Reactivity of Antibodies with Highly Glycosylated MUC1 Mucins from Colon Carcinoma Cells and Bile

Sikut, Rein; Sikut, Anu; Zhang, Ke; Baeckström, Dan; Hansson, Gunnar C.

doi:10.1159/000056513

Cited by 14 publications

(13 citation statements)

References 6 publications

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“…HMFG-2 reacts most strongly with the ϳ200-kDa species, while occasionally reacting with the Ͼ300-kDa form. B27.29, on the other hand, reacts most strongly with the largest form (while also recognizing the ϳ200-kDa form), and has been previously characterized as binding better to highly glycosylated MUC1 (30). Importantly, we found that the FLAG epitope was seemingly masked by glycosylation in the mammary gland, as we were unable to detect the Ͼ300-kDa form with anti-FLAG reagents (Fig.…”

Section: Mmtv-muc1mentioning

confidence: 64%

Transgenic MUC1 Interacts with Epidermal Growth Factor Receptor and Correlates with Mitogen-activated Protein Kinase Activation in the Mouse Mammary Gland

Schroeder

Thompson

Gardner

et al. 2001

Journal of Biological Chemistry

320

310

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MUC1 is a large (>400 kDa), heavily glycosylated transmembrane protein that is aberrantly expressed on greater than 90% of human breast carcinomas and subsequent metastases. The precise function of MUC1 overexpression in tumorigenesis is unknown, although various domains of MUC1 have been implicated in cell adhesion, cell signaling, and immunoregulation. Stimulation of the MDA-MB-468 breast cancer line as well as mouse mammary glands with epidermal growth factor results in the co-immunoprecipitation of MUC1 with a tyrosine-phosphorylated protein of ϳ180 kDa. We have generated transgenic lines overexpressing full-length (MMF), cytoplasmic tail deleted (⌬CT), or tandem repeat deleted (⌬TR)-human MUC1 under the control of the mouse mammary tumor virus promoter to further examine the role of MUC1 in signaling and tumorigenesis. Immunoprecipitation experiments revealed that fulllength transgenic MUC1 physically associates with all four erbB receptors, and co-localizes with erbB1 in the lactating gland. Furthermore, we detected a sharp increase in ERK1/2 activation in MUC1 transgenic mammary glands compared with Muc1 null and wild-type animals. These results point to a novel function of increased MUC1 expression, potentiation of erbB signaling through the activation of mitogenic MAP kinase pathways.The transmembrane mucin MUC1 (DF3, CD227, episialin, PEM) is a heavily O-glycosylated protein expressed on most secretory epithelium, including the mammary gland as well as some hematopoetic cells. MUC1 is expressed abundantly in the lactating mammary gland in addition to being overexpressed in greater than 90% of human breast carcinomas and metastases (1). In the normal mammary gland, MUC1 is expressed mainly on the apical surface of glandular epithelium and is believed to play a role in anti-adhesion and immune protection (2-4). In breast cancer, MUC1 is overexpressed, underglycosylated, and apical localization is lost (2). Mice lacking Muc1 demonstrate no overt phenotypic developmental abnormalities in the mammary gland, but when crossed with the tumorigenic MMTV 1 -mTag transgenic line (5), mammary gland tumor growth was significantly slowed. Additionally, these Muc1-null/MMTVmTag transgenics demonstrated a trend toward decreased metastasis, showing that the absence of Muc1 results in both reduced tumor growth and spread (6). MUC1 is transcribed as a large precursor gene product, which, upon translation, is cleaved in the endoplasmic reticulum, yielding two separate proteins that form a heterodimeric complex, bound together by noncovalent interactions (7). The larger of the two components (the "mucin-like" subunit) contains most of the extracellular domain, including the signal sequence, tandem repeats, as well as some degenerate repeats. The tandem repeats consist of 30 to 90 repeat sequences of 20 amino acids, rich in serine and threonine residues. Approximately 50 -90% of the mass of MUC1 is derived from O-glycosylation that occurs on these amino acids (8)). The second component of the heterodimer consists of an extr...

