Cloning of a Human UDP-N-Acetyl-α-d-Galactosamine:PolypeptideN-Acetylgalactosaminyltransferase That Complements Other GalNAc-Transferases in Complete O-Glycosylation of the MUC1 Tandem Repeat

Bennett, Eric; Hassan, Helle; Mandel, Ulla; Mirgorodskaya, Ekatarina; Roepstorff, Peter; Burchell, Joy; Taylor‐Papadimitriou, Joyce; Hollingsworth, Michael A.; Merkx, G.F.M.; Kessel, Ad Geurts van; Eiberg, Hans; Steffensen, Rudi; Clausen, Henrik

doi:10.1074/jbc.273.46.30472

Cited by 201 publications

(213 citation statements)

References 49 publications

(73 reference statements)

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“…3E). This HPLC and MS analysis revealed that a substantial amount of the tandem repeat sequence was modified by 1-3 mol of GalNAc, which is in agreement with the fact that GalNAc-T2 can attach GalNAc up to three of the five potential sites in the MUC1 tandem repeat sequence (Ser 8 , Thr 9 , and Thr 17 ) (38). Two minor peaks at 21.1 and 22.3 min were found when MUC1-YFP was expressed alone (Fig.…”

Section: Structural Analysis Of Muc1-yfp Expressed In Glycoengineeredsupporting

confidence: 64%

Engineering Mammalian Mucin-type O-Glycosylation in Plants

Yang

Drew

Jørgensen

et al. 2012

Journal of Biological Chemistry

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Mucin-type O-glycosylation is an important post-translational modification that confers a variety of biological properties and functions to proteins. This post-translational modification has a particularly complex and differentially regulated biosynthesis rendering prediction and control of where O-glycans are attached to proteins, and which structures are formed, difficult. Because plants are devoid of GalNAc-type O-glycosylation, we have assessed requirements for establishing human GalNAc O-glycosylation de novo in plants with the aim of developing cell systems with custom-designed O-glycosylation capacity. Transient expression of a Pseudomonas aeruginosa Glc(NAc) C4-epimerase and a human polypeptide GalNAc-transferase in leaves of Nicotiana benthamiana resulted in GalNAc O-glycosylation of co-expressed human O-glycoprotein substrates. A chimeric YFP construct containing a 3.5 tandem repeat sequence of MUC1 was glycosylated with up to three and five GalNAc residues when co-expressed with GalNAc-T2 and a combination of GalNAc-T2 and GalNAc-T4, respectively, as determined by mass spectrometry. O-Glycosylation was furthermore demonstrated on a tandem repeat of MUC16 and interferon α2b. In plants, prolines in certain classes of proteins are hydroxylated and further substituted with plant-specific O-glycosylation; unsubstituted hydroxyprolines were identified in our MUC1 construct. In summary, this study demonstrates that mammalian type O-glycosylation can be established in plants and that plants may serve as a host cell for production of recombinant O-glycoproteins with custom-designed O-glycosylation. The observed hydroxyproline modifications, however, call for additional future engineering efforts.

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Section: Structural Analysis Of Muc1-yfp Expressed In Glycoengineeredsupporting

confidence: 64%

Engineering Mammalian Mucin-type O-Glycosylation in Plants

Yang

Drew

Jørgensen

et al. 2012

Journal of Biological Chemistry

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“…In T47D cells, no functional core2 enzyme is expressed (6), and initial O-glycosylation can proceed to completion before inhibitory glycan substituents (sialylated core1) are formed in the trans-Golgi. One of the enzymes involved in initial O-glycosylation, the recently described rGalNAc-T4, which adds GalNAc site-specifically to the DTR motif, was found to prefer substrates carrying GalNAc at the proximal sites (24). However, we could demonstrate that the DTR motif is glycosylated in vivo to a significant extent even in the fraction of PAP20-GalNAc 2 pointing to the existence of other enzymes exhibiting similar site specificity, but no dependence on prior glycosylation.…”

Section: Discussioncontrasting

confidence: 43%

“…5). This finding implies that the DTR motif is a glycosylation target on VNTR-peptides with no or one GalNAc linked to the remaining sites and, accordingly, is in conflict with the reported preference of GalNAc 3 -substituted substrates described for the only known DTR glycosylating enzyme, rGalNAc-T4 (24). The intensity of the fragment ion at m/z 366.0 (indicating cleavage of Hex HexNAc) is weak compared with that of the corresponding fragment at m/z 204.0 (indicating cleavage of a HexNAc residue) and should be derived from trace amounts of a hexose containing glycopeptide (fragment) with a relative mass close to the selected precursor ion at m/z 765.1.…”

Section: Table II Pseudomolecular Ions Measured For Pap20 Glycopeptidcontrasting

confidence: 51%

High Density O-Glycosylation on Tandem Repeat Peptide from Secretory MUC1 of T47D Breast Cancer Cells

