Article

The Breast Tumor-associated Epitope Defined by Monoclonal Antibody 3E1.2 Is an O-linked Mucin Carbohydrate Containing N-Glycolylneuraminic Acid

Medical Innovations Limited, Labrador, Queensland, Australia.
Cancer Research (Impact Factor: 9.28). 12/1991; 51(21):5826-36.
Source: PubMed

ABSTRACT The breast cancer-associated epitope (mammary serum antigen or MSA) defined by monoclonal antibody (Mab) 3E1.2 is a neuraminidase-sensitive carbohydrate expressed on MUC-1-encoded molecules. However, the reactivity of Mab 3E1.2 is also reduced by protease treatment of the mucin, which suggests that 3E1.2 binds to multimers of the sialylated carbohydrate in a protein conformation-dependent manner. The common N-acetyl derivative of neuraminic acid (5-acetylneuraminic acid) is not involved in the epitope, since lectins specific for 5-acetylneuraminic acid (linked to GalNAc or Gal) are nonreactive with MSA-positive molecules. However, the N-glycolyl derivative, 5-glycolylneuraminic acid (Neu5Gc), forms a major part of the epitope since both free Neu5Gc and porcine stomach mucin (greater than 90% neuraminic acid as Neu5Gc) inhibit the binding of Mab 3E1.2, while bovine or ovine submaxillary mucins, fetuin, bovine gangliosides, and other carbohydrates do not. Indeed, the presence of Neu5Gc on human tumor mucin was confirmed by electrospray mass spectrometry. Neu5Gc is attached to an O-linked carbohydrate, since the expression of MSA by MCF-7 breast cancer cells is inhibited by the O-glycosylation inhibitor phenyl-N-acetyl-alpha-D-galactosaminide, but not by the N-glycosylation inhibitor tunicamycin, and the epitope is removed by treatment with O-glycanase but not N-glycanase F, endoglycosidase F, or endoglycosidase H, which are specific for N-linked glycans. This is likely to be a core glycan since 3E1.2 reacts after treatment of the mucin with trifluoromethanesulfonic acid, which removes most backbone and peripheral carbohydrates. Treatment with galactosidase or N-acetyl glucosaminidase enhances the binding of Mab 3E1.2, indicating that the Neu5Gc is not attached to galactose or N-acetyl galactosamine. Furthermore, the susceptibility of MSA to treatment with Arthrobacter urea-faciens neuraminidase [which is specific for alpha (2-6)-linked NeuNAc] and the loss in reactivity of GalNAc-specific lectins after periodate oxidation [alpha (2-3)-linked but not alpha (2-6)-linked NeuNAc protects GalNAc from periodate oxidation] indicate that the Neu5Gc may be attached alpha (2-6) to peptide-linked GalNAc. These results show that MSA is a Neu5Gc-containing O-linked core glycan, which represents a unique tumor-associated epitope not previously identified on human mucins.

