Purification and Characterization of UDP-<i>N</i>-Acetylgalactosamine: κ-Casein Polypeptide<i>N</i>-Acetylgalactosaminyltransferase from Mammary Gland of Lactating Cow

Takeuchi, Makoto; Yoshikawa, Masaaki; Sasaki, Ryuzo; Chiba, Hideo

doi:10.1080/00021369.1985.10866844

Cited by 5 publications

(5 citation statements)

References 6 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These results are in agreement with the expected values for enzymes located in the Golgi apparatus, considering that the pH range in this organelle has been estimated between 5.8 and 6.6 ( Wu et al, 2001 ). A comparison with the results on pH-optima described in other studies indicates that both xGalNAcT-isoforms have slightly lower pH-optima than mammalian isoforms, which were determined between 6.5 and 8.2 ( Takeuchi et al, 1985; Wang et al, 1992; Mendicino and Sangadala, 1998 ). Indeed, in our hands, activity tests using the recombinant human isoform 2 and MES-buffer at pH 5, 6 and 7 showed highest conversion at pH 7 (data not shown).…”

Section: Resultssupporting

confidence: 49%

“…The metal dependency of many glycosyltransferases of the GT-A superfamily is based on the formation of a complex between a divalent metal ion and the oxygen atoms of the two phosphate groups of the NDP-sugar ( Breton et al, 2006 ). Several studies describe Mn 2 + as the best activator for enzymatic activity of N -acetylgalactosaminyltransferases, with an optimal concentration between 2.5 and 10 mM ( Takeuchi et al, 1985; Wang et al, 1992 ), whereby reducing the concentration to 0.4 mM results in 50% loss of activity, whereas the increase to 20, 30 and 100 mM decreases the activity to 90%, 50% and 20%, respectively ( Wang et al, 1992; Mendicino and Sangadala, 1998 ). Fig.…”

Section: Resultsmentioning

confidence: 99%

“…xGalNAc-T6 showed a slightly higher affinity for Mn 2 + over Co 2 + (78% activity compared to Mn 2 + ), whereas xGalNAcT-16 preferred Co 2 + (Mn 2 + showed only 96% of the activity compared to Co 2 + ). Studies which compared the enzyme activity using Co 2 + instead of Mn 2 + described a decrease of 89% for murine ( Elhammer and Kornfeld, 1986 ), 39% for porcine ( Wang et al, 1992 ) and 0% for bovine N -acetylgalactosaminyltransferases ( Takeuchi et al, 1985 ). Interestingly, the stimulation of xGalNAc-T16 by Ni 2 + seems to be much higher compared to xGalNAcT-6 (the latter's activity being only 12% compared to the xGalNAc-T16 conversion rate).…”

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Biological and biochemical properties of two Xenopus laevis N-acetylgalactosaminyltransferases with contrasting roles in embryogenesis

Voglmeir

Laurent

Flitsch

et al. 2015

Comparative Biochemistry and Physiology Part B: Biochemistry an

View full text Add to dashboard Cite

The biosynthesis of mucin-type O-linked glycans in animals is initiated by members of the large family of polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts), which play important roles in embryogenesis, organogenesis, adult tissue homeostasis and carcinogenesis. Until now, the mammalian forms of these enzymes have been the best characterized. However, two N-acetylgalactosaminyltransferases (xGalNAc-T6 and xGalNAc-T16) from the African clawed frog (Xenopus laevis), which are most homologous to those encoded by the human GALNT6 and GALNT16 (GALNTL1) genes, were shown to have contrasting roles in TGF-β/BMP signaling in embryogenesis. In this study we have examined these two enzymes further and show differences in their in vivo function during X. laevis embyrogenesis as evidenced by in situ hybridization and overexpression experiments. In terms of enzymatic activity, both enzymes were found to be active towards the EA2 peptide, but display differential activity towards a peptide based on the sequence of ActR-IIB, a receptor relevant to TGF-β/BMP signaling. In summary, these data demonstrate that these two enzymes from different branches of the N-acetylgalactosaminyltransferase do not only display differential substrate specificities, but also specific and distinct expression pattern and biological activities in vivo.

show abstract

Section: Resultssupporting

confidence: 49%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Biological and biochemical properties of two Xenopus laevis N-acetylgalactosaminyltransferases with contrasting roles in embryogenesis

Voglmeir

Laurent

Flitsch

et al. 2015

Comparative Biochemistry and Physiology Part B: Biochemistry an

View full text Add to dashboard Cite

show abstract

“…The initial transfer of an N-acetylgalactosamine (GalNAc) 1 from UDP-GalNAc to the hydroxyl group of serine or threonine is catalyzed by UDP-GalNAc:polypeptide ␣1,O-N-acetylgalactosaminyl transferase (O-GalNAcT; EC 2.4.1.41) in the cisGolgi compartment (2). To date, at least three isozymes for the enzyme have been found by purification (3)(4)(5)(6)(7)(8)11) and cDNA cloning (7)(8)(9)(10)(11)(12)(13). Although the consensus amino acid sequence Asn-Xaa-Ser/Thr (Xaa Pro) is extensively known in the case of N-glycosylation sites, such a consensus primary amino acid sequence has not been identified among the mucin-type Oglycosylation sites.…”

mentioning

confidence: 99%

Discovery of the Shortest Sequence Motif for High Level Mucin-type O-Glycosylation

