Lectins from tunicates: structure-function relationships in innate immunity.
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Animals↗
Biomedical subjects
M S Quesenberry
Publications and source records attributed to M S Quesenberry.
Efficient immobilization of proteins by modification of plate surface with polystyrene derivatives.
Immobilization of proteins on microplate wells by simple adsorption (e.g., for ELISA) is convenient, but it can be inefficient, especially if proteins are hydrophilic or small in size. This problem was alleviated by the use of polyvinylbenzyl lactonoylamide (PVLA). PVLA is strongly adsorbed to the hydrophobic well surface, and its lactonamide part can be oxidized with periodate to generate aldehydo groups. Proteins are then immobilized covalently to the aldehydo groups by reductive amination under mild conditions. Using this method, henceforth termed the PVLA method, alkaline phosphatase (AP) was immobilized to microplates six- to sevenfold greater than by simple adsorption (as measured by activity). Similarly, the activity of immobilized mannose-binding protein A (MBP-A) was 4- to 8-fold higher by the PVLA method than by simple adsorption. The PVLA-coated plates needed as little as 200 ng of MBP-A per well to have a sufficient amount of MBP-A immobilized for the measurement of binding of 125I-labeled mannosylated bovine serum albumin (125I-Man-BSA), but unmodified plates required as much as 20 micrograms/well MBP-A to obtain the same response. Recommended conditions for the PVLA method are 40 microliters of 2 mg/ml of PVLA for coating, 1 mM NaIO4 for the generation of the aldehydo groups, and a 2-h reductive amination at 37 degrees C between pH 8 and 9 for the protein ligation.
Adsorption↗
Difference in the binding mode of two mannose-binding proteins: demonstration of a selective minicluster effect.
Serum-type and liver-type mannose-binding proteins (MBP) are both present in higher animals and both are composed of a carbohydrate-recognition domain (CRD) and a collagenous domain. Although known as mannose-binding proteins, these proteins bind N-acetylglucosamine and other related sugars quite well. An earlier specificity study using cloned CRD portions of both types of MBP from rat [Childs, R. A., Feizi, T., Yuen, C.-T., Drickamer, K., & Quesenberry, M. (1990) J. Biol. Chem. 265, 20770-20777] revealed that the liver MBP CRD binds the trimannosyl core structure of N-glycosides, whereas the serum MBP CRD does not. We studied the substrate preferences of these CRDs using both solid and solution phase assays, testing monosaccharides, glycoproteins, and synthetic cluster ligands. While there was no significant difference in the monosaccharide binding specificities of the two CRDs, they displayed very different affinities for natural glycoproteins and mannose-containing cluster glycosides. Most interestingly, synthetic cluster ligands with two terminal GlcNAc moieties have affinity equal to monovalent GlcNAc ligands toward both CRDs, whereas a series of structurally similar Man-terminated divalent ligand displays about 20-fold enhanced affinity toward liver CRD only. A plausible explanation is that the liver MBP CRD has two sugar binding sites per subunit, one of which binds only mannose, and the other, both mannose and N-acetylglucosamine. In contrast, the serum MBP CRD has only one site of the latter type. Results of isothermal titration calorimetry support this hypothesis.
Binding Sites↗
A rapid formaldehyde assay using purpald reagent: application under periodation conditions.
Measurement of formaldehyde is encountered in a broad range of applications including the wine and alcohol industry and environmental pollution surveillance. In carbohydrate structural chemistry, frequent use is made of formaldehyde by periodate oxidation of terminal vicinal diols. Popular methods for the detection of formaldehyde use reagents such as chromotropic acid (4,5-dihydroxynaphthalene-2,7-disulfonic acid) or acetylacetone. The chromotropic acid method requires heating of the sample under strongly acidic conditions, which is undesirable in many applications. The acetylacetone method yields a yellow color product, and is less specific and sensitive (Mimura et al., J. Hyg. Chem. 22, 39-41, 1976). The reaction of formaldehyde with Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole) works under alkaline conditions at room temperature, and the sensitivity is superior to other methods. The color development by this reagent, however, requires oxidation of the adduct with hydrogen peroxide, air oxygen, or dilute periodate. We found that low levels of periodate, commonly used to oxidize specifically terminal vicinal diols to yield formaldehyde, are compatible with color development with the Purpald reagent. We have investigated the conditions required for use of the Purpald reagent, especially in conjunction with periodate oxidation reactions. We have used the assay either in test tubes or with microplates, attaining sensitivity of as little as 1 nmol formaldehyde.
