Influence of acceptor substrate primary amino acid sequence on the activity of human UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase. Studies with the MUC1 tandem repeat.

Nishimori, Isao; Johnson, N; Sanderson, Sam D.; Perini, Fulvio; Mountjoy, K.; Cerny, Ronald L.; Gross, Michael L.; Hollingsworth, Michael A.

doi:10.1016/s0021-9258(17)33981-9

Cited by 62 publications

(9 citation statements)

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“…The change in glycosylation pattern seen in breast cancer also relates specifically to the studies on the MUC1 mucin as a potential target antigen in active specific immu-notherapy of breast cancer. The extracellular domain of the MUC1 glycoprotein is made up largely of tandem repeats of 20 amino acids: 25-100 depending on the allele (Gendler et al, 1990), and each repeat contains potential glycosylation sites (Nishimori et al, 1994;Stadie et al, 1995). The antigenic profile of the mucin is therefore dramatically altered when the composition of the O-glycans added is changed from being core-2 based to the simpler, shorter, and more heavily sialylated glycans found on the tumor mucin.…”

Section: Discussionmentioning

confidence: 99%

A Transfected Sialyltransferase That Is Elevated in Breast Cancer and Localizes to the medial/trans-Golgi Apparatus Inhibits the Development of core-2–based O-Glycans

Whitehouse

Burchell

Gschmeissner

et al. 1997

The Journal of Cell Biology

115

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The α2,3 sialyltransferase, α2,3 SAT (O), catalyzes the transfer of sialic acid to Galβ1,3 N-acetyld-galactosamine (GalNAc) (core-1) in mucin type O-glycosylation, and thus terminates chain extension. A Core-2 branch can also be formed from core-1 by the core-2 β1,6 N-acetyl-d-glucosamine transferase (β1,6 GlcNAc T) that leads to chain extension. Increased levels of the α2,3 SAT (O) and decreased levels of the core-2 β1,6 GlcNAc T are seen in breast cancer cells and correlate with differences in the structure of the O-glycans synthesized (Brockhausen et al., 1995; Lloyd et al., 1996). Since in mucin type O-glycosylation sugars are added individually and sequentially in the Golgi apparatus, the position of the transferases, as well as their activity, can determine the final structure of the O-glycans synthesized. A cDNA coding for the human α2,3 SAT (O) tagged with an immunoreactive epitope from the myc gene has been used to map the position of the glycosyltransferase in nontumorigenic (MTSV1-7) and malignant (T47D) breast epithelial cell lines. Transfectants were analyzed for expression of the enzyme at the level of message and protein, as well as for enzymic activity. In T47D cells, which do not express core-2 β1,6 GlcNAc T, the increased activity of the sialyltransferase correlated with increased sialylation of core-1 O-glycans on the epithelial mucin MUC1. Furthermore, in MTSV1-7 cells, which do express core-2 β1,6 GlcNAc T, an increase in sialylated core-1 structures is accompanied by a reduction in the ratio of GlcNAc: GalNAc in the O-glycans attached to MUC1, implying a decrease in branching. Using quantitative immunoelectron microscopy, the sialyltransferase was mapped to the medial- and trans-Golgi cisternae, with some being present in the TGN. The data represent the first fine mapping of a sialyltransferase specifically active in O-glycosylation and demonstrate that the structure of O-glycans synthesized by a cell can be manipulated by transfecting with recombinant glycosyltransferases.

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Section: Discussionmentioning

confidence: 99%

A Transfected Sialyltransferase That Is Elevated in Breast Cancer and Localizes to the medial/trans-Golgi Apparatus Inhibits the Development of core-2–based O-Glycans

Whitehouse

Burchell

Gschmeissner

et al. 1997

The Journal of Cell Biology

115

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“…The molecular parameters that govern the specificity and kinetics of mucin-type O-linked glycosylation of serine and threonine with N-acetylgalactosamine by uridine diphosphate-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases) 1 remain poorly understood. O-Glycosylation of glycoproteins is influenced by protein trafficking, parameters of substrate protein folding, and levels of enzyme activity (1,2); in contrast, the relative importance of acceptor substrate primary amino acid sequence to this reaction has been widely debated (3)(4)(5)(6)(7)(8)(9)(10)(11). A consensus primary amino acid sequence for O-glycosylation has not been found; however, there is substantial evidence that sequences flanking serine and threonine residues significantly affect catalytic activity of GalNAc transferases (4,5,(8)(9)(10)12).…”

mentioning

confidence: 99%

“…O-Glycosylation of glycoproteins is influenced by protein trafficking, parameters of substrate protein folding, and levels of enzyme activity (1,2); in contrast, the relative importance of acceptor substrate primary amino acid sequence to this reaction has been widely debated (3)(4)(5)(6)(7)(8)(9)(10)(11). A consensus primary amino acid sequence for O-glycosylation has not been found; however, there is substantial evidence that sequences flanking serine and threonine residues significantly affect catalytic activity of GalNAc transferases (4,5,(8)(9)(10)12).…”

