A conserved disulphide bond in sialyltransferases.
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Amino Acid Sequence↗
Biomedical subjects
K Drickamer
Publications and source records attributed to K Drickamer.
Expression and characterization of a carbohydrate-binding fragment of rat aggrecan.
The COOH-terminal portion of cartilage proteoglycan core protein, aggrecan, expressed by in vitro translation, binds carbohydrate-containing affinity columns. The in vitro expression approach has been used to define the sugar-binding portion of the core protein. The active fragment, which corresponds closely to the carbohydrate-recognition domains in the family of Ca(2+)-dependent (C-type) animal lectins, has been expressed in bacteria and characterized. The CD spectrum of the domain is very similar to the spectrum of the binding domain of serum mannose-binding protein, suggesting that its overall structure probably resembles the known three-dimensional structure of the mannose-binding domain. The binding specificity of the core protein fragment has been characterized using a solid-phase assay. The results suggest that the monosaccharide-binding site is also similar to that in other C-type carbohydrate-recognition domains.
Aggrecans↗
Evolution of Ca(2+)-dependent animal lectins.
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Amino Acid Sequence↗
Multivalent ligand binding by serum mannose-binding protein.
The serum-type mannose-binding protein (MBP) is a defense molecule that has carbohydrate-dependent bactericidal effects. It shares with mammalian and chicken hepatic lectins similarity in the primary structure of the carbohydrate-recognition domain, as well as the ligand-binding mode: a high affinity (KD approximately nM) is generated by clustering of approximately 30 terminal target sugar residues on a macromolecule, such as bovine serum albumin, although the individual monosaccharides have low affinity (KD 0.1-1 mM). On the other hand, MBP does not manifest any significant affinity enhancement toward small, di- and trivalent ligands, in contrast to the hepatic lectins whose affinity toward divalent ligands of comparable structures increased from 100- to 1000-fold. Such differences may be explained on the basis of different subunit organization between the hepatic lectins and MBP.
Acute-Phase Proteins↗
Structure of a C-type mannose-binding protein complexed with an oligosaccharide.
C-type (Ca(2+)-dependent) animal lectins such as mannose-binding proteins mediate many cell-surface carbohydrate-recognition events. The crystal structure at 1.7 A resolution of the carbohydrate-recognition domain of rat mannose-binding protein complexed with an oligomannose asparaginyl-oligosaccharide reveals that Ca2+ forms coordination bonds with the carbohydrate ligand. Carbohydrate specificity is determined by a network of coordination and hydrogen bonds that stabilizes the ternary complex of protein, Ca2+ and sugar. Two branches of the oligosaccharide crosslink neighbouring carbohydrate-recognition domains in the crystal, enabling multivalent binding to a single oligosaccharide chain to be visualized directly.
Acetylglucosamine↗
Engineering galactose-binding activity into a C-type mannose-binding protein.
Calcium-dependent or C-type carbohydrate-recognition domains are homologous protein modules found in a variety of animal lectins. Selective binding of sugars by these domains is essential for glycoprotein clearance, cell-cell adhesion and pathogen neutralization. Although various C-type carbohydrate-recognition domains share sequence identity ranging from 20 to 55%, their sugar-binding characteristics vary widely. The structure of a mannose-binding carbohydrate-recognition domain in complex with a saccharide ligand suggests that two glutamic acid-asparagine pairs are essential determinants of ligand binding by this domain. In C-type lectins that bind galactose with higher affinity than mannose, one of these pairs is replaced by glutamine-aspartic acid. Here we shift the sequence of the mannose-binding protein to correspond to that found in galactose-binding domains in order to test the importance of these residues in sugar-binding selectivity. This simple switch in the position of a single amide group alters the binding activity of the domain so that galactose becomes the preferred ligand.
Acetylgalactosamine↗
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↗
Specificity of lung surfactant protein SP-A for both the carbohydrate and the lipid moieties of certain neutral glycolipids.
