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Biomedical subjects

Kurt Drickamer

Publications and source records attributed to Kurt Drickamer.

At least 19 recordsLinked to original sources
PubMed2006

Lewis x antigen mediates adhesion of human breast carcinoma cells to activated endothelium. Possible involvement of the endothelial scavenger receptor C-type lectin.

Lewis x (Le(x), CD15), also known as SSEA-1 (stage specific embryonic antigen-1), is a trisaccharide with the structure Galbeta(1-4)Fucalpha(1-3)GlcNAc, which is expressed on glycoconjugates in human polymorphonuclear granulocytes and various tumors such as colon and breast carcinoma. We have investigated the role of Le(x) in the adhesion of MCF-7 human breast cancer cells and PMN to human umbilical endothelial cells (HUVEC) and the effects of two different anti-Le(x) mAbs (FC-2.15 and MCS-1) on this adhesion. We also analyzed the cytolysis of Le(x+)-cells induced by anti-Le(x) mAbs and complement when cells were adhered to the endothelium, and the effect of these antibodies on HUVEC. The results indicate that MCF-7 cells can bind to HUVEC, and that MCS-1 but not FC-2.15 mAb inhibit this interaction. Both mAbs can efficiently lyse MCF-7 cells bound to HUVEC in the presence of complement without damaging endothelial cells. We also found a Le(x)-dependent PMN interaction with HUVEC. Although both anti-Le(x) mAbs lysed PMN in suspension and adhered to HUVEC, PMN aggregation was only induced by mAb FC-2.15. Blotting studies revealed that the endothelial scavenger receptor C-type lectin (SRCL), which binds Le(x)-trisaccharide, interacts with specific glycoproteins of M (r ) approximately 28 kD and 10 kD from MCF-7 cells. The interaction between Le(x+)-cancer cells and vascular endothelium is a potential target for cancer treatment.

Antibodies, Monoclonal↗
PubMed2006

Widely divergent biochemical properties of the complete set of mouse DC-SIGN-related proteins.

The mouse genome sequence has been examined to identify the complete set of proteins related to the human glycanbinding receptor, DC-SIGN. In addition to five SIGNR proteins previously described, a pseudogene, encoding a hypothetical SIGNR6, and a further two expressed proteins, SIGNR7 and SIGNR8, have been identified. The ligand-binding properties of these novel proteins and of the previously described mouse SIGNs have been systematically investigated in order to define the mouse proteins that most resemble human DC-SIGN and DC-SIGNR. Results from screening of a glycan array demonstrate that only mouse SIGNR3 shares with human DC-SIGN the ability to bind both high mannose and fucose-terminated glycans in this format and to mediate endocytosis. The finding that neither SIGNR1 nor SIGNR5 binds with high affinity to specific ligands in a large panel of mammalian glycans is consistent with the suggestion that these receptors bind surface polysaccharides on bacterial and fungal pathogens in a manner analogous to serum mannose-binding protein. The data also reveal that two of the mouse SIGNs have unusual binding specificities that have not been previously described for members of the C-type lectin family; the newly identified SIGNR7 binds preferentially to the 6-sulfo-sialyl Lewis(x) oligosaccharide, whereas SIGNR2 binds almost exclusively to glycans that bear terminal GlcNAc residues. The results presented demonstrate that the mouse homologs of DC-SIGN have a diverse set of ligand-binding and intracellular trafficking properties, some of which are distinct from the properties of any of the human receptors.

Amino Acid Sequence↗
PubMed2006

Two categories of mammalian galactose-binding receptors distinguished by glycan array profiling.

Profiling of the four known galactose-binding receptors in the C-type lectin family has been undertaken in parallel on a glycan array. The results are generally consistent with those of previous assays using various different formats, but they provide a direct comparison of the properties of the four receptors, revealing that they fall into two distinct groups. The major subunit of the rat asialoglycoprotein receptor and the rat Kupffer cell receptor show similar broad preferences for GalNAc-terminated glycans, while the rat macrophage galactose lectin and the human scavenger receptor C-type lectin (SRCL) bind more restricted sets of glycans. Both of these receptors bind to Lewis x-type structures, but the macrophage galactose lectin also interacts strongly with biantennary galactose- and GalNAc-terminated glycans. Although the similar glycan-binding profiles for the asialoglycoprotein receptor and the Kupffer cell receptor might suggest that these receptors are functionally redundant, analysis of fibroblasts transfected with full-length Kupffer cell receptor reveals that they fail to endocytose glycosylated ligand.

