33 results about "N linked glycans" patented technology

Imino sugars, such as deoxynojirimycin (DNJ), are glucose analogues that selectively inhibit cellular α-glucosidase I and II (enzymes that process N-linked glycans in glycoprotein) and exhibit broad spectrum antiviral activities against many enveloped viruses. Previously we have reported a novel DNJ derivative, OSL-95II, with antiviral activity and reduced cytotoxicity. In order to develop imino sugars with more potent antiviral activity as well as improved toxicity profile, OSL-95II was modified by diversifying the nitrogen linked alkylated side chain. The antiviral activities were initially tested in bovine viral diarrhea virus (BVDV) infected MDBK cells, yielding several imino sugar derivatives with novel structure and superior antiviral activity and toxicity profile. Furthermore, these new compounds were shown to be active against Dengue virus (DV) and West Nile virus (WNV) infection in BHK cells where potent anti-DV activity having submicromolar EC50 values and SI of greater than 900. These compounds represent a new generation of iminio sugars and their analogues, having application in the clinical treatment of infection of DV and other members of flaviviridae.

The present disclosure provides recombinantly expressed insulin polypeptides that comprise an N-linked glycan motif. The N-linked glycan motif is not present in wild-type insulins and enables the recombinant expression of glycosylated insulin polypeptides (e.g., in yeast cells). Based on results obtained with synthetic glycosylated insulin conjugates we predict that when these recombinant glycosylated insulin polypeptides are administered to a mammal, at least one pharmacokinetic or pharmacodynamic property of the glycosylated insulin polypeptide will be sensitive to serum concentrations of glucose (or an exogenous saccharide such as alpha-methyl mannose). Exemplary insulin polypeptides, polynucleotides encoding these insulin polypeptides, glycosylated insulin polypeptides, pharmaceutical formulations and sustained release formulations are provided in addition to methods of use and preparation.

Disclosed are novel methods of treatment for retinal diseases and conditions including age-related macular degeneration, genetic-based retinal degenerations and retinal detachment. A novel glycan binding protein thought to be a cell surface receptor has been discovered in the retina. The retinal glycan binding receptor is shown to play an important role in promoting assembly of outer segment (OS) membranes by the photoreceptor cells of the eye, a process that is essential for vision. Based on the finding that certain sugars can bind with very high affinity to the retinal glycan receptor and stimulate its function, the invention provides novel therapeutic agents for treatment of retinal diseases that are multivalent N-linked glycans. Preferred pharmaceutical compositions in accordance with the present invention comprise active agents having the general formula: (Gal-GlcNAc)n-Man3-GlcNAc2, where n is 1-4. Particularly preferred multivalent glycans are galactosylated, biantennary (NA2), and asialo, galactosylated, triantennary (NA3) oligosaccharides.

The present invention relates to a method for gastric cancer diagnosis through the detection of glycan changes, and to a kit for gastric cancer diagnosis. More specifically, based on, among the changes in N-linked glycosylation of the gastric cancer patient-derived haptoglobin, which are detected through lectin and mass spectrometry, the increase in fucosylation, the increases or significant changes in particular glycan structures according to the classification of antennary, or the reduction in high-mannose structure of the N-linked glycan compared with normal persons, the discovered N-glycosylation structures can be favorably used for a method for gastric cancer diagnosis using lectin, as a diagnostic marker, or mass spectrometry, and a kit for diagnosing gastric cancer.

The invention belongs to the field of biotechnology, and in particular relates to a method for fast and high throughput detection of N-linked glycans in a glycoprotein sample based on membrane separation. A hydrophobic membrane porous plate and a hydrophilic membrane porous plate are used to process the glycoprotein sample, the glycoprotein sample processing flux is improved, and steps are simplified. In addition, by changing the reaction conditions of PNGase F enzyme, and the sample processing time is successfully shortened. The detection time of the N-linked glycans in the glycoprotein sample can be shortened from 2 days of a traditional method to within 4-6 hours. In addition, the method successfully solves the problem that in traditional experiments only a purified glycoprotein sample can be used to carry out the experiments, and by use of the method, cell culture supernatant can be directly used for experiments. A strong support is provided for fast qualitative analysis of the N-linked glycans in the glycoprotein for screening of early cell lines.