Binding specificity of the major surfactant protein SP-A from human and dog lung has been investigated. Radiobinding experiments have shown that both proteins bind in a Ca(2+)-dependent manner to galactose, mannose, fucose, and glucose linked to bovine serum albumin. These results are in accord with a previous study in which monosaccharides were linked to agarose (Haagsman, H. P., Hawgood, S., Sargeant, T., Buckley, D., White, R. T., Drickamer, K., and Benson, B. J. (1987) J. Biol. Chem. 262, 13877-13880). Chromatogram overlays in conjunction with in situ liquid secondary ion mass spectrometry (TLC-LSIMS) of several purified glycosphingolipids and neoglycolipids as well as binding assays with glycolipids immobilized on plastic wells, demonstrate recognition of galactose (human and dog SP-A), glucose, and lactose (human SP-A) in association with specific lipids. In addition, the occurrence of several neutral and acidic glycosphingolipids in human and rat extracellular surfactants and rat alveolar type II cells is described. Selected components among the neutral glycolipids are bound by radiolabeled human SP-A; these are identified by TLC-LSIMS as predominantly ceramide mono- and disaccharides (human surfactant) and ceramide tri- and tetrasaccharides (rat surfactant and type II cells). A recombinant carbohydrate recognition domain (CRD) of human SP-A inhibits the binding of human SP-A to galactosyl ceramide and to galactose- and mannose-bovine serum albumin, indicating that the CRD is directly involved in the binding of SP-A to these ligands. These results provide evidence for a novel type of binding specificity for proteins that have Ca(2+)-dependent CRDs and raise the possibility that glycosphingolipids are endogenous ligands for SP-A.
Animals↗
Contribution to ligand binding by multiple carbohydrate-recognition domains in the macrophage mannose receptor.
The extracellular portion of the macrophage mannose receptor is composed of several cysteine-rich domains, including a fibronectin type II repeat and eight segments related in sequence to Ca(2+)-dependent carbohydrate-recognition domains (CRDs) of animal lectins. Expression of portions of the receptor in vitro, in fibroblasts and in bacteria, has been used to determine which of the extracellular domains are involved in binding and endocytosis of ligand. The NH2-terminal cysteine-rich domain and the fibronectin type II repeat are not necessary for endocytosis of mannose-terminated glycoproteins. CRDs 1-3 have at most very weak affinity for carbohydrate, so the carbohydrate binding activity of the receptor resides in CRDs 4-8. CRD 4 shows the highest affinity binding and has multispecificity for a variety of monosaccharides. However, CRD 4 alone cannot account for the binding of the receptor to glycoproteins. At least 3 CRDs (4, 5, and 7) are required for high affinity binding and endocytosis of multivalent glycoconjugates. In this respect, the mannose receptor is like other carbohydrate-binding proteins, in which several CRDs, each with weak affinity for single sugars, are clustered to achieve high affinity binding to oligosaccharides. In the mannose receptor, these multiple weak interactions are achieved through several active CRDs in a single polypeptide chain rather than by oligomerization of polypeptides each containing a single CRD.
Animals↗
Expression and purification of the cytoplasmic tail of an endocytic receptor by fusion to a carbohydrate-recognition domain.
Gene fusion has been used to produce the cytoplasmic domain of an endocytic receptor. DNA sequences coding for the 52 COOH-terminal amino acids of the mannose receptor from human macrophages, including the 41-amino acid cytoplasmic tail, were fused to the codons specifying the carbohydrate-recognition domain (CRD) of rat mannose-binding protein. The fusion protein was expressed in Escherichia coli and purified in one step on mannose-Sepharose, making use of the carbohydrate-binding activity of the CRD. The tail peptide was released from the fusion protein using endoproteinase Arg-C. This method provides an alternative to chemical synthesis for the production of midlength peptides.
Amino Acid Sequence↗
Organization of the gene encoding the human macrophage mannose receptor (MRC1).
The gene for the human macrophage mannose receptor (MRC1) has been characterized by isolation of clones covering the entire coding region. Sequence analysis reveals that the gene is divided into 30 exons. The first three exons encode the signal sequence, the NH2-terminal cysteine-rich domain, and the fibronectin type II repeat, while the final exon encodes the transmembrane anchor and the cytoplasmic tail. The intervening 26 exons encode the eight carbohydrate-recognition domains and intervening spacer elements. However, no simple correlation between intron boundaries and functional carbohydrate-recognition domains is apparent. The pattern of intron positions as well as comparison of the sequences of the carbohydrate-recognition domains suggests that the duplication of these domains was an evolutionarily ancient event.
Amino Acid Sequence↗
Molecular mechanisms of complex carbohydrate recognition at the cell surface.
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Animals↗
Clearing up glycoprotein hormones.
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Animals↗
Structure of the calcium-dependent lectin domain from a rat mannose-binding protein determined by MAD phasing.
