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K Drickamer

Publications and source records attributed to K Drickamer.

At least 37 records · Page 2Linked to original sources
PubMed1996

Introduction of selectin-like binding specificity into a homologous mannose-binding protein.

The structures of the ligand-binding C-type carbohydrate-recognition domains of selectin cell adhesion molecules and of mannose-binding proteins (MBPs) are similar to each other even though these proteins bind very different carbohydrate ligands. Our current understanding of ligand binding by E-selectin is based on structural studies of unliganded E-selectin and of MBP-carbohydrate complexes, combined with results from mutagenesis of E-selectin. Five regions of E-selectin that differ in sequence from the corresponding regions of MBP have been introduced into the carbohydrate-recognition domain of MBP. Four of the changes have little effect on ligand binding. Insertion of one stretch of positively charged amino acids alters the sugar binding selectivity of the domain so that it now binds HL-60 cells and serum albumin derivatized with sialyl-Lewis X tetrasaccharide, thus mimicking the properties of E-selectin.

Amino Acid Sequence↗
PubMed1996

Selective sugar binding to the carbohydrate recognition domains of the rat hepatic and macrophage asialoglycoprotein receptors.

Asialoglycoprotein receptors on the surfaces of both hepatocytes and peritoneal macrophages bind terminal galactose residues of desialylated glycoproteins and mediate endocytosis and eventual degradation of these ligands. The hepatic receptor binds oligosaccharides with terminal N-acetylgalactosamine residues more tightly than ligands with terminal galactose residues, but the macrophage receptor shows no such differential binding affinity. Carbohydrate recognition domains from the macrophage receptor and the major subunit of the hepatic receptor have been expressed in a bacterial system and have been shown to retain the distinct binding selectivities of the receptors from which they derive. Binding of a series of N-acyl derivatives of galactosamine suggests that the 2-substituent of these sugars interacts with the surface of the hepatic receptor with highest affinity binding observed for the N-propionyl derivative. Chimeric sugar-binding domains have been used to identify three regions of the hepatic receptor that are essential for establishing selectivity for N-acetylgalactosamine over galactose. Based on these results and the orientation of N-acetylgalactosamine when bound to an homologous galactose-binding mutant of rat serum mannose-binding protein, a fourth region likely to interact with N-acetylgalactosamine has been identified and probed by site-directed mutagenesis. The results of these studies define a binding pocket for the 2-substituent of N-acetylgalactosamine in the hepatic asialoglycoprotein receptor.

Amino Acid Sequence↗
PubMed1996

Structural analysis of monosaccharide recognition by rat liver mannose-binding protein.

The structural basis of carbohydrate recognition by rat liver mannose-binding protein (MBP-C) has been explored by determining the three-dimensional structure of the C-type carbohydrate-recognition domain (CRD) of MBP-C using x-ray crystallography. The structure was solved by molecular replacement using rat serum mannose-binding protein (MBP-A) as a search model and was refined to maximum Bragg spacings of 1.7 A. Despite their almost identical folds, the dimeric structures formed by the two MBP CRDs differ dramatically. Complexes of MBP-C with methyl glycosides of mannose, N-acetylglucosamine, and fucose were prepared by soaking MBP-C crystals in solutions containing these sugars. Surprisingly, the pyranose ring of mannose is rotated 180 degrees relative to the orientation observed previously in MBP-A, but the local interactions between sugar and protein are preserved. For each of the bound sugars, vicinal, equatorial hydroxyl groups equivalent to the 3- and 4-OH groups of mannose directly coordinate Ca2+ and form hydrogen bonds with residues also serving as Ca2+ ligands. Few interactions are observed between other parts of the sugar and the protein. A complex formed between free galactose and MBP-C reveals a similar mode of binding, with the anomeric hydroxyl group serving as one of the Ca2+ ligands. A second binding site for mannose has also been observed in one of two copies in the asymmetric unit at a sugar concentration of 1.3 M. These structures explain how MBPs recognize a wide range of monosaccharides and suggest how fine specificity differences between MBP-A and MBP-C may be achieved.

Amino Acid Sequence↗
PubMed1996

Differential ligand binding by two subunits of the rat liver asialoglycoprotein receptor.