Amino Acid Sequence↗
PubMed2006

Collagen binding by the mannose receptor mediated through the fibronectin type II domain.

The macrophage mannose receptor is the prototype for a family of receptors each having an extracellular region consisting of an N-terminal cysteine-rich domain related to the R-type carbohydrate-recognition domain of ricin, a fibronectin type II domain and eight to ten domains related to C-type carbohydrate-recognition domains. The mannose receptor acts as a molecular scavenger, clearing harmful glycoconjugates or micro-organisms through recognition of their defining carbohydrate structures. Cell-adhesion assays, as well as collagen-binding assays, have now been used to show that the mannose receptor can also bind collagen and that the fibronectin type II domain mediates this activity. Neither of the two types of sugar-binding domain in the receptor is involved in collagen binding. Fibroblasts expressing the mannose receptor adhere to type I, type III and type IV collagens, but not to type V collagen, and the adherence is inhibited by isolated mannose receptor fibronectin type II domain. The fibronectin type II domain shows the same specificity for collagen as the whole receptor, binding to type I, type III and type IV collagens. This is the first activity assigned to the fibronectin type II domain of the mannose receptor. The results suggest additional roles for this multifunctional receptor in mediating collagen clearance or cell-matrix adhesion.

Amino Acid Sequence↗
PubMed2006

All but the shortest polymorphic forms of the viral receptor DC-SIGNR assemble into stable homo- and heterotetramers.

Polymorphisms that affect the length of the extracellular neck region of the endothelial receptor DC-SIGNR (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin-related protein) have been linked to differences in susceptibility to infection by enveloped viruses. We have characterized the effects of these polymorphisms on the ability of DC-SIGNR to form tetramers containing the clusters of sugar-binding sites needed for binding to viral envelope glycoproteins. Chemical cross-linking and analytical ultracentrifugation experiments have been used to show that only the smallest form of DC-SIGNR is defective in homotetramer assembly. A novel affinity-tagging approach has been employed to demonstrate that, contrary to previous speculation, heterotetramers can be assembled efficiently from DC-SIGNR polypeptides of different lengths. The heterotetramers are stable and can be detected in fibroblasts transfected with multiple forms of DC-SIGNR. These results provide a molecular basis for interpreting the way polymorphisms affect interactions with viruses.

Animals↗
PubMed2006

Polymorphisms in human langerin affect stability and sugar binding activity.

Langerhans cells are specialized skin dendritic cells that take up and degrade antigens for presentation to the immune system. Langerin, a cell surface C-type lectin of Langerhans cells, can be internalized and accumulates in Birbeck granules, subdomains of the endosomal recycling compartment that are specific to Langerhans cells. Langerin binds and mediates uptake and degradation of glycoconjugates containing mannose and related sugars. Analysis of the human genome has identified three single nucleotide polymorphisms that result in amino acid changes in the carbohydrate-recognition domain of langerin. The effects of the amino acid changes on the activity of langerin were examined by expressing each of the polymorphic forms. Expression of full-length versions of the four common langerin haplotypes in fibroblasts revealed that all of these forms can mediate endocytosis of neoglycoprotein ligands. However, sugar binding assays and differential scanning calorimetry performed on fragments from the extracellular domain showed that two of the amino acid changes reduce the affinity of the carbohydrate-recognition domain for mannose and decrease the stability of the extracellular domain. In addition, analysis of sugar binding by langerin containing the rare W264R mutation, previously identified in an individual lacking Birbeck granules, shows that this mutation abolishes sugar binding activity. These findings suggest that certain langerin haplotypes may differ in their binding to pathogens and thus might be associated with susceptibility to infection.

Amino Acid Sequence↗
PubMed2005

A focused microarray approach to functional glycomics: transcriptional regulation of the glycome.