The presently disclosed subject matter provides methods using mass spectrometry for direct profiling of N-linked glycans from a biological sample. In addition, the embodiments of the present invention also disclose novel methods, known as targeted analyte detection (TAD), for improving the detection limit of MALDI-MS. These methods take advantage of the carrier effect of the added standard analytes, which occurs due to the generic sigmoidal shape of the calibration curve. The functionality of TAD depends on the relative enhancement of sensitivity over the increase of the standard deviation at the analysis of target analytes with spiking in exogenous concentration. At certain ranges of exogenous concentration, the increment in the sensitivity overcomes the standard deviation, resulting in an improved LOD. Theoretically, exogenous concentrations approximately at 1 LODorig would generate the optimum LOD improvement. TAD is a cost-effective LOD improvement method, which is not limited to a certain group of analytes, or detection methods or instruments. It can be applied to enhance the detection of any analyte with different detection methods, provided that the analyte of interest can be extracted or is available in synthetic form.

The invention discloses a subcellular structure characteristic N-linked carbohydrate chain and the application thereof and belongs to the technical field of glycobiology. Bladder YTS1 cells are used as the experiment material, the protein components of four subcellular structures (microsome, chondriosome, cell nucleus and cytoplasm) are obtained with the differential centrifugation method, and subcellular structure difference carbohydrate chain distribution is displayed through the change of the type and structure of subcellular structure protein galactosylated modification identified through a united lectin chip and a mass spectrum (PS) respectively. Subcellular structure characteristic N-linked glycan of ten kinds of human bladder YTS1 cells is obtained. By means of characteristic N-linked glycan, indirect detection and qualitative or quantitative detection of other target substances in corresponding subcellular structures can be achieved.

The invention provides compositions comprising one or more isoforms of an erythropoietin (EPO) comprising glycans linked thereto, characterized in that said glycans comprise LewisX structures and on average at least 6 sialic acid moieties per EPO molecule. The invention further provides methods for obtaining a composition comprising one or more isoforms of an erythropoietin (EPO) comprising glycans linked thereto wherein said glycans comprise on average at least 6 sialic acids per EPO molecule and from 0 to 2 Lewis x structures, said method comprising: a) providing a eukaryotic cell containing a nucleic acid sequence encoding an adenoviral E1A protein in expressible format and further containing a nucleic acid encoding an EPO in expressible format, wherein said cell further contains a nucleic acid sequence encoding a sialyltransferase, preferably an alpha-2,6-sialyltransferase or an alpha-2,3-sialyltransferase, under control of a heterologous promoter; b) culturing said cell in a serum-free culture medium and allow expression of an EPO in said cell; c) harvesting the expressed EPO from said cell and / or from the culture medium; and d) purifying and fractionating the EPO to obtain fractions which have an increased average sialic acid content of the N-linked glycans per EPO molecule, to obtain a composition comprising one or more iso forms of an EPO comprising glycans linked thereto wherein said glycans comprise on average at least 6 sialic acids per EPO molecule and from 0 to 2 Lewis x structures.

Provided are compositions comprising one or more isoforms of an erythropoietin (“EPO”) comprising glycans linked thereto, wherein the glycans have Lewis x structures and on average at least six sialic acid moieties per EPO molecule. Further provided are methods for obtaining a composition comprising one or more isoforms of EPO comprising glycans linked thereto, wherein the glycans comprise on average at least six sialic acids per EPO molecule and from zero to two Lewis x structures, the method comprising: a) providing a eukaryotic cell containing a nucleic acid sequence encoding an adenoviral E1A protein in expressible format and a nucleic acid encoding EPO in expressible format, wherein the cell further contains a nucleic acid sequence encoding a sialyltransferase, e.g., an α-2,6-sialyltransferase or an α-2,3-sialyltransferase, under control of a heterologous promoter; b) culturing the cell in a serum-free culture medium and allowing expression of EPO in the cell; c) harvesting the expressed EPO from the cell and / or from the culture medium; and d) purifying and fractionating the EPO to obtain fractions that have an increased average sialic acid content of the N-linked glycans per EPO molecule, to obtain a composition comprising one or more isoforms of an EPO comprising glycans linked thereto, wherein the glycans comprise on average at least six sialic acids per EPO molecule and from zero to two Lewis x structures.

The present invention relates recombinant human α1-antitrypsin (rhAAT) comprising N-linked glycans, wherein at least 10% of said N-linked glycans are tetra-antennary glycans; and the degree of capping with sialic acid on said N-linked glycans (Z / A) is at least 50%. The invention further relates to rhAAT for use as a medicament, in particular for use in the prevention and / or treatment of a disease associated with AAT deficiency, and / or a disease involving neutrophil-mediated tissue damage.

