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C R Bertozzi

Publications and source records attributed to C R Bertozzi.

At least 37 records · Page 2Linked to original sources
PubMed2001

Metabolic selection of glycosylation defects in human cells.

Changes in glycosylation are often associated with disease progression, but the genetic and metabolic basis of these events is rarely understood in detail at a molecular level. We describe a metabolism-based approach to the selection of mutants in glycoconjugate biosynthesis that provides insight into regulatory mechanisms for oligosaccharide expression and metabolic flux. Unnatural intermediates are used to challenge a specific pathway, and cell surface expression of their metabolic products provides a readout of flux in that pathway and a basis for selecting genetic mutants. The approach was applied to the sialic acid metabolic pathway in human cells, yielding novel mutants with phenotypes related to the inborn metabolic defect sialuria and metastatic tumor cells.

Amino Acid Sequence↗
PubMed2001

Chemical and biological strategies for engineering cell surface glycosylation.

Oligosaccharides play a crucial role in many of the recognition, signaling, and adhesion events that take place at the surface of cells. Abnormalities in the synthesis or presentation of these carbohydrates can lead to misfolded and inactive proteins, as well as to several debilitating disease states. However, their diverse structures, which are the key to their function, have hampered studies by biologists and chemists alike. This review presents an overview of techniques for examining and manipulating cell surface oligosaccharides through genetic, enzymatic, and chemical strategies.

Animals↗
PubMed2000

Sulfation of N-acetylglucosamine by chondroitin 6-sulfotransferase 2 (GST-5).

Based on sequence homology with a previously cloned human GlcNAc 6-O-sulfotransferase, we have identified an open reading frame (ORF) encoding a novel member of the Gal/GalNAc/GlcNAc 6-O-sulfotransferase (GST) family termed GST-5 on the human X chromosome (band Xp11). GST-5 has recently been characterized as a novel GalNAc 6-O-sulfotransferase termed chondroitin 6-sulfotransferase-2 (Kitagawa, H., Fujita, M., Itio, N., and Sugahara K. (2000) J. Biol. Chem. 275, 21075-21080). We have coexpressed a human GST-5 cDNA with a GlyCAM-1/IgG fusion protein in COS-7 cells and observed four-fold enhanced [(35)S]sulfate incorporation into this mucin acceptor. All mucin-associated [(35)S]sulfate was incorporated as GlcNAc-6-sulfate or Galbeta1-->4GlcNAc-6-sulfate. GST-5 was also expressed in soluble epitope-tagged form and found to catalyze 6-O-sulfation of GlcNAc residues in synthetic acceptor structures. In particular, GST-5 was found to catalyze 6-O-sulfation of beta-benzyl GlcNAc but not alpha- or beta-benzyl GalNAc. In the mouse genome we have found a homologous ORF that predicts a novel murine GlcNAc 6-O-sulfotransferase with 88% identity to the human enzyme. This gene was mapped to mouse chromosome X at band XA3.1-3.2. GST-5 is the newest member of an emerging family of carbohydrate 6-O-sulfotransferases that includes chondroitin 6-sulfotransferase (GST-0), keratan-sulfate galactose 6-O-sulfotransferase (GST-1), the ubiquitously expressed GlcNAc 6-O-sulfotransferase (GST-2), high endothelial cell GlcNAc 6-O-sulfotransferase (GST-3), and intestinal GlcNAc 6-O-sulfotransferase (GST-4).

Acetylglucosamine↗
PubMed2000

Minimal sulfated carbohydrates for recognition by L-selectin and the MECA-79 antibody.

Sulfated forms of sialyl-Le(X) containing Gal-6-SO(4) or GlcNAc-6-SO(4) have been implicated as potential recognition determinants on high endothelial venule ligands for L-selectin. The optimal configuration of sulfate esters on the N-acetyllactosamine (Galbeta1-->4GlcNAc) core of sulfosialyl-Le(X), however, remains unsettled. Using a panel of sulfated lactose (Galbeta1-->4Glc) neoglycolipids as substrates in direct binding assays, we found that 6',6-disulfolactose was the preferred structure for L-selectin, although significant binding to 6'- and 6-sulfolactose was observed as well. Binding was EDTA-sensitive and blocked by L-selectin-specific monoclonal antibodies. Surprisingly, 6', 6-disulfolactose was poorly recognized by MECA-79, a carbohydrate- and sulfate-dependent monoclonal antibody that binds competitively to L-selectin ligands. Instead, MECA-79 bound preferentially to 6-sulfolactose. The difference in preferred substrates between L-selectin and MECA-79 may explain the variable activity of MECA-79 as an inhibitor of lymphocyte adhesion to high endothelial venules in lymphoid organs. Our results suggest that both Gal-6-SO(4) and GlcNAc-6-SO(4) may contribute to L-selectin recognition, either as components of sulfosialyl-Le(X) capping groups or in internal structures. By contrast, only GlcNAc-6-SO(4) appears to contribute to MECA-79 binding.

