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Ajit Varki - One of the best experts on this subject based on the ideXlab platform.
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Metabolism of N-Glycolylneuraminic Acid in Human and Nonhuman Cells and Potential Relationships to Human Disease
Glycoscience: Biology and Medicine, 2021Co-Authors: Anne K. Bergfeld, Annie N. Samraj, Ajit VarkiAbstract:N-Glycolylneuraminic Acid (Neu5Gc) is a sialic Acid commonly found at the outermost position of glycan chains at the surface of most mammalian cells. The only known pathway for synthesis of Neu5Gc is the conversion of CMP-N-acetylneuraminic Acid (CMP-Neu5Ac) to CMP-Neu5Gc, catalyzed by the cytidine monophospho-N-acetylneuraminic Acid hydroxylase (Cmah). These CMP-activated sialic Acids serve as precursors for glycan assembly in the Golgi apparatus. A degradative pathway to eliminate Neu5Gc and regulate Neu5Gc levels in mammalian cells involves enzymatic conversion of Neu5Gc to N-glycolylglucosamine-6-phosphate (GlcNGc-6P) via N-glycolylmannosamine (ManNGc) and GlcNGc. Irreversible de-N-glycolylation of GlcNGc-6P generates the common cellular metabolites glucosamine-6-phosphate and glycolate and therewith eliminates the N-glycolyl group from sialic Acid and amino sugar biosynthetic pathways. Humans lack endogenous Neu5Gc biosynthesis due to an inactivating deletion in the human CMAH gene. This has been confirmed by the absence of detectable Neu5Gc in the Cmah-/- mouse model harboring the humanlike Cmah allele. However, humans incorporate exogenous Neu5Gc from animal-derived food products (primarily meats of mammalian origin) into endogenous cellular glycoconjugates despite a polyclonal anti-Neu5Gc antibody response, which makes Neu5Gc the first known “xeno-autoantigen” in humans. The metabolic pathways and potential implications for human disease are discussed.
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In vivo exploration of the significance of N-Glycolylneuraminic Acid exclusion from the vertebrate brain (787.3)
The FASEB Journal, 2014Co-Authors: Yuko Naito-matsui, Pascal Gagneux, Leela R. L. Davies, Hiromu Takematsu, Hsun-hua Chou, Pam Tangvoranuntakul, Aaron F. Carlin, Andrea Verhagen, Ajit VarkiAbstract:Sialic Acids are Acidic sugars often occupying the non-reducing terminus of glycans. In mammals, the major sialic Acids are N-acetylneuraminic Acid (Neu5Ac) and N-Glycolylneuraminic Acid (Neu5Gc). The nucleotide donor CMP-Neu5Gc is biosynthesized from CMP-Neu5Ac by CMP-Neu5Ac hydroxylase (Cmah). Regulation of Cmah expression results in markedly varying ratios of Neu5Ac and Neu5Gc among different tissues in different animal species. However, the brain shows extremely low Neu5Gc expression (
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N-Glycolylneuraminic Acid deficiency worsens cardiac and skeletal muscle pathophysiology in α-sarcoglycan-deficient mice
Glycobiology, 2013Co-Authors: Paul T. Martin, Ajit Varki, Marybeth Camboni, Bethannie Golden, Kumaran Chandrasekharan, Chiou-miin Wang, Paul M.l. JanssenAbstract:Roughly 3 million years ago, an inactivating deletion occurred in CMAH, the human gene encoding CMP-Neu5Ac (cytidine-5′-monophospho-N-acetylneuraminic Acid) hydroxylase (Chou HH, Takematsu H, Diaz S, Iber J, Nickerson E, Wright KL, Muchmore EA, Nelson DL, Warren ST, Varki A. 1998. A mutation in human CMP-sialic Acid hydroxylase occurred after the Homo-Pan divergence. Proc Natl Acad Sci USA. 95:11751–11756). This inactivating deletion is now homozygous in all humans, causing the loss of N-Glycolylneuraminic Acid (Neu5Gc) biosynthesis in all human cells and tissues. The CMAH enzyme is active in other mammals, including mice, where Neu5Gc is an abundant form of sialic Acid on cellular membranes, including those in cardiac and skeletal muscle. We recently demonstrated that the deletion of mouse Cmah worsened the severity of pathophysiology measures related to muscular dystrophy in mdx mice, a model for Duchenne muscular dystrophy (Chandrasekharan K, Yoon JH, Xu Y, deVries S, Camboni M, Janssen PM, Varki A, Martin PT. 2010. A human-specific deletion in mouse Cmah increases disease severity in the mdx model of Duchenne muscular dystrophy. Sci Transl Med. 2:42–54). Here, we demonstrate similar changes in cardiac and skeletal muscle pathology and physiology resulting from Cmah deletion in α-sarcoglycan-deficient (Sgca−/−) mice, a model for limb girdle muscular dystrophy 2D. These experiments demonstrate that loss of mouse Cmah can worsen disease severity in more than one form of muscular dystrophy and suggest that Cmah may be a general genetic modifier of muscle disease.