Glycosylation is the most common posttranslational modification of proteins, yet genes relevant to the synthesis of glycan structures and function are incompletely represented and poorly annotated on the commercially available arrays. To fill the need for expression analysis of such genes, we employed the Affymetrix technology to develop a focused and highly annotated glycogene-chip representing human and murine glycogenes, including glycosyltransferases, nucleotide sugar transporters, glycosidases, proteoglycans, and glycan-binding proteins. In this report, the array has been used to generate glycogene-expression profiles of nine murine tissues. Global analysis with a hierarchical clustering algorithm reveals that expression profiles in immune tissues (thymus [THY], spleen [SPL], lymph node, and bone marrow [BM]) are more closely related, relative to those of nonimmune tissues (kidney [KID], liver [LIV], brain [BRN], and testes [TES]). Of the biosynthetic enzymes, those responsible for synthesis of the core regions of N- and O-linked oligosaccharides are ubiquitously expressed, whereas glycosyltransferases that elaborate terminal structures are expressed in a highly tissue-specific manner, accounting for tissue and ultimately cell-type-specific glycosylation. Comparison of gene expression profiles with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) profiling of N-linked oligosaccharides suggested that the alpha1-3 fucosyltransferase 9, Fut9, is the enzyme responsible for terminal fucosylation in KID and BRN, a finding validated by analysis of Fut9 knockout mice. Two families of glycan-binding proteins, C-type lectins and Siglecs, are predominately expressed in the immune tissues, consistent with their emerging functions in both innate and acquired immunity. The glycogene chip reported in this study is available to the scientific community through the Consortium for Functional Glycomics (CFG) (http://www.functionalglycomics.org).

Animals↗
PubMed2005

Selective binding of the scavenger receptor C-type lectin to Lewisx trisaccharide and related glycan ligands.

The scavenger receptor C-type lectin (SRCL) is an endothelial receptor that is similar in organization to type A scavenger receptors for modified low density lipoproteins but contains a C-type carbohydrate-recognition domain (CRD). Fragments of the receptor consisting of the entire extracellular domain and the CRD have been expressed and characterized. The extracellular domain is a trimer held together by collagen-like and coiled-coil domains adjacent to the CRD. The amino acid sequence of the CRD is very similar to the CRD of the asialoglycoprotein receptor and other galactose-specific receptors, but SRCL binds selectively to asialo-orosomucoid rather than generally to asialoglycoproteins. Screening of a glycan array and further quantitative binding studies indicate that this selectivity results from high affinity binding to glycans bearing the Lewis(x) trisaccharide. Thus, SRCL shares with the dendritic cell receptor DC-SIGN the ability to bind the Lewis(x) epitope. However, it does so in a fundamentally different way, making a primary binding interaction with the galactose moiety of the glycan rather than the fucose residue. SRCL shares with the asialoglycoprotein receptor the ability to mediate endocytosis and degradation of glycoprotein ligands. These studies suggest that SRCL might be involved in selective clearance of specific desialylated glycoproteins from circulation and/or interaction of cells bearing Lewis(x)-type structures with the vascular endothelium.

Animals↗
PubMed2004

Extended neck regions stabilize tetramers of the receptors DC-SIGN and DC-SIGNR.

The human cell surface receptors DC-SIGN (dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin) and DC-SIGNR (DC-SIGN-related) bind to oligosaccharide ligands found on human tissues as well as on pathogens including viruses, bacteria, and parasites. The extracellular portion of each receptor contains a membrane-distal carbohydrate-recognition domain (CRD) and forms tetramers stabilized by an extended neck region consisting of 23 amino acid repeats. Cross-linking analysis of full-length receptors expressed in fibroblasts confirms the tetrameric state of the intact receptors. Hydrodynamic studies on truncated receptors demonstrate that the portion of the neck of each protein adjacent to the CRD is sufficient to mediate the formation of dimers, whereas regions near the N terminus are needed to stabilize the tetramers. Some of the intervening repeats are missing from polymorphic forms of DC-SIGNR. Two different crystal forms of truncated DC-SIGNR comprising two neck repeats and the CRD reveal that the CRDs are flexibly linked to the neck, which contains alpha-helical segments interspersed with non-helical regions. Differential scanning calorimetry measurements indicate that the neck and CRDs are independently folded domains. Based on the crystal structures and hydrodynamic data, models for the full extracellular domains of the receptors have been generated. The observed flexibility of the CRDs in the tetramer, combined with previous data on the specificity of these receptors, suggests an important role for oligomerization in the recognition of endogenous glycans, in particular those present on the surfaces of enveloped viruses recognized by these proteins.