Animals↗
PubMed2000

A "traceless" Staudinger ligation for the chemoselective synthesis of amide bonds.

[reaction: see text] Here we report a novel modification of our previously reported "Staudinger ligation" that generates an amide bond from an azide and a specifically functionalized phosphine. This method for the selective formation of an amide bond, which does not require the orthogonal protection of distal functional groups, should find general utility in synthetic and biological chemistry.

Amides↗
PubMed2000

Cell surface engineering by a modified Staudinger reaction.

Selective chemical reactions enacted within a cellular environment can be powerful tools for elucidating biological processes or engineering novel interactions. A chemical transformation that permits the selective formation of covalent adducts among richly functionalized biopolymers within a cellular context is presented. A ligation modeled after the Staudinger reaction forms an amide bond by coupling of an azide and a specifically engineered triarylphosphine. Both reactive partners are abiotic and chemically orthogonal to native cellular components. Azides installed within cell surface glycoconjugates by metabolism of a synthetic azidosugar were reacted with a biotinylated triarylphosphine to produce stable cell-surface adducts. The tremendous selectivity of the transformation should permit its execution within a cell's interior, offering new possibilities for probing intracellular interactions.

Acetylation↗
PubMed2000

Tyrosine sulfation: a modulator of extracellular protein-protein interactions.

Tyrosine sulfation is a post-translational modification of many secreted and membrane-bound proteins. Its biological roles have been unclear. Recent work has implicated tyrosine sulfate as a determinant of protein-protein interactions involved in leukocyte adhesion, hemostasis and chemokine signaling.

Amino Acid Sequence↗
PubMed2000

Biosynthetic incorporation of unnatural sialic acids into polysialic acid on neural cells.

In this study we demonstrate that polysialyltransferases are capable of accepting unnatural substrates in terminally differentiated human neurons. Polysialyltransferases catalyze the glycosylation of the neural cell adhesion molecule (NCAM) with polysialic acid (PSA). The unnatural sialic acid analog, N-levulinoyl sialic acid (SiaLev), was incorporated into cell surface glycoconjugates including PSA by the incubation of cultured neurons with the metabolic precursor N-levulinoylmannosamine (ManLev). The ketone group within the levulinoyl side chain of SiaLev was then used as a chemical handle for detection using a biotin probe. The incorporation of SiaLev residues into PSA was demonstrated by protection from sialidases that can cleave natural sialic acids but not those bearing unnatural N-acyl groups. The presence of SiaLev groups on the neuronal cell surface did not impede neurite outgrowth or significantly affect the distribution of PSA on neuronal compartments. Since PSA is important in neural plasticity and development, this mechanism for modulating PSA structure might be useful for functional studies.

Cell Compartmentation↗
PubMed1999

A chemically synthesized version of the insect antibacterial glycopeptide, diptericin, disrupts bacterial membrane integrity.

Insects protect themselves against bacterial infection by secreting a battery of antimicrobial peptides into the hemolymph. Despite recent progress, important mechanistic questions, such as the precise bacterial targets, the nature of any cooperation that occurs between peptides, and the purpose of multiple peptide isoforms, remain largely unanswered. We report herein the chemical synthesis and preliminary mechanistic investigation of diptericin, an 82 residue glycopeptide that contains regions similar to two different types of antibacterial peptides. A revised, highly practical synthesis of the precursor N(alpha)-Fmoc-Thr(Ac(3)-alpha-D-GalNAc) allowed us to produce sufficient quantities of the glycopeptide for mechanistic assays. The synthetic, full-length polypeptide proved to be active in growth inhibition assays with an IC(50) of approximately 250 nM, a concentration similar to that found in the insect hemolymph. Biological analysis of diptericin fragments indicated that the main determinant of antibacterial activity lay in the C-terminal region that is similar to the attacin peptides, although the N-terminal segment, related to the proline-rich family of antibacterial peptides, augmented that activity by 100-fold. In all assays, activity appeared glycosylation independent. Circular dichroism of unglycosylated diptericin indicated that the peptide lacked structure both in plain buffer and in the presence of liposomes. Diptericin increased the permeability of the outer and inner membranes of Escherichia coli D22 cells, suggesting possible mechanisms of action. The ability to access glycopeptides of this type through chemical synthesis will facilitate further mechanistic studies.