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Canine and feline parvoviruses preferentially recognize the non-human cell surface sialic Acid N-Glycolylneuraminic Acid
Virology, 2013Co-Authors: Jonas Lofling, Sangbom M. Lyi, Colin R. Parrish, Ajit VarkiAbstract:Feline panleukopenia virus (FPV) is a pathogen whose canine-adapted form (canine parvovirus (CPV)) emerged in 1978. These viruses infect by binding host transferrin receptor type-1 (TfR), but also hemagglutinate erythrocytes. We show that hemagglutination involves selective recognition of the non-human sialic Acid N-Glycolylneuraminic Acid (Neu5Gc) but not N-acetylneuraminic Acid (Neu5Ac), which differs by only one oxygen atom from Neu5Gc. Overexpression of α2-6 sialyltransferase did not change binding, indicating that both α2-3 and α2-6 linkages are recognized. However, Neu5Gc expression on target cells did not enhance CPV or FPV infection in vitro. Thus, the conserved Neu5Gc-binding preference of these viruses likely plays a role in the natural history of the virus in vivo. Further studies must clarify relationships between virus infection and host Neu5Gc expression. As a first step, we show that transcripts of CMAH (which generates Neu5Gc from Neu5Ac) are at very low levels in Western dog breed cells.
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metabolism of vertebrate amino sugars with n glycolyl groups elucidating the intracellular fate of the non human sialic Acid n glycolylneuraminic Acid
Journal of Biological Chemistry, 2012Co-Authors: Anne K. Bergfeld, Sandra Diaz, Oliver M. T. Pearce, Tho Pham, Ajit VarkiAbstract:The two major mammalian sialic Acids are N-acetylneuraminic Acid and N-Glycolylneuraminic Acid (Neu5Gc). The only known biosynthetic pathway generating Neu5Gc is the conversion of CMP-N-acetylneuraminic Acid into CMP-Neu5Gc, which is catalyzed by the CMP-Neu5Ac hydroxylase enzyme. Given the irreversible nature of this reaction, there must be pathways for elimination or degradation of Neu5Gc, which would allow animal cells to adjust Neu5Gc levels to their needs. Although humans are incapable of synthesizing Neu5Gc due to an inactivated CMAH gene, exogenous Neu5Gc from dietary sources can be metabolically incorporated into tissues in the face of an anti-Neu5Gc antibody response. However, the metabolic turnover of Neu5Gc, which apparently prevents human cells from continued accumulation of this immunoreactive sialic Acid, has not yet been elucidated. In this study, we show that pre-loaded Neu5Gc is eliminated from human cells over time, and we propose a conceivable Neu5Gc-degrading pathway based on the well studied metabolism of N-acetylhexosamines. We demonstrate that murine tissue cytosolic extracts harbor the enzymatic machinery to sequentially convert Neu5Gc into N-glycolylmannosamine, N-glycolylglucosamine, and N-glycolylglucosamine 6-phosphate, whereupon irreversible de-N-glycolylation of the latter results in the ubiquitous metabolites glycolate and glucosamine 6-phosphate. We substantiate this finding by demonstrating activity of recombinant human enzymes in vitro and by studying the fate of radiolabeled pathway intermediates in cultured human cells, suggesting that this pathway likely occurs in vivo. Finally, we demonstrate that the proposed degradative pathway is partially reversible, showing that N-glycolylmannosamine and N-glycolylglucosamine (but not glycolate) can serve as precursors for biosynthesis of endogenous Neu5Gc.
Masaharu Naiki - One of the best experts on this subject based on the ideXlab platform.