Animals↗
PubMed2004

Structural basis for distinct ligand-binding and targeting properties of the receptors DC-SIGN and DC-SIGNR.

Both the dendritic cell receptor DC-SIGN and the closely related endothelial cell receptor DC-SIGNR bind human immunodeficiency virus and enhance infection. However, biochemical and structural comparison of these receptors now reveals that they have very different physiological functions. By screening an extensive glycan array, we demonstrated that DC-SIGN and DC-SIGNR have distinct ligand-binding properties. Our structural and mutagenesis data explain how both receptors bind high-mannose oligosaccharides on enveloped viruses and why only DC-SIGN binds blood group antigens, including those present on microorganisms. DC-SIGN mediates endocytosis, trafficking as a recycling receptor and releasing ligand at endosomal pH, whereas DC-SIGNR does not release ligand at low pH or mediate endocytosis. Thus, whereas DC-SIGN has dual ligand-binding properties and functions both in adhesion and in endocytosis of pathogens, DC-SIGNR binds a restricted set of ligands and has only the properties of an adhesion receptor.

Binding Sites↗
PubMed2004

Phylogenetic analysis of the vertebrate galectin family.

Galectins form a family of structurally related carbohydrate binding proteins (lectins) that have been identified in a large variety of metazoan phyla. They are involved in many biological processes such as morphogenesis, control of cell death, immunological response, and cancer. To elucidate the evolutionary history of galectins and galectin-like proteins in chordates, we have exploited three independent lines of evidence: (i) location of galectin encoding genes (LGALS) in the human genome; (ii) exon-intron organization of galectin encoding genes; and (iii) sequence comparison of carbohydrate recognition domains (CRDs) of chordate galectins. Our results suggest that a duplication of a mono-CRD galectin gene gave rise to an original bi-CRD galectin gene, before or early in chordate evolution. The N-terminal and C-terminal CRDs of this original galectin subsequently diverged into two different subtypes, defined by exon-intron structure (F4-CRD and F3-CRD). We show that all vertebrate mono-CRD galectins known to date belong to either the F3- or F4- subtype. A sequence of duplication and divergence events of the different galectins in chordates is proposed.

Animals↗
PubMed2004

Localization of the serine protease-binding sites in the collagen-like domain of mannose-binding protein: indirect effects of naturally occurring mutations on protease binding and activation.

Mutations in the collagen-like domain of serum mannose-binding protein (MBP) interfere with the ability of the protein to initiate complement fixation through the MBP-associated serine proteases (MASPs). The resulting deficiency in the innate immune response leads to susceptibility to infections. Studies have been undertaken to define the region of MBP that interacts with MASPs and to determine how the naturally occurring mutations affect this interaction. Truncated and modified MBPs and synthetic peptides that represent segments of the collagen-like domain of MBP have been used to demonstrate that MASPs bind on the C-terminal side of the hinge region formed by an interruption in the Gly-X-Y repeat pattern of the collagen-like domain. The binding sites for MASP-2 and for MASP-1 and -3 overlap but are not identical. The two most common naturally occurring mutations in MBP result in substitution of acidic amino acids for glycine residues in Gly-X-Y triplets on the N-terminal side of the hinge. Circular dichroism analysis and differential scanning calorimetry demonstrate that the triple helical structure of the collagen-like domain is largely intact in the mutant proteins, but it is more easily unfolded than in wild-type MBP. Thus, the effect of the mutations is to destabilize the collagen-like domain, indirectly disrupting the binding sites for MASPs. In addition, at least one of the mutations has a further effect on the ability of MBP to activate MASPs.