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    • "N-Glycolylneuraminic acid (Neu5Gc) is a sialic acid which is synthesized by animals, but not by humans or birds because of the lack of the enzyme CMP-Neu5Ac hydroxylase which converts Neu5Ac to Neu5Gc [1]. However, it can be incorporated in human cells after entering the body [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]. According to Bardor et al. [11], incorporation of Neu5Gc involves a pinocytic/endocytic procedure and the action of lysosomal sialidase as well as lysosomal sialic acid transporter. "
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    ABSTRACT: Background. N-Glycolylneuraminic acid (Neu5Gc) is a sialic acid synthesized by animals, but not by humans or birds. However, it can be incorporated in human cells and can trigger immune response. In the present study, we detected anti-Neu5Gc antibodies in samples of the general population and of patients suffering from hypothyroidism/Hashimoto’s disease, which is known to have autoimmune origin. Methods. Antibodies were measured using enzyme-immunosorbent techniques. Results. Serum anti-Neu5Gc IgG antibodies were higher in patients with hypothyroidism (mean: 14.8 ± 15.9 μg/mL, median: 10.0 μg/mL, p = 0.0003, Mann-Whitney) and even higher in the group with Hashimoto’s thyroiditis (mean: 31.1 ± 16.3 μg/mL, median: 27.2 μg/mL, p = 0.0000, Mann-Whitney) compared to the general population (mean: 5.3 ± 4.7 μg/mL, median : 4 μg/mL). All anti-TPO positive samples had anti-Neu5Gc antibody concentration higher than the mean value of the general population while anti-TPO concentration was increased as anti-Neu5Gc concentration increased. Low concentrations of IgA and IgM antibodies were measured in both general population and patient groups. Conclusion.The increased values of anti-Neu5Gc antibodies in patients with hypothyroidism/Hashimoto’s disease and the correlation of anti-TPO incidence with increased anti-Neu5Gc concentration raise the possibility of an association between anti-Neu5Gc antibody development and autoimmune hypothyroidism.
    BioMed Research International 06/2014; 2014. DOI:10.1155/2014/963230 · 2.71 Impact Factor
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    • "The N-glycolyl (NeuGc)-neuraminic acid variant of gangliosides is widely expressed in most mammalian tissues, but is rarely found in normal human cells. However, the presence of NeuGc-sialic acid has been reported in human tumors (Higashi et al., 1984, 1988; Hirabagashi et al., 1987; Miyake et al., 1990; Devine et al., 1991; Watarai et al., 1995; Marquina et al., 1996; Malykh et al., 2001). Chickens are another vertebrate species where NeuGc-glycoconjugates are not expressed in normal tissues and therefore a strong antibody response against this sialic acid variant can be obtained in immunized animals (Fujii et al., 1982; Ledeen and Yu, 1982). "
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    ABSTRACT: The heavy-chain variable regions (VH) from 14F7 MAb, an IgG1 antibody specific for GM3(NeuGc) ganglioside, and its anti-idiotype, the 4G9 MAb, were cloned and sequenced. Comparison with previously reported sequences showed that VH 14F7 belongs to the J558(VHI) gene family and that it is highly mutated. VH 4G9 belongs to the Q52(VHII) gene family. The HCDR3 14F7 sequence contains three basic residues that could be involved in the binding to 4G9 MAb, which bears acidic residues in its HCDR3. Studies performed in the syngeneic model showed that 14F7 MAb requires both coupling to KLH and the use of Freund's adjuvant to induce an effective anti-idiotypic IgG (Ab2) response. In contrast, P3 MAb, a germline gene-encoded Ab1 that also recognizes the GM3(NeuGc) ganglioside through a basic motif in its H-CDRs, has been reported to be immunogenic in syngeneic mice, even when injected in saline. In addition, when Leghorn chickens were immunized with 14F7 or P3 MAbs emulsified in Freund's adjuvant, only P3-immunized animals were able to develop antibodies that recognized NeuGc-containing gangliosides, antigens which are not present in the normal tissues of this animal species. This phenomenon could be due to the lack of idiotypic connectivity of 14F7MAb.
    Immunobiology 02/2007; 212(1):57-70. DOI:10.1016/j.imbio.2006.08.005 · 3.18 Impact Factor
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    • "In the course of the following decade, many researchers demonstrated the expression of Neu5Gc on gangliosides and glycoproteins isolated from various human tumours, using mainly immunochemical techniques [17]. In the nineties, the presence of this monosaccharide in human cancerous tissues was also detected by mass spectrometry [18] [19] [20] [21]. "
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    ABSTRACT: N-Glycolylneuraminic acid (Neu5Gc) is an abundant sialic acid, occurring in the glycoconjugates of most deuterostome animals. Homo sapiens is a notable exception, since Neu5Gc is effectively absent from normal human tissues. This is due to a deletion in the human gene coding for CMP-Neu5Ac hydroxylase, the enzyme usually responsible for Neu5Gc biosynthesis. Despite this mutation, persistent reports in the literature suggest that Neu5Gc occurs in the glycoconjugates of many human tumours, where it might be responsible for the formation of so-called Hanganutziu-Deicher antibodies. However, the variety of systems studied and the various experimental approaches adopted have yielded a complex picture of Neu5Gc occurrence in human neoplasias. The aim of this paper is therefore to provide a critical review of the evidence for Neu5Gc in human tumours, paying particular attention to the analytical methods employed. The possible clinical applications of Neu5Gc-containing glycoconjugates and Hanganutziu-Deicher antibodies in the diagnosis and treatment of breast cancer and melanoma are also discussed. In view of the lack of CMP-Neu5Ac hydroxylase in human cells, alternative metabolic pathways for the biosynthesis of glycoconjugate-bound Neu5Gc are considered.
    Biochimie 08/2001; 83(7):623-34. · 3.12 Impact Factor
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