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Failure of Human Immunoresponse to N-Glycolylneuraminic Acid Epitope Contained in Recombinant Human Erythropoietin
Nephron, 1996Co-Authors: Akira Noguchi, Chege J. Mukuria, Eiko Suzuki, Masaharu NaikiAbstract:Recombinant human erythropoietin (rHuEPO) was produced by Chinese hamster ovary cells and commercially distributed to hospitals by two pharmaceutical companies in Japan (‘ESPO’ by Kirin Brewery Co. Ltd., and Sankyo Co. Ltd., and ‘EPOGIN’ by Chugai Pharmaceutical Co. Ltd.) These products contained about 1% N-Glycolylneuraminic Acid (Neu5Gc) in total sialic Acid content. Since humans do not synthesize Neu5Gc, successive injection of Neu5Gc-containing products was feared to lead to allergic-like symptoms. Therefore, serum levels of antibodies to Neu5Gc epitope in 90 patients who received repeated i.v. injections of ESPO or EPOGIN were determined by an enzyme immunoassay using Neu5Gcα2-3Galβ1 -4Glc-Cer, GM3(Neu5Gc), as an antigen and compared with those in 100 healthy persons. Either no or low antibody levels were detected in both groups with no significant difference. In 40 patients who received s.c. injections of ESPO or EPOGIN, serum HD antibody levels were determined before and after weekly therapeutic injections carried out for one to several months, but no significant elevations were detected in all patients. The above results indicated that therapeutic administration of rHuEPO to patients with chronic renal failure is safe from allergic-like side effects associated with the production of Neu5Gc-specific antibodies, and it was concluded that Neu5Gc epitope of rHuEPO is minimally antigenic in humans.
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Immunogenicity of N-Glycolylneuraminic Acid-containing carbohydrate chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells.
Journal of biochemistry, 1995Co-Authors: Akira Noguchi, Chege J. Mukuria, Eiko Suzuki, Masaharu NaikiAbstract:Recombinant human erythropoietin (EPO) produced by Chinese hamster ovary cells and distributed by two different pharmaceutical companies were confirmed to contain about 1% N-Glycolylneuraminic Acid (Neu5Gc) in total sialic Acid content. Since chickens, like humans, do not synthesize Neu5Gc, they were used to determine the immunogenicity of Neu5Gc epitope in EPO. Chickens immunized with EPO did not produce significant titer of antibody that was specific to GM3(Neu5Gc) as compared to antibody titers produced in chickens immunized with fetuin containing 7% Neu5Gc or GM3(Neu5Gc) containing 100% Neu5Gc. Results obtained by an ELISA inhibition test showed that EPO, compared to GM3(Neu5Gc), reacted almost one thousand times less strongly with a human Hanganutziu-Deicher (HD) antibody. This study implies that an increase of Neu5Gc content in a molecule enhances its HD antigenicity. The response to Neu5Gc in patients receiving therapeutic injections of EPO is currently under investigation.
Michael P. Jennings - One of the best experts on this subject based on the ideXlab platform.
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Detection of N-Glycolylneuraminic Acid biomarkers in sera from patients with ovarian cancer using an engineered N-Glycolylneuraminic Acid-specific lectin SubB2M
Biochemical and biophysical research communications, 2018Co-Authors: Lucy K. Shewell, Adrienne W Paton, James C. Paton, J.j. Wang, Christopher J. Day, Michael P. JenningsAbstract:N-Glycolylneuraminic Acid (Neu5Gc)-containing glycans are a prominent form of aberrant glycosylation found in human tumor cells and have been proposed as cancer biomarkers. The B subunit of the subtilase cytotoxin (SubB) produced by Shiga toxigenic Escherichia coli recognises Neu5Gc containing glycans. We have previously engineered this lectin, SubB2M, for greater specificity and enhanced recognition of Neu5Gc-containing glycans. Here we further explore the utility of SubB2M to detect Neu5Gc tumor biomarkers in sera from patients with ovarian cancer. Using surface plasmon resonance (SPR) we show that SubB2M can detect the established ovarian cancer biomarker, CA125, in a highly sensitive and specific fashion in the context of human serum. These studies established conditions for screening serum samples from patients with ovarian cancer for Neu5Gc glycans. We found that serum from patients with all stages of ovarian cancer had significantly elevated mean levels of Neu5Gc glycans compared to normal controls. Serum from patients with late stage disease (stages IIIC, IV) had uniformly elevated levels of Neu5Gc glycans. Detection of Neu5Gc-glycans using SubB2M has the potential to be used as a diagnostic ovarian cancer biomarker, as well as a tool for monitoring treatment and disease progression in late stage disease.