The invention provides compositions comprising one or more isoforms of an erythropoietin (EPO) comprising glycans linked thereto, characterized in that said glycans comprise LewisX structures and on average at least 6 sialic acid moieties per EPO molecule. The invention further provides methods for obtaining a composition comprising one or more isoforms of an erythropoietin (EPO) comprising glycans linked thereto wherein said glycans comprise on average at least 6 sialic acids per EPO molecule and from 0 to 2 Lewis x structures, said method comprising: a) providing a eukaryotic cell containing a nucleic acid sequence encoding an adenoviral E1A protein in expressible format and further containing a nucleic acid encoding an EPO in expressible format, wherein said cell further contains a nucleic acid sequence encoding a sialyltransferase, preferably an alpha-2,6-sialyltransferase or an alpha-2,3-sialyltransferase, under control of a heterologous promoter; b) culturing said cell in a serum-free culture medium and allow expression of an EPO in said cell; c) harvesting the expressed EPO from said cell and / or from the culture medium; and d) purifying and fractionating the EPO to obtain fractions which have an increased average sialic acid content of the N-linked glycans per EPO molecule, to obtain a composition comprising one or more iso forms of an EPO comprising glycans linked thereto wherein said glycans comprise on average at least 6 sialic acids per EPO molecule and from 0 to 2 Lewis x structures.

The present invention discloses mannosyl-glycoprotein endo-beta-N-acetylglucosamidase (E.C.3.2.1.96, endo-N-acetyl-beta-D-glucosaminidase acting on the di-N-acetylchitobiosyl part of N-linked glycans) from filamentous fungi such as Trichoderma reesei.

Disclosed are novel methods of treatment for retinal diseases and conditions including age-related macular degeneration, genetic-based retinal degenerations and retinal detachment. A novel glycan binding protein thought to be a cell surface receptor has been discovered in the retina. The retinal glycan binding receptor is shown to play an important role in promoting assembly of outer segment (OS) membranes by the photoreceptor cells of the eye, a process that is essential for vision. Based on the finding that certain sugars can bind with very high affinity to the retinal glycan receptor and stimulate its function, the invention provides novel therapeutic agents for treatment of retinal diseases that are multivalent N-linked glycans. Preferred pharmaceutical compositions in accordance with the present invention comprise active agents having the general formula: (Gal-GlcNAc)n-Man3-GlcNAc2, where n is 1-4. Particularly preferred multivalent glycans are galactosylated, biantennary (NA2), and asialo, galactosylated, triantennary (NA3) oligosaccharides.

Disclosed herein are glycan-modified anti-CD4 monoclonal antibodies with N-linked glycans attached to the variable region. Expression vectors and cell lines useful for the production of such antibodies, and use of such antibodies for HIV prevention and therapy are also disclosed.

The present invention provides recombinant cells that contain a genetic modification to at least one mannosyl transferase gene. As a result of the modification the cells produce a glycoprotein or glycopeptide that has an N-linked glycan profile that is simplified or more easily humanized. The glycoprotein or glycopeptide can have at least 25% fewer high mannose structures on than the glycoprotein or glycopeptide produced by a reference cell. In some embodiments the modification is a deletion or disruption of a mannosyl transferase gene, which can be in an alg3 gene. Therefore, the proteins produced are more useful for the production of therapeutic glycoproteins than those produced by species having foreign or plant-like patterns of glycosylation. The invention also provides compositions of the glycoproteins or glycopeptides and methods of making them.

Disclosed herein are glycan-modified anti-CD4 monoclonal antibodies with N-linked glycans attached to the variable region. Expression vectors and cell lines useful for the production of such antibodies, and use of such antibodies for HIV prevention and therapy are also disclosed.

The invention provides a polynucleotide encoding an envelope protein comprising gp120 of human immunodeficiency virus-1 (HIV-1) having a mutation at amino acid residues 197-199 whereby a single N-linked glycan is removed. In a typical embodiment, the mutation replaces the asparagine (N) at residue 197 with another amino acid, such as glutamine (Q), creating an N197Q mutation. Because the N197 glycan is highly conserved among HIV-1 subtypes, this approach is applicable across HIV-1 isolates. The invention provides polypeptides, polynucleotides encoding the polypeptides, vectors, and recombinant viruses containing the polynucleotides, antigen-presenting cells (APCs) presenting the polypeptides, immune cells directed against HIV, and pharmaceutical compositions. The invention additionally provides methods, including methods for preventing and treating infection, for killing infected cells, for inhibiting viral replication, for enhancing secretion of antiviral and / or immunomodulatory lymphokines, and for enhancing production of neutralizing antibody.