Amino Acid Sequence↗
PubMed1999

Engineering novel cell surface receptors for virus-mediated gene transfer.

The absence of viral receptors is a major barrier to efficient gene transfer in many cells. To overcome this barrier, we developed an artificial receptor based on expression of a novel sugar. We fed cells an unnatural monosaccharide, a modified mannosamine that replaced the acetyl group with a levulinate group (ManLev). ManLev was metabolized and incorporated into cell-surface glycoconjugates. The synthetic sugar decorated the cell surface with a unique ketone group that served as a foundation on which we built an adenovirus receptor by covalently binding biotin hydrazide to the ketone. The artificial receptor enhanced adenoviral vector binding and gene transfer to cells that are relatively resistant to adenovirus infection. These data are the first to suggest the feasibility of a strategy that improves the efficiency of gene transfer by using the biosynthetic machinery of the cell to engineer novel sugars on the cell surface.

3T3 Cells↗
PubMed1999

Carbohydrate sulfotransferases: mediators of extracellular communication.

Sulfated carbohydrates mediate diverse extracellular recognition events in both normal and pathological processes. The sulfotransferases that generate specific carbohydrate 'sulfoforms' have recently been recognized as key modulators of these processes and therefore represent potential therapeutic targets.

Animals↗
PubMed1998

Metabolic delivery of ketone groups to sialic acid residues. Application To cell surface glycoform engineering.

The development of chemical strategies for decorating cells with defined carbohydrate epitopes would greatly facilitate studies of carbohydrate-mediated cell surface interactions. This report describes a general strategy for engineering the display of chemically defined oligosaccharides on cell surfaces that combines the concepts of metabolic engineering and selective chemical reactivity. Using a recently described method (Mahal, L. K., Yarema, K. J., and Bertozzi, C. R. (1997) Science 276, 1125-1128), we delivered a uniquely reactive ketone group to endogenous cell surface sialic acid residues by treating cells with the ketone-bearing metabolic precursor N-levulinoylmannosamine (ManLev). The ketone undergoes highly selective condensation reactions with complementary nucleophiles such as aminooxy and hydrazide groups. The detailed quantitative parameters of ManLev metabolism in human and nonhuman-derived cell lines were determined to establish a foundation for the modification of cell surfaces with novel epitopes at defined cell-surface densities. Ketones within the glycoconjugates on ManLev-treated cells were then reacted with synthetic aminooxy and hydrazide-functionalized carbohydrates. The remodeled cells were endowed with novel lectin binding profiles as determined by flow cytometry analysis. The simplicity and generality of this method make it well suited for use in the study of carbohydrate-mediated cell surface interactions.

Animals↗
PubMed1998

Chemoselective ligation reactions with proteins, oligosaccharides and cells.

Traditional chemical synthesis does not lend itself to the easy, rapid construction of even moderately sized biomolecules, because it requires elaborate protection schemes. Furthermore, many biological studies would be aided by the ability to assemble biomolecules under physiological conditions. These challenges have motivated the development of chemoselective ligation, the selective covalent coupling of mutually and uniquely reactive functional groups under mild, aqueous conditions. This technique has attracted significant attention recently for the synthesis of biological macromolecules of defined homogeneous composition, the design of self-assembling drugs and the chemical remodeling of cell surfaces.

Animals↗
PubMed1998

Identification of an N-acetylglucosamine-6-0-sulfotransferase activity specific to lymphoid tissue: an enzyme with a possible role in lymphocyte homing.