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Specificity and utility of SubB2M, a new N-Glycolylneuraminic Acid lectin
Biochemical and biophysical research communications, 2018Co-Authors: Jing Wang, Adrienne W Paton, James C. Paton, Lucy K. Shewell, Christopher J. Day, Michael P. JenningsAbstract:The B subunit of the subtilase cytotoxin (SubB) recognises N-Glycolylneuraminic Acid (Neu5Gc) containing glycans, the most prominent form of aberrant glycosylation in human cancers. We have previously engineered SubB by construction of a SubBΔS106/ΔT107 mutant (SubB2M) for greater specificity and enhanced recognition of Neu5Gc containing glycans. In this study, we further explore the utility of SubB2M as a Neu5Gc lectin by showing its improved specificity and recognition for Neu5Gc containing glycans over the wild-type SubB protein and an anti-Neu5Gc IgY antibody in a N-acetylneuraminic Acid (Neu5Ac)/Neu5Gc glycan array and by surface plasmon resonance. Far-western blot analysis showed that SubB2M preferentially binds to bovine serum glycoproteins over human serum glycoproteins. SubB2M was also able to detect Neu5Gc containing bovine glycoproteins spiked into normal human serum with greater sensitivity than the wild-type SubB and the anti-Neu5Gc IgY antibody. These results suggest that SubB2M will be a useful tool for the testing of serum and other bodily fluids for cancer diagnosis and prognosis.
Darius Ghaderi - One of the best experts on this subject based on the ideXlab platform.
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novel mechanism for the generation of human xeno autoantibodies against the nonhuman sialic Acid n glycolylneuraminic Acid
Journal of Experimental Medicine, 2010Co-Authors: Rachel E Taylor, Shengshu Huang, Hai Yu, Darius Ghaderi, Vered Padlerkaravani, Christopher J Gregg, Ricardo U Sorensen, Xi Chen, Jaime Inostroza, Victor NizetAbstract:The nonhuman sialic Acid N-Glycolylneuraminic Acid (Neu5Gc) is metabolically incorporated into human tissues from certain mammalian-derived foods, and this occurs in the face of an anti-Neu5Gc “xeno-autoantibody” response. Given evidence that this process contributes to chronic inflammation in some diseases, it is important to understand when and how these antibodies are generated in humans. We show here that human anti-Neu5Gc antibodies appear during infancy and correlate with weaning and exposure to dietary Neu5Gc. However, dietary Neu5Gc alone cannot elicit anti-Neu5Gc antibodies in mice with a humanlike Neu5Gc deficiency. Other postnatally appearing anti-carbohydrate antibodies are likely induced by bacteria expressing these epitopes; however, no microbe is known to synthesize Neu5Gc. Here, we show that trace exogenous Neu5Gc can be incorporated into cell surface lipooligosaccharides (LOS) of nontypeable Haemophilus influenzae (NTHi), a human-specific commensal/pathogen. Indeed, infant anti-Neu5Gc antibodies appear coincident with antibodies against NTHi. Furthermore, NTHi that express Neu5Gc-containing LOS induce anti-Neu5Gc antibodies in Neu5Gc-deficient mice, without added adjuvant. Finally, Neu5Gc from baby food is taken up and expressed by NTHi. As the flora residing in the nasopharynx of infants can be in contact with ingested food, we propose a novel model for how NTHi and dietary Neu5Gc cooperate to generate anti-Neu5Gc antibodies in humans.
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implications of the presence of n glycolylneuraminic Acid in recombinant therapeutic glycoproteins
Nature Biotechnology, 2010Co-Authors: Rachel E Taylor, Darius Ghaderi, Vered Padlerkaravani, Sandra Diaz, Ajit VarkiAbstract:Recombinant glycoproteins produced in animal cell lines often bear the nonhuman sialic Acid N-Glycolylneuraminic Acid (Neu5Gc). Ghaderi et al. show that two monoclonal antibodies in clinical use differ with respect to addition of Neu5Gc and propose that drug developers should consider the consequences of the Neu5Gc modification.