The present invention provides recombinant cells that contain a modification causing altered expression or function of at least one mannosyl transferase enzyme. As a result of the modification the cells produce a glycoprotein or glycopeptide that has an N-linked glycan profile that is simplified or humanized. The glycoprotein or glycopeptide can have at least 25% fewer high mannose structures on than the glycoprotein or glycopeptide produced by a cell that does not have the modification. In some embodiments the modification is a deletion, knock out, or disruption of a gene encoding a mannosyl transferase, which can be in an Alg3 gene. Therefore, the proteins produced avoid many of the problems associated with the therapeutic use of glycoproteins from species having foreign or plant-like patterns of glycosylation. The invention also provides compositions of the glycoproteins or glycopeptides and methods of making them.

Provided are compositions comprising one or more isoforms of an erythropoietin (“EPO”) comprising glycans linked thereto, wherein the glycans have Lewis x structures and on average at least six sialic acid moieties per EPO molecule. Further provided are methods for obtaining a composition comprising one or more isoforms of EPO comprising glycans linked thereto, wherein the glycans comprise on average at least six sialic acids per EPO molecule and from zero to two Lewis x structures, the method comprising: a) providing a eukaryotic cell containing a nucleic acid sequence encoding an adenoviral E1A protein in expressible format and a nucleic acid encoding EPO in expressible format, wherein the cell further contains a nucleic acid sequence encoding a sialyltransferase, e.g., an α-2,6-sialyltransferase or an α-2,3-sialyltransferase, under control of a heterologous promoter; b) culturing the cell in a serum-free culture medium and allowing expression of EPO in the cell; c) harvesting the expressed EPO from the cell and / or from the culture medium; and d) purifying and fractionating the EPO to obtain fractions that have an increased average sialic acid content of the N-linked glycans per EPO molecule, to obtain a composition comprising one or more isoforms of an EPO comprising glycans linked thereto, wherein the glycans comprise on average at least six sialic acids per EPO molecule and from zero to two Lewis x structures.

A fixed-length alpha-numeric code for representing N-linked glycan structures commonly found in secreted glycoproteins from mammalian cell cultures. The code employs a pre-assigned alpha-numeric index to represent the monosaccharides attached in different branches to the core glycan structure. The present branch-centric representation allows visualization of the structure while the numerical nature of the code makes it machine readable. A difference operator can be defined to quantitatively differentiate between glycan structures for further analysis. The code can be incorporated in a retrievable format into an information management system. A method is also provided for representing the structure of at least a portion of an oligosaccharide, using the fixed-length alpha-numeric code.

The present invention relates to the use of N-linked glycan profiles of blood or blood component proteins as biomarkers for diagnosing diabetes mellitus and for monitoring the efficacy of anti-diabetic therapy. Specifically, the present invention relates to detecting changes in the amounts of N-linked glycans as diagnostic biomarkers for diabetes mellitus and as indicators of the efficacy of anti-diabetic therapy over time.

The present invention relates to the use of N-linked glycan profiles of blood or blood component proteins as biomarkers for diagnosing diabetes mellitus and for monitoring the efficacy of anti-diabetic therapy. Specifically, the present invention relates to detecting changes in the amounts of N-linked glycans as diagnostic biomarkers for diabetes mellitus and as indicators of the efficacy of anti-diabetic therapy over time.

Imino sugars, such as deoxynojirimycin (DNJ), are glucose analogues that selectively inhibit cellular α-glucosidase I and II (enzymes that process N-linked glycans in glycoprotein) and exhibit broad spectrum antiviral activities against many enveloped viruses. Previously we have reported a novel DNJ derivative, OSL-95II, with antiviral activity and reduced cytotoxicity. In order to develop imino sugars with more potent antiviral activity as well as improved toxicity profile, OSL-95II was modified by diversifying the nitrogen linked alkylated side chain. The antiviral activities were initially tested in bovine viral diarrhea virus (BVDV) infected MDBK cells, yielding several imino sugar derivatives with novel structure and superior antiviral activity and toxicity profile. Furthermore, these new compounds were shown to be active against Dengue virus (DV) and West Nile virus (WNV) infection in BHK cells where potent anti-DV activity having submicromolar EC50 values and SI of greater than 900. These compounds represent a new generation of iminio sugars and their analogues, having application in the clinical treatment of infection of DV and other members of flaviviridae.

