpha1-6)]Man bind to PAL in low-energy conformations identical with that of ConA, Man(alpha1-6)Man is required to adopt a different conformation. Man(alpha1-2)Man can bind only in a single binding mode, in sharp contrast to ConA, which creates a higher affinity for this disaccharide by allowing two binding modes.     

Characterization of the endomannosidase pathway for the processing of N-linked oligosaccharides in glucosidase II-deficient and parent mouse lymphoma cells.

Studies on N-linked oligosaccharide processing in the mouse lymphoma glucosidase II-deficient mutant cell line (PHAR2.7) as well as the parent BW5147 cells indicated that the former maintain their capacity to synthesize complex carbohydrate units through the use of the deglucosylation mechanism provided by endomannosidase. The in vivo activity of this enzyme was evident in the mutant cells from their production of substantial amounts of glucosylated mannose saccharides, predominantly Glc2Man; moreover, in the presence of 1-deoxymannojirimycin or kifunensine to prevent processing by mannosidase I, N-linked Man8GlcNAc2 was observed entirely in the form of the characteristic isomer in which the terminal mannose of the alpha 1,3-linked branch is missing (isomer A). In contrast, parent lymphoma cells, as well as HepG2 cells in the presence of 1-deoxymannojirimycin accumulated Man9GlcNAc2 as the primary deglucosylated N-linked oligosaccharide and contained only about 16% of their Man8GlcNAc2 as isomer A. In the presence of the glucosidase inhibitor castanospermine the mutant released Glc3Man instead of Glc2Man, and the parent cells converted their deglucosylation machinery to the endomannosidase route. Despite the mutant's capacity to accommodate a large traffic through this pathway no increase in the in vitro determined endomannosidase activity was evident. The exclusive utilization of endomannosidase by the mutant for the deglucosylation of its predominant N-linked Glc2Man9GlcNAc2 permitted an exploration of the in vivo site of this enzyme's action. Pulse-chase studies utilizing sucrose-D2O density gradient centrifugation indicated that the Glc2Man9GlcNAc2 to Man8GlcNAc2 conversion is a relatively late event that is temporally separated from the endoplasmic reticulum-situated processing of Glc3Man9GlcNAc2 to Glc2Man9GlcNAc2 and in contrast to the latter takes place in the Golgi compartment.     

Rat epididymal alpha-D-mannosidase: purification, carbohydrate composition, substrate specificity, and antibody production

Two alpha-D-mannosidases have previously been identified in rat epididymis. This communication reports the purification and characterization of the "acid" alpha-D-mannosidase. The enzyme was purified over 1000-fold to near homogeneity by acetone and (NH4)2SO4 precipitation followed by ion-exchange and hydroxylapatite chromatography. The molecular weight of the enzyme was estimated to be 220,000 by gel filtration. Polyacrylamide gel electrophoresis of the native enzyme under two conditions of buffer and pH showed a single band when stained for protein while electrophoresis under denaturing conditions resulted in bands of apparent Mr 60,000 and 31,000. The enzyme is a glycoprotein containing about 5.6% hexose. In addition to mannose (3.1%) and glucosamine (2.0%), the enzyme also contained small amounts of glucose, fucose, and galactose. Chemical analysis indicated the absence of sialic acid. The substrate specificity of the purified enzyme was investigated using linear and branched mannose-containing oligosaccharides. The enzyme cleaved linear oligosaccharides [Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc and Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc] very efficiently. However, little or no activity was observed toward high mannose oligosaccharides (Man9GlcNAc through Man5GlcNAc) or the branched trimannosyl derivative Man3GlcNAc. This specificity is very similar to that observed with rat kidney lysosomal alpha-D-mannosidase. Additional evidence that the epididymal enzyme is essentially a lysosomal alpha-D-mannosidase is the fact that polyclonal antibody prepared against the purified epididymal enzyme cross-reacted with lysosomal alpha-D-mannosidase from several rat tissues and with acidic alpha-D-mannosidase of a human cell line, results suggesting that the antibody will be useful in studying the biosynthesis and turnover of lysosomal alpha-D-mannosidases in at least two species.     

Endoplasmic reticulum-associated degradation of mammalian glycoproteins involves sugar chain trimming to Man6-5GlcNAc2

Endoplasmic reticulum-associated degradation of misfolded or misprocessed glycoproteins in mammalian cells is prevented by inhibitors of class I alpha-mannosidases implicating mannose trimming from the precursor oligosaccharide Glc3Man9GlcNAc2 as an essential step in this pathway. However, the extent of mannose removal has not been determined. We show here that glycoproteins subject to endoplasmic reticulum-associated degradation undergo reglucosylation, deglucosylation, and mannose trimming to yield Man6GlcNAc2 and Man5GlcNAc2. These structures lack the mannose residue that is the acceptor of glucose transferred by UDP-Glc:glycoprotein glucosyltransferase. This could serve as a mechanism for removal of the glycoproteins from folding attempts catalyzed by cycles of reglucosylation and calnexin/calreticulin binding and result in targeting of these molecules for proteasomal degradation.     

Interleukin-2 carbohydrate recognition modulates CTLL-2 cell proliferation

Interleukin-2 (IL-2) specifically recognizes high-mannose type glycans with five or six mannosyl residues. To determine whether the carbohydrate recognition activity of IL-2 contributes to its physiological activity, the inhibitory effects of high-mannose type glycans on IL-2-dependent CTLL-2 cell proliferation were investigated. Man(5)GlcNAc(2)Asn added to CTLL-2 cell cultures inhibited not only phosphorylation of tyrosine kinases but also IL-2-dependent cell proliferation. We found that a complex of IL-2, IL-2 receptor alpha, beta, gamma subunits, and tyrosine kinases was formed in rhIL-2-stimulated CTLL-2 cells. Among the components of this complex, only the IL-2 receptor alpha subunit was stained with Galanthus nivalis agglutinin which specifically recognizes high-mannose type glycans. This staining was diminished after digestion of the glycans with endo-beta-N-acetylglucosaminidase H or D, suggesting that at least a N-glycan containing Man(5)GlcNAc(2) is linked to the extracellular portion of the IL-2 receptor alpha subunit. Our findings indicate that IL-2 binds the IL-2 receptor alpha subunit through Man(5)GlcNAc(2) and a specific peptide sequence on the surface of CTLL-2 cells. When IL-2 binds to the IL-2Ralpha subunit, this may trigger formation of the high affinity complex of IL-2-IL-2Ralpha, -beta, and -gamma subunits, leading to cellular signaling.     

Bovine serum conglutinin is a lectin which binds non-reducing terminal N-acetylglucosamine, mannose and fucose residues.

Carbohydrate recognition by bovine serum conglutinin has been investigated by inhibition and direct binding assays using glycoproteins and polysaccharides from Saccharomyces cerevisiae (baker's yeast), and neoglycolipids derived from N-acetylglucosamine oligomers, mannobiose and human milk oligosaccharides. The results clearly show that conglutinin is a lectin which binds terminal N-acetylglucosamine, mannose and fucose residues as found in chitobiose (GlcNAc beta 1-4GlcNAc), mannobiose (Man alpha 1-3Man) and lacto-N-fucopentaose II [Fuc alpha 1-4(Gal beta 1-3)GlcNAc beta 1-3Gal beta 1-4Glc] respectively.