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sensitive and specific detection of the non human sialic Acid n glycolylneuraminic Acid in human tissues and biotherapeutic products
PLOS ONE, 2009Co-Authors: Sandra Diaz, Ajit Varki, Hai Yu, Darius Ghaderi, Vered Padlerkaravani, Xi Chen, Nancy Hurtadoziola, Els Brinkmanvan Der C M Linden, Nissi M. VarkiAbstract:Author(s): Diaz, Sandra L; Padler-Karavani, Vered; Ghaderi, Darius; Hurtado-Ziola, Nancy; Yu, Hai; Chen, Xi; Brinkman-Van der Linden, Els CM; Varki, Ajit; Varki, Nissi M | Abstract: Humans are genetically defective in synthesizing the common mammalian sialic Acid N-Glycolylneuraminic Acid (Neu5Gc), but can metabolically incorporate it from dietary sources (particularly red meat and milk) into glycoproteins and glycolipids of human tumors, fetuses and some normal tissues. Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies. These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies. Thus, there is need for the sensitive and specific detection of Neu5Gc in human tissues and biotherapeutic products. Unlike monoclonal antibodies that recognize Neu5Gc only in the context of underlying structures, chicken immunoglobulin Y (IgY) polyclonal antibodies can recognize Neu5Gc in broader contexts. However, prior preparations of such antibodies (including our own) suffered from some non-specificity, as well as some cross-reactivity with the human sialic Acid N-acetylneuraminic Acid (Neu5Ac).We have developed a novel affinity method utilizing sequential columns of immobilized human and chimpanzee serum sialoglycoproteins, followed by specific elution from the latter column by free Neu5Gc. The resulting mono-specific antibody shows no staining in tissues or cells from mice with a human-like defect in Neu5Gc production. It allows sensitive and specific detection of Neu5Gc in all underlying glycan structural contexts studied, and is applicable to immunohistochemical, enzyme-linked immunosorbent assay (ELISA), Western blot and flow cytometry analyses. Non-immune chicken IgY is used as a reliable negative control. We show that these approaches allow sensitive detection of Neu5Gc in human tissue samples and in some biotherapeutic products, and finally show an example of how Neu5Gc might be eliminated from such products, by using a human cell line grown under defined conditions.We report a reliable antibody-based method for highly sensitive and specific detection of the non-human sialic Acid Neu5Gc in human tissues and biotherapeutic products that has not been previously described.
Sandra Diaz - One of the best experts on this subject based on the ideXlab platform.
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metabolism of vertebrate amino sugars with n glycolyl groups elucidating the intracellular fate of the non human sialic Acid n glycolylneuraminic Acid
Journal of Biological Chemistry, 2012Co-Authors: Anne K. Bergfeld, Sandra Diaz, Oliver M. T. Pearce, Tho Pham, Ajit VarkiAbstract:The two major mammalian sialic Acids are N-acetylneuraminic Acid and N-Glycolylneuraminic Acid (Neu5Gc). The only known biosynthetic pathway generating Neu5Gc is the conversion of CMP-N-acetylneuraminic Acid into CMP-Neu5Gc, which is catalyzed by the CMP-Neu5Ac hydroxylase enzyme. Given the irreversible nature of this reaction, there must be pathways for elimination or degradation of Neu5Gc, which would allow animal cells to adjust Neu5Gc levels to their needs. Although humans are incapable of synthesizing Neu5Gc due to an inactivated CMAH gene, exogenous Neu5Gc from dietary sources can be metabolically incorporated into tissues in the face of an anti-Neu5Gc antibody response. However, the metabolic turnover of Neu5Gc, which apparently prevents human cells from continued accumulation of this immunoreactive sialic Acid, has not yet been elucidated. In this study, we show that pre-loaded Neu5Gc is eliminated from human cells over time, and we propose a conceivable Neu5Gc-degrading pathway based on the well studied metabolism of N-acetylhexosamines. We demonstrate that murine tissue cytosolic extracts harbor the enzymatic machinery to sequentially convert Neu5Gc into N-glycolylmannosamine, N-glycolylglucosamine, and N-glycolylglucosamine 6-phosphate, whereupon irreversible de-N-glycolylation of the latter results in the ubiquitous metabolites glycolate and glucosamine 6-phosphate. We substantiate this finding by demonstrating activity of recombinant human enzymes in vitro and by studying the fate of radiolabeled pathway intermediates in cultured human cells, suggesting that this pathway likely occurs in vivo. Finally, we demonstrate that the proposed degradative pathway is partially reversible, showing that N-glycolylmannosamine and N-glycolylglucosamine (but not glycolate) can serve as precursors for biosynthesis of endogenous Neu5Gc.
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implications of the presence of n glycolylneuraminic Acid in recombinant therapeutic glycoproteins
Nature Biotechnology, 2010Co-Authors: Rachel E Taylor, Darius Ghaderi, Vered Padlerkaravani, Sandra Diaz, Ajit VarkiAbstract:Recombinant glycoproteins produced in animal cell lines often bear the nonhuman sialic Acid N-Glycolylneuraminic Acid (Neu5Gc). Ghaderi et al. show that two monoclonal antibodies in clinical use differ with respect to addition of Neu5Gc and propose that drug developers should consider the consequences of the Neu5Gc modification.