The rat liver asialoglycoprotein receptor consists of two types of subunits, a predominant polypeptide designated rat hepatic lectin 1 (RHL-1) and a minor polypeptide, RHL-2/3, that comes in two differentially glycosylated forms. The exact stoichiometry and arrangement of the subunits in the RHL oligomer are not known. The carbohydrate-recognition domain of RHL-2/3 has been prepared by limited proteolysis of the liver receptor so that its properties can be compared with those of the corresponding domain of RHL-1 previously produced in a bacterial expression system. Binding studies indicate that while RHL-1 binds N-acetylgalactosamine with approximately 60-fold higher affinity than it binds galactose, RHL-2/3 has only 2-fold selectivity for N-acetylgalactosamine. In general, the pattern of monosaccharide-binding specificity for RHL-2/3 is similar to RHL-1, but the discrimination of various sugars relative to galactose is reduced substantially. Limited proteolysis and crosslinking studies demonstrate that RHL-2/3 is easily removed from the RHL oligomer in detergent solution and that RHL-1 remains at least trimeric following removal of RHL-2/3. These studies suggest that RHL-1 forms a ligand-binding core while RHL-2/3 acts more as an accessory subunit contributing to selective binding of certain oligosaccharide structures.

Amino Acid Sequence↗
PubMed1996

Structural basis of lectin-carbohydrate recognition.

Lectins are responsible for cell surface sugar recognition in bacteria, animals, and plants. Examples include bacterial toxins; animal receptors that mediate cell-cell interactions, uptake of glycoconjugates, and pathogen neutralization; and plant toxins and mitogens. The structural basis for selective sugar recognition by members of all of these groups has been investigated by x-ray crystallography. Mechanisms for sugar recognition have evolved independently in diverse protein structural frameworks, but share some key features. Relatively low affinity binding sites for monosaccharides are formed at shallow indentations on protein surfaces. Selectivity is achieved through a combination of hydrogen bonding to the sugar hydroxyl groups with van der Waals packing, often including packing of a hydrophobic sugar face against aromatic amino acid side chains. Higher selectivity of binding is achieved by extending binding sites through additional direct and water-mediated contacts between oligosaccharides and the protein surface. Dramatically increased affinity for oligosaccharides results from clustering of simple binding sites in oligomers of the lectin polypeptides. The geometry of such oligomers helps to establish the ability of the lectins to distinguish surface arrays of polysaccharides in some instances and to crosslink glycoconjugates in others.

Carbohydrate Metabolism↗
PubMed1995

Increasing diversity of animal lectin structures.

The number of animal proteins known to recognize carbohydrates and the number of their biological roles continue to increase. Comparisons of primary structures show that some of the newly described lectins are akin to previously investigated lectins, whereas others represent new structural groups. Progress has been made in understanding structure-function relationships for several lectins in both the old and the new categories.

Animals↗
PubMed1994

Trimeric structure of a C-type mannose-binding protein.

BACKGROUND: Mannose-binding proteins (MBPs) are C-type (Ca(2+)-dependent) animal lectins found in serum. They recognize cell-surface oligosaccharide structures characteristic of pathogenic bacteria and fungi, and trigger the neutralization of these organisms. Like most lectins, MBPs display weak intrinsic affinity for monovalent sugar ligands, but bind avidly to multivalent ligands. RESULTS: We report physical studies in solution and the crystal structure determined at 1.8 A Bragg spacings of a trimeric fragment of MBP-A, containing the carbohydrate-recognition domain (CRD) and the neck domain that links the carboxy-terminal CRD to the collagen-like portion of the intact molecule. The neck consists of a parallel triple-stranded coiled coil of alpha-helices linked by four residues to the CRD. The isolated neck peptide does not form stable helices in aqueous solution. The previously characterized carbohydrate-binding sites lie at the distal end of the trimer and are separated from each other by 53 A. CONCLUSIONS: The carbohydrate-binding sites in MBP-A are too far apart for a single trimer to bind multivalently to a typical mammalian high-mannose oligosaccharide. Thus MBPs can recognize pathogens selectively by binding avidly only to the widely spaced, repetitive sugar arrays on pathogenic cell surfaces. Sequence alignments reveal that other C-type lectins are likely to have a similar oligomeric structure, but differences in their detailed organization will have an important role in determining their interactions with oligosaccharides.

Amino Acid Sequence↗
PubMed1994

Binding of sugar ligands to Ca(2+)-dependent animal lectins. I. Analysis of mannose binding by site-directed mutagenesis and NMR.