Amino Acid Sequence↗
PubMed2003

Crystal structure of the CUB1-EGF-CUB2 region of mannose-binding protein associated serine protease-2.

Serum mannose-binding proteins (MBPs) are C-type lectins that recognize cell surface carbohydrate structures on pathogens, and trigger killing of these targets by activating the complement pathway. MBPs circulate as a complex with MBP-associated serine proteases (MASPs), which become activated upon engagement of a target cell surface. The minimal functional unit for complement activation is a MASP homodimer bound to two MBP trimeric subunits. MASPs have a modular structure consisting of an N-terminal CUB domain, a Ca(2+)-binding EGF-like domain, a second CUB domain, two complement control protein modules and a C-terminal serine protease domain. The CUB1-EGF-CUB2 region mediates homodimerization and binding to MBP. The crystal structure of the MASP-2 CUB1-EGF-CUB2 dimer reveals an elongated structure with a prominent concave surface that is proposed to be the MBP-binding site. A model of the full six-domain structure and its interaction with MBPs suggests mechanisms by which binding to a target cell transmits conformational changes from MBP to MASP that allow activation of its protease activity.

Amino Acid Sequence↗
PubMed2003

Oligolysine-based oligosaccharide clusters: selective recognition and endocytosis by the mannose receptor and dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin.

Dendritic cells are potent antigen-presenting cells that express several membrane lectins, including the mannose receptor and DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin). To identify highly specific ligands for these dendritic cell receptors, oligosaccharides were converted into glycosynthons (Os1) and were used to prepare oligolysine-based glycoclusters, Os-[Lys(Os)]n-Ala-Cys-NH2. Clusters containing two to six dimannosides as well as clusters containing four or five pentasaccharides (Lewisa or Lewisx) or hexasaccharides (Lewisb) were synthesized. The thiol group of the appended cysteine residue allows easy tagging by a fluorescent probe or convenient substitution with an antigen. Surface plasmon resonance was used to determine the affinity of the different glycoclusters for purified mannose receptor and DC-SIGN, whereas flow cytometry and confocal microscopy analysis allowed assessment of cell uptake of fluoresceinyl-labeled glycoclusters. Dimannoside clusters are recognized by the mannose receptor with an affinity constant close to 106 liter.mol-1 but have a very low affinity for DC-SIGN (less than 104 liter x mol-1). Conversely, Lewis clusters have a higher affinity toward DC-SIGN than toward the mannose receptor. Dimannoside clusters are efficiently taken up by human dendritic cells as well as by rat fibroblasts expressing the mannose receptor but not by HeLa cells or rat fibroblasts expressing DC-SIGN; DC-SIGN-expressing cells take up Lewis clusters. The results suggest that ligands containing dimannoside clusters can be used specifically to target the mannose receptor, whereas ligands containing Lewis clusters will be targeted to DC-SIGN.

Adoptive Transfer↗
PubMed2003

Molecular characterization of the rat Kupffer cell glycoprotein receptor.

The Kupffer cell receptor for glycoproteins has been reported to have a role in clearance of galactose- and fucose-terminated glycoproteins from circulation. Although the gene and a cDNA encoding the receptor have been described, there has been little study of the receptor protein. To address some questions about possible ligands and functions for this receptor, fragments representing portions of the extracellular domain have been expressed and characterized. The extracellular domain consists of a trimer stabilized by an extended coiled-coil of alpha-helices. The receptor displays monosaccharide-binding characteristics similar to the hepatic asialoglycoprotein receptor, but with somewhat less selectivity. The two best monosaccharide ligands are GalNAc and galactose. alpha-Methyl fucoside is a particularly poor ligand. Analysis of Kupffer cell receptor binding to glycoproteins and oligosaccharides released from them reveals highest affinity for desialylated, complex N-linked glycans. The best glycoprotein ligands contain multiple highly branched oligosaccharides. A human ortholog of the rat receptor gene does not encode a full-length protein and is not expressed in liver. These characteristics suggest that the receptor may have functions parallel to those of the hepatocyte asialoglycoprotein receptor in some (but not all) mammalian species.

Amino Acid Sequence↗