Calcium-dependent (C-type) animal lectins participate in many cell surface recognition events mediated by protein-carbohydrate interactions. The C-type lectin family includes cell adhesion molecules, endocytic receptors, and extracellular matrix proteins. Mammalian mannose-binding proteins are C-type lectins that function in antibody-independent host defense against pathogens. The crystal structure of the carbohydrate-recognition domain of a rat mannose-binding protein, determined as the holmium-substituted complex by multiwavelength anomalous dispersion (MAD) phasing, reveals an unusual fold consisting of two distinct regions, one of which contains extensive nonregular secondary structure stabilized by two holmium ions. The structure explains the conservation of 32 residues in all C-type carbohydrate-recognition domains, suggesting that the fold seen here is common to these domains. The strong anomalous scattering observed at the Ho LIII edge demonstrates that traditional heavy atom complexes will be generally amenable to the MAD phasing method.
Acute-Phase Proteins↗
Physical characterization and crystallization of the carbohydrate-recognition domain of a mannose-binding protein from rat.
A portion of rat mannose-binding protein A (MBP-A), a Ca(2+)-dependent animal lectin, has been overproduced in a bacterial expression system, biochemically characterized, and crystallized. A fragment corresponding to the COOH-terminal 115 residues of native MBP-A, produced by subtilisin digestion of the bacterially expressed protein, contains the carbohydrate-recognition domain (CRD). Gel filtration, chemical cross-linking, and crystallographic self-rotation function analyses indicate that the subtilisin fragment is a dimer, although the complete bacterially expressed fragment, containing the neck and CRD of MBP-A, is a trimer. Crystals of the minimal CRD, obtained only as a complex with a Man6GlcNAc2Asn glycopeptide, diffract to Bragg spacings of at least 1.7 A. Several trivalent lanthanide ions (Ln3+) can substitute for Ca2+, as assessed by their ability to support carbohydrate binding and to protect the CRD from proteolysis in a manner similar to that observed for Ca2+. These assays indicate that Ln2+ binds about 30 times more tightly than Ca2+ to the CRD, and that two Ca2+ or Ln3+ bind to each monomer, a result confirmed by determination of the Ho3+ positions in a Ho(3+)-containing crystal of the CRD. Crystals grown in the presence of Ln3+ belong to different space groups from those obtained with Ca2+ and are therefore not useable for traditional crystallographic phase determination methods, but are well-suited for high resolution structure determination by multiwavelength anomalous dispersion phasing.
Animals↗
Evolutionary conservation of intron position in a subfamily of genes encoding carbohydrate-recognition domains.
The structure of the gene encoding a chicken liver receptor, the chicken hepatic lectin, which mediates endocytosis of glycoproteins has been established. The coding sequence is divided into six exons separated by five introns. The first three exons correspond to separate functional domains of the receptor polypeptide (cytoplasmic tail, transmembrane sequence, and extracellular neck region), while the final three exons encode the Ca(2+)-dependent carbohydrate-recognition domain. These results, as well as computer-assisted multiple sequence comparisons, establish this receptor as the evolutionary homolog of the mammalian asialoglycoprotein receptors. It is interesting that the chicken receptor falls into a subfamily of proteins along with the mammalian asialoglycoprotein receptors, since the saccharide-binding specificity of the chicken receptor resembles more closely that of a different set of calcium-dependent animal lectins, which includes the mannose-binding proteins. The portions of the genes encoding the carbohydrate-recognition domains of these proteins lack introns. The results suggest that divergence of intron-containing and intron-lacking carbohydrate-recognition domains preceded shuffling events in which other functional domains were associated with the carbohydrate-recognition domains. This was followed by further divergence, generating a variety of saccharide-binding specificities.
Amino Acid Sequence↗
Ligand-binding characteristics of rat serum-type mannose-binding protein (MBP-A). Homology of binding site architecture with mammalian and chicken hepatic lectins.
Sugar-binding characteristics of rat serum mannose-binding protein (MBP) were studied using the carbohydrate-recognition domain of this protein expressed from a cloned cDNA. To assess the binding affinity of various test compounds, they were added as inhibitors in a binding assay in which 125I-MBP was incubated with yeast cells and the extent of binding was estimated from the radioactivity associated with the pelleted cells. The results of such inhibition assays suggest that MBP has a small binding site which is probably of the trough-type. The 3- and 4-OH of the target sugar are indispensable, while the 6-OH is not required. These characteristics are shared by the rat hepatic lectin and chicken hepatic lectin, both of which are C-type lectins containing carbohydrate-recognition domains highly homologous to that of MBP. Apparently, the related primary structures of these lectins give rise to similar gross architecture of their binding sites, despite the fact that each exhibits different sugar binding specificities.
Animals↗