Chromatography, Gel↗
Synthesis of neoglycoconjugates by transglycosylation with Arthrobacter protophormiae endo-beta-N-acetylglucosaminidase. Demonstration of a macro-cluster effect for mannose-binding proteins.
The transglycosylation activity of endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (endo-A) can be enhanced dramatically by inclusion of organic solvent in the reaction mixture (see accompanying article; Fan, J.-Q., Takegawa, K., Iwahara, S., Kondo, A., Kato, I., Abeygunawardana, C., and Lee, Y. C. (1995) J. Biol. Chem. 270, 17723-17729). This finding was extended to synthesis of important intermediates for preparation of neoglycoconjugates. When 0.2 M GlcNAc-O-(CH2)6NH2, GlcNAc-O-CH2CH = CH2, GlcNAc-O-(CH2)3-CH = CH2, GlcNAc-O-(CH2)3NHCOCH = CH2, GlcNAc-S-CH2CN, GlcNAc-S-(CH2)3CH3, or GlcNAc-S-CH2-CONHCH2CH(OMe)2 were used as acceptors in 30% acetone-containing media, the transglycosylation was accomplished with about 80% yield. The transglycosylation yields to benzyl beta-GlcNAc (67%), 4-methyl-umbelliferyl beta-GlcNAc (66%), p-nitrophenyl beta-GlcNAc (33%), and (GlcNAc-beta-S-CH2CH2CH2)2 (43%) were lower, because their poor solubilities allowed only 0.05 M or lower concentrations in the reaction mixture. A micromole-scale synthesis of Man9GlcNAc2-O-(CH2)3-NHCOCH = CH2 (Man9GlcNAc2-NAP) was accomplished with 90% yield, and the structure of the transglycosylation product was confirmed by 1H NMR. Man9GlcNAc2-NAP was co-polymerized with acrylamide. The ratio of sugar side chain to acrylamide in this glycopolymer was 1:44 and the molecular weight of glycopolymer was estimated to be between 1,500,000 and 2,000,000 by high performance gel filtration chromatography. The glycopolymer was shown to be a much more efficient inhibitor of binding by recombinant rat mannose binding protein-carbohydrate recognition domains (MBP-CRD) from serum (I50 = 3.5 microM Man9GlcNAc2-sugar chain) and liver (I50 = 74.5 nM) than soybean agglutinin.
Acetylglucosaminidase↗
Role of conserved and nonconserved residues in the Ca(2+)-dependent carbohydrate-recognition domain of a rat mannose-binding protein. Analysis by random cassette mutagenesis.
The carbohydrate-recognition domain of rat serum mannose-binding protein A has been subjected to random cassette mutagenesis. Mutant domains, expressed in bacteria, were initially screened for binding to invertase-coated nitrocellulose and then analyzed further for Ca2+ affinity, saccharide binding, resistance to proteolysis, and oligomerization. The results are consistent with previous evolutionary and structural studies. Six out of seven completely inactive mutants have changes in residues directly involved in ligating Ca2+. Most changes in conserved residues which form part of the hydrophobic core characteristic of Ca(2+)-dependent (C-type) animal lectins result in decreased affinity for Ca2+, even though these residues are distant from the Ca2+ sites. Changes can be made in large portions of the surface without affecting saccharide binding. The results indicate that the precise arrangement of the regular portion of the domain containing the hydrophobic core is necessary for formation of a stable Ca(2+)-ligated structure under physiological conditions. The data also suggest that the saccharide-binding site is likely to be in close proximity to the bound Ca2+.