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sensitive and specific detection of the non human sialic Acid n glycolylneuraminic Acid in human tissues and biotherapeutic products
PLOS ONE, 2009Co-Authors: Sandra Diaz, Ajit Varki, Hai Yu, Darius Ghaderi, Vered Padlerkaravani, Xi Chen, Nancy Hurtadoziola, Els Brinkmanvan Der C M Linden, Nissi M. VarkiAbstract:Author(s): Diaz, Sandra L; Padler-Karavani, Vered; Ghaderi, Darius; Hurtado-Ziola, Nancy; Yu, Hai; Chen, Xi; Brinkman-Van der Linden, Els CM; Varki, Ajit; Varki, Nissi M | Abstract: Humans are genetically defective in synthesizing the common mammalian sialic Acid N-Glycolylneuraminic Acid (Neu5Gc), but can metabolically incorporate it from dietary sources (particularly red meat and milk) into glycoproteins and glycolipids of human tumors, fetuses and some normal tissues. Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies. These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies. Thus, there is need for the sensitive and specific detection of Neu5Gc in human tissues and biotherapeutic products. Unlike monoclonal antibodies that recognize Neu5Gc only in the context of underlying structures, chicken immunoglobulin Y (IgY) polyclonal antibodies can recognize Neu5Gc in broader contexts. However, prior preparations of such antibodies (including our own) suffered from some non-specificity, as well as some cross-reactivity with the human sialic Acid N-acetylneuraminic Acid (Neu5Ac).We have developed a novel affinity method utilizing sequential columns of immobilized human and chimpanzee serum sialoglycoproteins, followed by specific elution from the latter column by free Neu5Gc. The resulting mono-specific antibody shows no staining in tissues or cells from mice with a human-like defect in Neu5Gc production. It allows sensitive and specific detection of Neu5Gc in all underlying glycan structural contexts studied, and is applicable to immunohistochemical, enzyme-linked immunosorbent assay (ELISA), Western blot and flow cytometry analyses. Non-immune chicken IgY is used as a reliable negative control. We show that these approaches allow sensitive detection of Neu5Gc in human tissue samples and in some biotherapeutic products, and finally show an example of how Neu5Gc might be eliminated from such products, by using a human cell line grown under defined conditions.We report a reliable antibody-based method for highly sensitive and specific detection of the non-human sialic Acid Neu5Gc in human tissues and biotherapeutic products that has not been previously described.
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mechanism of uptake and incorporation of the non human sialic Acid n glycolylneuraminic Acid into human cells
Journal of Biological Chemistry, 2005Co-Authors: Muriel Bardor, Sandra Diaz, Dzung H Nguyen, Ajit VarkiAbstract:N-Glycolylneuraminic Acid (Neu5Gc) is a widely expressed sialic Acid in mammalian cells. Although humans are genetically deficient in producing Neu5Gc, small amounts are present in human cells in vivo. A dietary origin was suggested by human volunteer studies and by observing that free Neu5Gc is metabolically incorporated into cultured human carcinoma cells by unknown mechanisms. We now show that free Neu5Gc uptake also occurs in other human and mammalian cells. Inhibitors of certain non-clathrin-mediated endocytic pathways reduce Neu5Gc accumulation. Studies with human mutant cells show that the lysosomal sialic Acid transporter is required for metabolic incorporation of free Neu5Gc. Incorporation of glycosidically bound Neu5Gc from exogenous glycoconjugates (relevant to human gut epithelial exposure to dietary Neu5Gc) requires the transporter as well as the lysosomal sialidase, which presumably acts to release free Neu5Gc. Thus, exogenous Neu5Gc reaches lysosomes via pinocytic/endocytic pathways and is exported in free form into the cytosol, becoming available for activation and transfer to glycoconjugates. In contrast, N-glycolylmannosamine (ManNGc) apparently traverses the plasma membrane by passive diffusion and becomes available for conversion to Neu5Gc in the cytosol. This mechanism can also explain the metabolic incorporation of chemically synthesized unnatural sialic Acids, as reported by others. Finally, to our knowledge, this is the first example of delivery to the cytosol of an extracellular small molecule that cannot cross the plasma membrane, utilizing fluid pinocytosis and a specific lysosomal transporter. The approach could, thus, potentially be generalized to any small molecule that has a specific lysosomal transporter but not a plasma membrane transporter.