Yoshida

Suzuki

Ikenaga

et al. 1997

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The consensus primary amino acid sequence for mucin-type O-glycosylation sites has not been identified. To determine the shortest motif sequence required for high level mucin-type O-glycosylation, we prepared more than 100 synthetic peptides and assayed in vitro O-GalNAc transfer to serine or threonine in these peptides using a bovine colostrum UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyl transferase (O-GalNAcT). We chose the sequence PDAASAAP from human erythropoietin (hEPO) for further systematic substitutions because it accepted GalNAc and was a fairly simple sequence consisting only of four kinds of amino acids. Several substitutions showed that threonine is ϳ40-fold better than serine as the glycosylated amino acid and a proline at position ؉3 on the C-terminal side is very important. To define the effect of proline residues around the glycosylation site, we analyzed a series of peptides containing one to three proline residues in a parent peptide AAATAAA. The results clearly indicated that prolines at positions ؉1 and ؉3 had a positive effect. The O-GalNAc transfer level of AAATPAP was increased approximately 90-fold from AAATAAA. The deletion of amino acids from the N-terminal side of the glycosylation site suggested that five amino acids from position ؊1 to ؉3 were especially important for glycosylation. Moreover, the influence of all 20 amino acids at positions ؊1, ؉2, and ؉4 was analyzed. Uncharged amino acids were preferred at position ؊1, and small or positively charged amino acids were preferred at position ؉2. No preference was observed at position ؉4. We propose a mucin-type O-glycosylation motif, XTPXP, which may be suitable as a signal for protein O-glycosylation. The features observed in this study also appear to be very useful for prediction of mucin-type O-glycosylation sites in glycoproteins.

show abstract

“…The majority of these are natural metabolites (or close analogues thereof) and are, therefore, unlikely to have much potential as drugs. For example, a range of nucleotide triphosphates (including ATP, ADP, AMP, CTP, GTP and UTP) inhibit ppGalNAc-T activity purified from the mammary gland of cows [68]. It is not clear what isoform(s) were being inhibited in this study (which was performed before the diversity of the pp-GalNAc-T family was known).…”

Section: Potential As a Drug Targetmentioning

confidence: 96%

UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase- 6 (pp-GalNAc-T6): Role in Cancer and Prospects as a Drug Target

Banford¹,

Timson

2016

CCDT

View full text Add to dashboard Cite

UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyl transferase-6 (pp-GalNAc-T6) is a member of the N-acetyl-D-galactosamine transferase family. It catalyzes the addition of N-acetyl-D-galactosamine to proteins, often the first step in Oglycosylation of proteins. Glycosylated proteins play important roles in vivo in the cell membrane. These are often involved in cell-cell adhesion, cytoskeleton regulation and immune recognition. pp-GalNAc-T6 has been shown to be upregulated in a number of types of cancer. Abnormally glycosylated forms of mucin 1 (substrate of the enzyme), are used clinically as a biomarker for breast cancer. There is potential for other products of the pp-GalNAc-T6 catalyzed reaction to be used. It is also possible that pp-GalNAc-T6 itself could be used as a biomarker, since levels of this protein tend to be low in nonmalignant tissues. pp-GalNAc-T6 has been implicated in malignant transformation and metastasis of cancer cells. As such, it has considerable potential as a target for chemotherapy. To date, no selective inhibitors of the enzyme have been identified. However, general inhibitors of the enzyme family result in reduced cell surface Olinked glycosylation and induce apoptosis in cultured cells. Thus, a selective inhibitor of pp-GalNAc-T6 is likely to target cancer cells and could be developed into a novel anticancer therapy.

show abstract

Purification and Characterization of UDP-N-Acetylgalactosamine: κ-Casein PolypeptideN-Acetylgalactosaminyltransferase from Mammary Gland of Lactating Cow

Cited by 5 publications

References 6 publications

Biological and biochemical properties of two Xenopus laevis N-acetylgalactosaminyltransferases with contrasting roles in embryogenesis

Biological and biochemical properties of two Xenopus laevis N-acetylgalactosaminyltransferases with contrasting roles in embryogenesis

Discovery of the Shortest Sequence Motif for High Level Mucin-type O-Glycosylation

UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase- 6 (pp-GalNAc-T6): Role in Cancer and Prospects as a Drug Target

Contact Info

Product

Resources

About