Amino Acids↗
Molecular mechanisms of complex carbohydrate recognition at the cell surface.
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Animals↗
Determination of the minimum carbohydrate-recognition domain in two C-type animal lectins.
Comparison of the primary structures of numerous Ca(2+)-dependent animal lectins reveals the presence of a common sequence motif which has been suggested to form the carbohydrate-recognition domain in these proteins. The extent of the functional carbohydrate-recognition domains in two rat C-type lectins, mannose-binding protein A and the major subunit of the asialoglycoprotein receptor (rat hepatic lectin 1), has been defined by expressing truncated fragments of the proteins in an in vitro transcription and translation system. The shortest fully functional fragments constitute the COOH-terminal 120 amino acids of mannose-binding protein A and 135 amino acids of rat hepatic lectin 1. These segments correspond closely to protease-resistant protein cores which can be isolated from the native lectins. The NH2-terminal boundary of each minimum carbohydrate-recognition domain falls near the site of an intron in the corresponding gene.
Amino Acid Sequence↗
Differential recognition of core and terminal portions of oligosaccharide ligands by carbohydrate-recognition domains of two mannose-binding proteins.
Two different mannose-binding proteins (MBP-A and MBP-C), which show 56% sequence identity, are present in rat serum and liver. It has previously been shown that MBP-A binds to a range of monosaccharide-bovine serum albumin conjugates, and that, among oligosaccharide ligands tested, preferential binding is to terminal nonreducing N-acetylglucosamine residues of complex type N-linked oligosaccharides. In order to compare the binding specificity of MBP-C, an expression system has been developed for production of a fragment of this protein which contains the COOH-terminal carbohydrate-recognition domain. After radioiodination, the domain has been used to probe natural glycoproteins, neoglycoproteins, and neoglycolipids. Like MBP-A, MBP-C binds several different monosaccharides conjugated to bovine serum albumin, including mannose, fucose, and N-acetylglucosamine, although binding to the last of these is relatively weaker than observed for MBP-A. The results of binding to natural glycoproteins and to neoglycolipids containing oligosaccharides derived from these proteins are most compatible with the interpretation that MBP-C interacts primarily with the trimannosyl core of complex N-linked oligosaccharides, with additional ligands being terminal fucose and perhaps also peripheral mannose residues of high mannose type oligosaccharides. This binding specificity is thus quite distinct from that of MBP-A. The presence of multiple MBPs with distinct binding specificities in preparations derived from serum and liver explains conflicting conclusions which have been reached about carbohydrate recognition by these proteins.
Amino Acid Sequence↗
Major and minor forms of the rat liver asialoglycoprotein receptor are independent galactose-binding proteins. Primary structure and glycosylation heterogeneity of minor receptor forms.
Preparations of the rat liver asialoglycoprotein receptor (rat hepatic lectin, RHL), which is responsible for the selective uptake of partially deglycosylated serum glycoproteins, have been found to contain multiple polypeptide species. A method has been developed for separating the predominant species (RHL-1) from the minor species (RHL-2/3) using conditions in which RHL-1 retains its galactose-binding activity. Endoglycosidase digestion and lectin blotting have been utilized to demonstrate that RHL-2 and RHL-3 differ by the presence of different carbohydrate structures attached to a common peptide backbone. Antisera specific for RHL-1 and RHL-2/3 have been prepared and utilized to analyze the results of cross-linking experiments performed on purified receptor and hepatocyte membranes. The results show that RHL-1 and RHL-2/3 polypeptides are each associated into homooligomers but are physically unlinked to each other. The structure of the RHL-2/3 polypeptide has been established by protein and cDNA sequence analysis, which reveals that this protein is homologous to RHL-1 throughout its length but contains one major insertion of 18 amino acids near its NH2 terminus. The COOH-terminal portion of the RHL-2/3 polypeptide has been demonstrated to contain a galactose-recognition domain by expression in an in vitro transcription/translation system. The results of these experiments indicate that RHL-1 and RHL-2/3 polypeptides are self-associated into two distinct molecules, each of which has galactose-binding activity.
Amino Acid Sequence↗