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Section: Mmtv-muc1mentioning

confidence: 64%

Transgenic MUC1 Interacts with Epidermal Growth Factor Receptor and Correlates with Mitogen-activated Protein Kinase Activation in the Mouse Mammary Gland

Schroeder

Thompson

Gardner

et al. 2001

Journal of Biological Chemistry

320

310

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“…MUC1 was detected using the MAb 214D4 directed against the extracellular tandem repeat region of the human MUC1 mucin. This MAb reacts well with the high-glycosylated forms found in the intestine (31). Although antibodies against repetitive sequences are not that reliable for semiquantitative estimates, a dramatically increased amount (estimated to more than 1,000-fold) of MUC1 mucin was found in the CFM mice compared with the non-CF mice (Fig.…”

Section: Resultsmentioning

confidence: 87%

Increased levels of mucins in the cystic fibrosis mouse small intestine, and modulator effects of the Muc1 mucin expression

Malmberg¹,

Noaksson²,

Phillipson³

et al. 2006

American Journal of Physiology-Gastrointestinal and Liver Physi

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The mouse model (Cftr(tm1UNC)/Cftr(tm1UNC)) for cystic fibrosis (CF) shows mucus accumulation and increased Muc1 mucin mRNA levels due to altered splicing (Hinojosa-Kurtzberg AM, Johansson MEV, Madsen CS, Hansson GC, and Gendler SJ. Am J Physiol Gastrointest Liver Physiol 284: G853-G862, 2003). However, it is not known whether Muc1 is a major mucin contributing to the increased mucus and why CF/Muc1-/- mice show lower mucus accumulation. To address this, we have purified mucins from the small intestine of CF mice using guanidinium chloride extraction, ultracentrifugation, and gel filtration and analyzed them by slot blot, gel electrophoresis, proteomics, and immunoblotting. Normal and CF mice with wild-type (WT) Muc1 or Muc1-/- or that are transgenic for human MUC1 (MUC1.Tg, on a Muc1-/- background) were analyzed. The total amount of mucins, both soluble and insoluble in guanidinium chloride, increased up to 10-fold in the CF mice compared with non-CF animals, whereas the CF mice lacking Muc1 showed intermediate levels between the CF and non-CF mice. However, the levels of Muc3 (orthologue of human MUC17) were increased in the CF/Muc1-/- mice compared with the CF/MUC1.Tg animals. The amount of MUC1 mucin was increased several magnitudes in the CF mice, but MUC1 did still not appear to be a major mucin. The amount of insoluble mucus of the large intestine was also increased in the CF mice, an effect that was partially restored in the CF/Muc1-/- mice. The thickness of the firmly adherent mucus layer of colon in the Muc1-/- mice was significantly lower than that of WT mice. The results suggest that MUC1 is not a major component in the accumulated mucus of CF mice and that MUC1 can influence the amount of other mucins in a still unknown way.

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“…The MUC1 on colon cancer cells is highly glycosylated and therefore glycosylation-sensitive antibodies rarely stain colon cells or colon tumors. 25,26 This suggests that our antibody PH1-IgG1 recognizes MUC1 in a differential glycosyla- …”

Section: Discussionmentioning

confidence: 93%

A Human Immunoglobulin G1 Antibody Originating from an in Vitro-Selected Fab Phage Antibody Binds Avidly to Tumor-Associated MUC1 and Is Efficiently Internalized

Henderikx¹,

Neer²,

Jacobs³

et al. 2002

The American Journal of Pathology

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Reactivity of Antibodies with Highly Glycosylated MUC1 Mucins from Colon Carcinoma Cells and Bile

Cited by 14 publications

References 6 publications

Transgenic MUC1 Interacts with Epidermal Growth Factor Receptor and Correlates with Mitogen-activated Protein Kinase Activation in the Mouse Mammary Gland

Transgenic MUC1 Interacts with Epidermal Growth Factor Receptor and Correlates with Mitogen-activated Protein Kinase Activation in the Mouse Mammary Gland

Increased levels of mucins in the cystic fibrosis mouse small intestine, and modulator effects of the Muc1 mucin expression

A Human Immunoglobulin G1 Antibody Originating from an in Vitro-Selected Fab Phage Antibody Binds Avidly to Tumor-Associated MUC1 and Is Efficiently Internalized

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