The presently disclosed subject matter provides methods using mass spectrometry for direct profiling of N-linked glycans from a biological sample. In addition, the embodiments of the present invention also disclose novel methods, known as targeted analyte detection (TAD), for improving the detection limit of MALDI-MS. These methods take advantage of the carrier effect of the added standard analytes, which occurs due to the generic sigmoidal shape of the calibration curve. The functionality of TAD depends on the relative enhancement of sensitivity over the increase of the standard deviation at the analysis of target analytes with spiking in exogenous concentration. At certain ranges of exogenous concentration, the increment in the sensitivity overcomes the standard deviation, resulting in an improved LOD. Theoretically, exogenous concentrations approximately at 1 LODorig would generate the optimum LOD improvement. TAD is a cost-effective LOD improvement method, which is not limited to a certain group of analytes, or detection methods or instruments. It can be applied to enhance the detection of any analyte with different detection methods, provided that the analyte of interest can be extracted or is available in synthetic form.

A fixed-length alpha-numeric code for representing N-linked glycan structures commonly found in secreted glycoproteins from mammalian cell cultures. The code employs a pre-assigned alpha-numeric index to represent the monosaccharides attached in different branches to the core glycan structure. The present branch-centric representation allows visualization of the structure while the numerical nature of the code makes it machine readable. A difference operator can be defined to quantitatively differentiate between glycan structures for further analysis. The code can be incorporated in a retrievable format into an information management system. A method is also provided for representing the structure of at least a portion of an oligosaccharide, using the fixed-length alpha-numeric code.

The present invention relates recombinant human α1-antitrypsin (rhAAT) comprising N-linked glycans, wherein at least 10% of said N-linked glycans are tetra-antennary glycans; and the degree of capping with sialic acid on said N-linked glycans (Z / A) is at least 50%. The invention further relates to rhAAT for use as a medicament, in particular for use in the prevention and / or treatment of a disease associated with AAT deficiency, and / or a disease involving neutrophil-mediated tissue damage.

A method for detecting a site which can be modified with an N-linked glycan chain and to which an N-linked glycan chain is actually linked in a glycoprotein; and a method for determining the state of an N-linked glycan chain addition at the site are provided. A glycoprotein having an N-linked glycan chain linked thereto is subjected to an N-linked glycan chain removal treatment with a peptide N-glycanase, subsequently a site capable of being modified with an N-linked glycan chain, in which an Asn residue has been changed to an Asp residue by the action of the peptide N-glycanase, is treated with an endo-type peptidase capable of recognizing an Asp residue to thereby produce peptide fragments, and subsequently the mass of the fragments is detected. In this manner, a site which can be modified with an N-linked glycan chain and to which an N-linked glycan chain is actually linked can be detected. Furthermore, the proportion or state of of the N-linked glycan chain addition at the site can be determined from the intensity of a signal generated upon the detection.

The invention discloses a characteristic N-linked sugar chain of a subcellular structure and an application thereof, belonging to the technical field of glycobiology. In the present invention, the bladder YTS1 cells are used as experimental materials, and the protein components of four subcellular structures (microsomes, mitochondria, nuclei and cytoplasm) are obtained by using differential centrifugation, and the method of combining lectin chip and mass spectrometry (MS) The glycosylation modification types and structural changes of identified subcellular structural proteins showed the distribution of sugar chains in subcellular structural differences. The present invention obtains the characteristic N-linked glycans of the subcellular structure of 10 kinds of human bladder YTS1 cells; using the characteristic N-linked glycans, the indirect detection, qualitative or quantitative of other target substances in the corresponding subcellular structure can be realized detection.

The invention provides a high-efficiency separation preparation method for preparing an N-linked polysaccharide from ovalbumin. The main component of the N-linked polysaccharide is an N-linked polysaccharide component linked with only one amino acid asparagine (Asn). The method comprises the following steps: carrying out enzymatic hydrolysis on the ovalbumin by using a non-specific protease, centrifuging the obtained enzymatic hydrolysis solution, concentrating the centrifuged solution, removing polypeptides and residual proteins obtained after the ovalbumin enzymatic hydrolysis on the concentrated solution through using a one-dimensional chromatographic column, and collecting the target fraction; and centrifuging the collected fraction, concentrating the centrifuged fraction, and allowing the concentrated fraction to go through a two-dimensional chromatographic column to remove salt, 2-4 amino acids-linked N-connected polysaccharide components and micro-molecular amino acids in the fraction in order to obtain the N-linked polysaccharide component only linked with asparagines. A result of high resolution mass spectrum identification of the Asn linked N-linked polysaccharide component prepared in the invention shows that the N-linked polysaccharide component has clear composition, and the extraction and separation method has the advantages of high repeatability and good maneuverability, and is suitable for large-scale separation and preparation of the N-linked polysaccharide component from the ovalbumin.