Müller¹,

Alving

Peter‐Katalinić

et al. 1999

Journal of Biological Chemistry

151

113

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The site-specific O-glycosylation of MUC1 tandem repeat peptides from secretory mucin of T47D breast cancer cells was analyzed. After affinity isolation on immobilized BC3 antibody, MUC1 was partially deglycosylated by enzymatic treatment with ␣-sialidase/␤-galactosidase and fragmented by proteolytic cleavage with the Arg-C-specific endopeptidase clostripain. The PAP20 glycopeptides were isolated by reversed phase high pressure liquid chromatography and subjected to the structural analyses by quadrupole time-offlight electrospray ionization mass spectrometry and to the sequencing by Edman degradation. All five positions of the repeat peptide were revealed as O-glycosylation targets in the tumor cell, including the Thr within the DTR motif. The degree of substitution was estimated to average 4.8 glycans per repeat, which compares to 2.6 glycosylated sites per repeat for the mucin from milk (Mü ller, S., Goletz, S., Packer, N., Gooley, A. A., Lawson, A. M., and Hanisch, F.-G. (1997) J. Biol. Chem. 272, 24780-24793). In addition to a modification by glycosylation, the immunodominant DTR motif on T47D-MUC1 is altered by amino acid replacements (PAPGSTAPAAHGVTSAPESR), which were revealed in about 50% of PAP20 peptides. The high incidence of these replacements and their detection also in other cancer cell lines imply that the conserved tandem repeat domain of MUC1 is polymorphic with respect to the peptide sequence.Due to the structural complexity of O-linked glycans, this characteristic posttranslational modification of mucin peptides is a polygenic regulated phenomenon and hence is prone to multiple, differentiation-dependent alterations. According to numerous reports, mucin O-glycosylation can now be regarded as a diagnostically relevant indicator of tumor-associated changes that are characterized by 1) the de novo expression of novel glycotopes the ectopic or incompatible expression of carbohydrate blood groups, or 2) by deletion/truncation of glycan chains (1).Also, the widely distributed epithelial mucin MUC1 has been described to be aberrantly processed in cancer cells (2-4). In breast cancer, the nonexpression of the core2 enzyme, Gal␤1-3GalNAc/␤-6-N-acetylglucosaminyltransferase (5), leads to the truncation of polylactosamine-type chains found on the lactation-associated mucin (6) and to the accumulation of core-type chains (2-4). The preponderance of sialylated core1-trisaccharide on carcinoma-associated MUC1, which can be regarded as a biosynthetic dead end product, has been shown to originate from the simultaneous up-regulation and overexpression of Gal␤1-3GalNAc/␣-3-sialyltransferase (7). Moreover, not only the chain length of the glycans but also their density has been described to be reduced on breast cancer cell-specific MUC1 (4).Reduced glycosylation has been assumed to permit the immune system access to the peptide core of the tumor-associated mucin (8). The preferred target site for most peptide-specific mouse antibodies generated to the tumor mucin, to synthetic variable number of tandem repeats (V...

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“…Higher expression of GalNAc-T1, GalNAc-T2 and GalNAc-T3 has also been described in colorectal carcinoma when compared with the normal colonic epithelium [27]. Different levels of expression of GalNAc-T3 were detected in patients with colorectal [12], lung [28], pancreatic [29], gastric [30], gallbladder [31], prostate [32] and extrahepatic bile duct carcinomas [33], and it was identified as an independent factor of prognosis. GalNAc-T6, which exhibits a high sequence homology to GalNAc-T3, has been recently described to be expressed in most ductal breast carcinomas but not in normal breast epithelium.…”

Section: Introductionmentioning

confidence: 99%

N-Acetylgalactosaminyltransferase-14 as a potential biomarker for breast cancer by immunohistochemistry

Guo

Wang

et al. 2010

BMC Cancer

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BackgroundThe post-translational modification of proteins, including glycosylation, differs between normal and tumor cells. The UDP-N-acetyl-D-galactosamine polypeptide N-acetylgalactosaminyltransferases (GalNAc-Tases) family of enzymes regulates the initial steps of mucin O-glycosylation and is responsible for the altered glycosylation state observed in cancer cells. Recently it was found that GalNAc-T14 mRNA is heterogeneously expressed in breast carcinomas compared to normal tissue, however the expression profile of GalNAc-T14 protein in breast carcinomas compared to normal tissue is still unknown. In this study, we assessed the expression profile of GalNAc-T14 protein in malignant and non-malignant breast tissues by immunohistochemistry to evaluate whether GalNAc-T14 might be a potential biomarker for breast cancer.MethodsIn formalin-fixed tissues, the expression level of GalNAc-T14 protein was evaluated by immunohistochemistry assay in breast tissues. Expression profiles were assessed in normal tissues, benign fibroadenomas and several types of carcinomas.ResultsOur results showed that GalNAc-T14 was heterogeneously expressed in breast carcinomas compared to non-malignant tissue. GalNAc-T14 expression was observed in 47/56 (83.9%) carcinoma samples, 7/48 (14.6%) non-malignant breast tissue samples. GalNAc-T14 expression level was associated with histological grade. For this enzyme a significant association with invasive ductal type, mucinous adenocarcinoma and ductal carcinoma in situ (DCIS) type was found.ConclusionOur results provide evidence that GalNAc-T14 may be a potential biomarker for breast cancer by immunohistochemistry. GalNAc-T14 expression level was associated with histological grade. GalNAc-T14 expression can provide new insights about breast cancer glycobiology.

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Cloning of a Human UDP-N-Acetyl-α-d-Galactosamine:PolypeptideN-Acetylgalactosaminyltransferase That Complements Other GalNAc-Transferases in Complete O-Glycosylation of the MUC1 Tandem Repeat

Cited by 201 publications

References 49 publications

Engineering Mammalian Mucin-type O-Glycosylation in Plants

Engineering Mammalian Mucin-type O-Glycosylation in Plants

High Density O-Glycosylation on Tandem Repeat Peptide from Secretory MUC1 of T47D Breast Cancer Cells

N-Acetylgalactosaminyltransferase-14 as a potential biomarker for breast cancer by immunohistochemistry

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