The Ca(2+)-dependent carbohydrate-recognition domain (CRD) of rat serum mannose-binding protein has been subjected to site-directed mutagenesis to determine the importance of individual residues in ligation of mannose and related sugars. The effects of the mutations were assessed by direct binding assays, competition binding studies, partial proteolysis, and NMR analysis of sugar-CRD titrations. As suggested by the crystal structure of the mannose-binding CRD complexed with oligosaccharide ligand, asparagine and glutamic acid residues that interact with hydroxyl groups 3 and 4 of the sugar, as well as with one of the two bound Ca2+, are critical for ligand binding. In addition, the beta-carbon of His189 contributes substantially to the binding affinity, apparently through a van der Waals contact with C-4 of the sugar ligand. van der Waals contacts between the imidazole ring of His189 and the 2 hydroxyl group of mannose, and between Ile207 and C-6 of mannose, observed in the crystal structure, contribute less to stability of the ligand complex. The effects of changes at positions 189 and 207 on the ability of the CRD to distinguish between alpha-and beta-methyl L-fucosides suggest that fucose may bind in an alternative orientation compared to the arrangement originally proposed based on the mannose-CRD complex.

Amino Acid Sequence↗
PubMed1994

Binding of sugar ligands to Ca(2+)-dependent animal lectins. II. Generation of high-affinity galactose binding by site-directed mutagenesis.

Changes have been introduced into the Ca(2+)-dependent carbohydrate-recognition domain (CRD) of rat serum mannose-binding protein by site-directed mutagenesis to model the binding sites of homologous galactose-binding CRDs. Binding assays reveal that galactose-binding activity nearly identical to that of the CRD from the asialoglycoprotein receptor can be introduced into the mannose-binding site by 3 single amino acid changes and insertion of a segment of 5 amino acids. Separate changes are required to establish high-affinity binding to galactose and create high selectivity by exclusion of mannose from the binding site. The mutagenesis studies and NMR analysis of sugar-CRD titrations demonstrate that an important component of high-affinity galactose binding is interaction between the B face of the sugar and tryptophan. The binding properties of the C-type CRD from the cartilage proteoglycan, aggrecan, can also be modeled based on the mannose-binding CRD frame-work. This lower affinity binding site involves stacking of a phenylalanine residue against the sugar ligand.

Amino Acid Sequence↗
PubMed1993

Determinants of oligomeric structure in the chicken liver glycoprotein receptor.

The oligomeric state of the chicken liver receptor (chicken hepatic lectin), which mediates endocytosis of glycoproteins terminating with N-acetylglucosamine, has been investigated using physical methods as well as chemical cross-linking. Receptor isolated from liver and from transfected rat fibroblasts expressing the full-length polypeptide is a homotrimer immediately following solubilization in non-ionic detergent, but forms the previously observed hexamer during purification. These results are most consistent with the presence of a trimer of receptor polypeptides in liver membranes and in transfected cells. Analysis of truncated receptors reveals that the C-terminal extracellular portion of this type-II transmembrane protein does not form stable oligomers when isolated from the membrane anchor and cytoplasmic tail. The behaviour of chimeric receptors, in which the cytoplasmic tail of the glycoprotein receptor is replaced with the corresponding segments of rat liver asialoglycoprotein receptor or the beta-subunit of Na+,K(+)-ATPase, or with unrelated sequences from globin, indicates that the cytoplasmic tail influences oligomer stability. Replacement of N-terminal portions of the receptor with corresponding segments of influenza virus neuraminidase results in formation of tetramers, suggesting that the membrane anchor and flanking sequences are important determinants of oligomer formation.

Amino Acid Sequence↗
PubMed1993

Structural requirements for high affinity binding of complex ligands by the macrophage mannose receptor.

The mannose receptor of macrophage and hepatic endothelial cells discriminates between endogenous and exogenous sugar-bearing structures. Previous competition studies have indicated that the receptor binds the monosaccharides mannose, fucose, and N-acetylglucosamine but displays much higher affinity for multivalent oligosaccharides, such as those found on the surface of potentially pathogenic microorganisms. The hydrodynamic properties of the receptor have been examined, revealing that the receptor is a monomer. This result suggests that multiple carbohydrate recognition domains (CRDs) in the extracellular domain of a single receptor polypeptide cooperate to achieve high affinity binding of complex ligands. In order to determine the importance of individual CRDs, properties of receptor segments containing groups of CRDs expressed in insect cells have been examined. The results indicate that two of the CRDs (4 and 5) form a protease-resistant, ligand-binding core but that five CRDs in tandem (4-8) are required to match the affinity of the intact receptor for yeast mannan. A consequence of the organization of the receptor is that both valency and geometry of glycoconjugates are important determinants of binding affinity.

Animals↗