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confidence: 99%

“…The repeated nature of mucin-like tandem repeats and the presence of multiple acceptor substrate sites for GalNAc transferases provide a useful paradigm to evaluate the influence of acceptor substrate sequence on GalNAc transferase activity. O-Glycosylation of the tandem repeat domain of MUC1 by total GalNAc transferase activity has previously been investigated in human tumor cells (8,9). Two-thirds of MUC1 is composed of multiple copies of the tandem repeat (from 18 to over 100, depending on allele length, which is highly polymorphic) of the sequence GVTSAP-DTRPAPGSTAPPAH.…”

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confidence: 99%

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Structural Analysis of Peptide Substrates for Mucin-Type O-Glycosylation

et al. 1998

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The structures of three nine-residue peptide substrates that show differential kinetics of O-linked glycosylation catalyzed by distinct recombinant uridine diphosphate-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases) were investigated by NMR spectroscopy. A combined use of NMR data, molecular modeling techniques, and kinetic data may explain some structural features required for O-glycosylation of these substrates by two GalNAc transferases, GalNAc-T1 and GalNAc-T3. In the proposed model, the formation of an extended backbone structure at the threonine residue to be glycosylated is likely to enhance the O-glycosylation process. The segment of extended structure includes the reactive residue in a beta-like or an inverse gamma-turn conformation and flanking residues in a beta-strand conformation. The hydroxyl group of the threonine to be glycosylated is exposed to solvent, and both the amide proton and carbonyl oxygen of the peptide backbone are exposed to solvent. The exchange rate of the amide proton for the reactive threonine correlated well with substrate efficiency, leading us to hypothesize that this proton may serve as a donor for hydrogen bonding with the active site of the enzyme. The oxygens of the residue to be glycosylated and several flanking residues may also be involved in a set of hydrogen bonds with the GalNAc-T1 and -T3 transferases.

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“…Antibody activity was estimated using either antigen-coated plates, or cell-coated plates. For testing of HMFG1-␤-glucosidase, plates were coated with a peptide containing the sequence Pro-Asp-Thr-Arg-Pro (PDTRP), which is the epitope on PEM recognised by the antibody HMFG1 (Nishimori et al, 1994), diluted in 1% bovine serum albumin (BSA) in PBS. For testing of the H17E2-␤glucosidase, plates were coated with KB cells.…”

Section: Testing Of the Fractions For Enzyme And Antibody Activitymentioning

confidence: 99%

In vitro cytotoxicity following specific activation of amygdalin by β-glucosidase conjugated to a bladder cancer-associated monoclonal antibody

Syrigos

Rowlinson-Busza

Epenetos³

1998

Int. J. Cancer

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We describe a novel version of antibody‐directed enzyme prodrug therapy (ADEPT), with the use of amygdalin as prodrug. Amygdalin is a naturally occurring cyanogenic glycoside, which can be cleaved by sweet almond β‐glucosidase to yield free cyanide. If amygdalin could be activated specifically at the tumour site, then malignant cells would be killed without the systemic toxicity usually associated with chemotherapy. To this end, we have conjugated β‐glucosidase to a tumour‐associated monoclonal antibody (MAb) (HMFG1) and the conjugate has been tested in vitro for specificity and cytotoxicity subsequent to activation of amygdalin. Amygdalin was cytotoxic to HT1376 bladder cancer cells only at high concentrations, whereas the combination of amygdalin with HMFG1‐β‐glucosidase enhanced the cytotoxic effect of amygdalin by 36‐fold. When 2 concentrations of HMFG1‐β‐glucosidase were compared, the toxic effect was dose dependent. The cytotoxicity of amygdalin was also enhanced by the MAb–enzyme conjugate even when the unbound conjugate was removed from the medium prior to exposure to amygdalin and the cells were washed. In addition to the cytotoxic effect, we also demonstrated specificity, using a MAb–enzyme conjugate that does not recognise the HT1376 bladder cancer cells. Finally, we studied the cytotoxic effect of the conjugate in co‐culture of HMFG1‐positive and ‐negative cell lines (HT1376 and U87MG cells). We demonstrated that the rate of surviving cells corresponds well to the percentage of U87MG (HMFG1‐negative) cells in the flask. Our findings indicate that ADEPT is more effective than non‐directed enzyme activation of a prodrug and can result in a non‐toxic cancer therapy. Int. J. Cancer 78:712–719, 1998. © 1998 Wiley‐Liss, Inc.

show abstract

Influence of acceptor substrate primary amino acid sequence on the activity of human UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase. Studies with the MUC1 tandem repeat.

Cited by 62 publications

References 15 publications

A Transfected Sialyltransferase That Is Elevated in Breast Cancer and Localizes to the medial/trans-Golgi Apparatus Inhibits the Development of core-2–based O-Glycans

A Transfected Sialyltransferase That Is Elevated in Breast Cancer and Localizes to the medial/trans-Golgi Apparatus Inhibits the Development of core-2–based O-Glycans

Structural Analysis of Peptide Substrates for Mucin-Type O-Glycosylation

In vitro cytotoxicity following specific activation of amygdalin by β-glucosidase conjugated to a bladder cancer-associated monoclonal antibody

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