BACKGROUND: The leukocyte adhesion molecule L-selection participates in the initial attachment of blood-borne lymphocytes to high endothelial venules (HEVs) during lymphocyte homing to secondary lymphoid organs, and contributes to leukocyte adhesion and extravasation in HEV-like vessels at sites of chronic inflammation. The L-selection ligands on lymph mode HEVs are mucin-like glycoproteins adorned with the unusual sulfated carbohydrate epitope, 6-sulfo sialyl Lewis x. Sulfation of this epitope on the N-acetylglucosamine (GlcNAc) residue confers high-avidity L-selection binding, and is thought to be restricted in the vasculature to sites of sustained lymphocyte recruitment. The GlcNAc-6-0 sulfotransferase that installs the sulfate ester may be a key modulator of lymphocyte recruitment to secondary lymphoid organs and sites of chronic inflammation and is therefore a potential target for anti-inflammatory therapy. RESULTS: A GlcNAc-6-0-sulfotransferase activity was identified within porcine lymph nodes and characterized using a rapid, sensitive, and quantitative assay. We synthesized two unnatural oligosaccharide substrates, GlcNAc beta 1-->6Gal alpha-R and Gal beta 1-->4GlcNAc beta 1-->6Gal alpha-R, that incorporate structural motifs from the native L-selection ligands into an unnatural C-glycosyl hydrocarbon scaffold. The sulfotransferase incorporated greater than tenfold more sulfate into the disaccharide than the trisaccharide, indicating a requirement for a terminal GlcNAc. Activity across tissues was highly restricted to the HEVs within peripheral lymph node. CONCLUSIONS: The restricted expression of the GlcNAc-6-0-sulfotransferase activity to lymph node HEVs strongly suggestions a role in the biosynthesis of L-selection ligands. In addition, similar sulfated epitopes are known to be expressed on HEV-like vessels of chronically inflamed tissues; indicating that this sulfotransferase may also contribute to inflammatory lymphocyte recruitment. We identified a concise disaccharide motif, GlcNAc beta 1-->6Gal alpha-R, that preserved both recognition and specificity determinants for the GlcNAc-6-0-sulfotransferase. The absence of activity on the trisaccharide Gal beta 1-->6Gal alpha-R indicates a requirement for a substrate with a terminal GlcNAc residue, suggesting that sulfation precedes further biosynthetic assembly of L-selection ligands.

Animals↗
PubMed1998

Chemical approaches to glycobiology and emerging carbohydrate-based therapeutic agents.

The contributions of cell surface oligosaccharides to critical biological processes such as leukocyte-endothelial cell adhesion, bacterial and viral infection, and immunological recognition of tumor cells and foreign tissue are now understood in significant molecular detail. These discoveries at the forefront of biological research have motivated the design of synthetic glycoconjugates as tools for the fundamental study of glycobiology and as candidates for future generations of therapeutic and pharmaceutical reagents. During the past two years, significant progress has been made in the design and synthesis of carbohydrate-based inhibitors of selectins, receptors involved in the attachment of leukocytes to endothelial cells at sites of inflammation. Monomeric and multivalent oligosaccharides that bind to bacterial and viral receptors have been shown to abrogate infection by agents such as Helicobacter pilori, influenza virus and HIV. The identification of certain cell surface oligosaccharides as potent antigens has prompted their use in tumor vaccines, and inspired new approaches to the management of tissue rejection subsequent to xenotransplantation. To better understand how cell surface oligosaccharides function within their native context, novel chemical approaches to modulating cell surface oligosaccharides structures are now being developed. These stratergies for cell surface 'glycoform remodeling' promise to facilitate the investigation of carbohydrate mediated cell-cell interactions.

Drugs, Investigational↗
PubMed1997

Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis.

Cell surface oligosaccharides can be engineered to display unusual functional groups for the selective chemical remodeling of cell surfaces. An unnatural derivative of N-acetyl-mannosamine, which has a ketone group, was converted to the corresponding sialic acid and incorporated into cell surface oligosaccharides metabolically, resulting in the cell surface display of ketone groups. The ketone group on the cell surface can then be covalently ligated under physiological conditions with molecules carrying a complementary reactive functional group such as the hydrazide. Cell surface reactions of this kind should prove useful in the introduction of new recognition epitopes, such as peptides, oligosaccharides, or small organic molecules, onto cell surfaces and in the subsequent modulation of cell-cell or cell-small molecule binding events. The versatility of this technology was demonstrated by an example of selective drug delivery. Cells were decorated with biotin through selective conjugation to ketone groups, and selectively killed in the presence of a ricin A chain-avidin conjugate.

Avidin↗