Cloning and Expression of Mouse UDP-GalNAc:PolypeptideN-Acetylgalactosaminyltransferase-T3

Zara, Jane; Hagen, Fred K.; Hagen, Karl S.; Wuyckhuyse, B.C. Van; Tabak, Lawrence A.

doi:10.1006/bbrc.1996.1613

Cited by 60 publications

(41 citation statements)

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“…GalNAc-T11 is the first isoform exhibiting selectively high levels of expression in another organ, the kidney, and there is no expression in any cell compartment of salivary glands as evaluated by immunohistology. Northern analysis of kidney mRNA indicates that GalNAc-T1, -T2, and -T3 are expressed at low to moderate levels (4,5,44,45) and putative GalNAc-T8 is expressed at high levels (46), whereas GalNAc-T4, -T5, -T6, and -T7 and putative GalNAc-T9 and GalNAc-T10 are not expressed (6 -8, 10 -12, 46, 47). Immunohistological analysis showed weak expression of GalNAc-T1; moderate expression of GalNAc-T2; no expression of GalNAc-T3, -T4, and -T6; and high expression of GalNAc-T11 (Fig.…”

Section: Discussionmentioning

confidence: 99%

Functional Conservation of Subfamilies of Putative UDP-N-acetylgalactosamine:Polypeptide N-Acetylgalactosaminyltransferases inDrosophila, Caenorhabditis elegans, and Mammals

Schwientek¹,

Bennett²,

Flores³

et al. 2002

Journal of Biological Chemistry

172

147

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The completed fruit fly genome was found to contain up to 15 putative UDP-N-acetyl-␣-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) genes. Phylogenetic analysis of the putative catalytic domains of the large GalNAc-transferase enzyme families of Drosophila melanogaster (13 available), Caenorhabditis elegans (9 genes), and mammals (12 genes) indicated that distinct subfamilies of orthologous genes are conserved in each species. In support of this hypothesis, we provide evidence that distinctive functional properties of Drosophila and human GalNAc-transferase isoforms were exhibited by evolutionarily conserved members of two subfamilies (dGalNAc-T1 (l(2)35Aa) and GalNAc-T11; dGalNAc-T2 (CG6394) and GalNAc-T7). dGalNAc-T1 and novel human GalNAc-T11 were shown to encode functional GalNActransferases with the same polypeptide acceptor substrate specificity, and dGalNAc-T2 was shown to encode a GalNAc-transferase with similar GalNAc glycopeptide substrate specificity as GalNAc-T7. Previous data suggested that the putative GalNAc-transferase encoded by l(2)35Aa had a lethal phenotype (Flores, C., and Engels, W. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2964-2969), and this was substantiated by sequencing of three lethal alleles l(2)35Aa HG8

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Section: Discussionmentioning

confidence: 99%

Functional Conservation of Subfamilies of Putative UDP-N-acetylgalactosamine:Polypeptide N-Acetylgalactosaminyltransferases inDrosophila, Caenorhabditis elegans, and Mammals

Schwientek¹,

Bennett²,

Flores³

et al. 2002

Journal of Biological Chemistry

172

147

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“…Sequences comprising the conserved domains used in Figs. 1 and 2 begin with the consensus sequence FNXXXSD in the putative catalytic domain (aa position 84 in ppGaNTase-mT1 (11), 104 in ppGaNTase-hT2 (12), 150 in ppGaNTase-mT3 (13), 102 in ppGaNTase-mT4 (14), 454 in ppGaNTase-rT5 (15), 142 in ppGaNTase-hT6 (16), 175 in ppGaNTase-rT7 (17), 149 in ppGaNTase-hT8 (18), 119 in ppGaNTase-hT9 (19), 113 in ppGaNTase-rT10 (4), 119 in ppGaNTase-hT11 (7), 103 in ppGaNTase-h12 (20), 83 in ppGaNTase-h13 (10), 79 in ppGaNTase-h14 (21), 114 in PGANT35A (6), 116 in PGANT1, 152 in PGANT2, 118 in PGANT3, 161 in PGANT4, 142 in PGANT5, 170 in PGANT6, 110 in PGANT7, 95 in PGANT8, 176 in CG30463, 75 in CG10000, 75 in CG7304, 103 in CG31776, and 18 in CG7579 and end at a conserved residue (amino acid position 421 in ppGaNTase-mT1, 436 in ppGaNTase-hT2, 495 in ppGaNTase-mT3, 434 in ppGaNTase-mT4, 792 in ppGaNTase-rT5, 487 in ppGaNTase-hT6, 520 in ppGaNTase-rT7, 490 in ppGaNTase-hT8, 458 in ppGaNTase-hT9, 445 in ppGaNTase-rT10, 454 in ppGaNTase-hT11, 435 in ppGaNTase-hT12, 420 in ppGaNTase-hT13, 410 in ppGaNTase-hT14, 452 in PGANT35A, 463 in PGANT1, 484 in PGANT2, 459 in PGANT3, 497 in PGANT4, 479 in PGANT5, 504 in PGANT6, 454 in PGANT7, 432 in PGANT8, 512 in CG30463, 425 in CG10000, 420 in CG7304, 440 in CG31776, and 351 in CG7579). The segment of conserved sequences is ϳ340 amino acids in length in the various isoforms and corresponds to the putative catalytic domain based on structural modeling and mutagenesis studies (22).…”

Section: Methodsmentioning

confidence: 99%

Functional Characterization and Expression Analysis of Members of the UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferase Family from Drosophila melanogaster

Hagen

Tran

Gerken

et al. 2003

Journal of Biological Chemistry

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Here we report the cloning and functional characterization of eight members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase gene family from Drosophila melanogaster (polypeptide GalNAc transferase ‫؍‬ pgant1-8). Full-length cDNAs were isolated from a Drosophila embryonic library based on homology to known ppGaNTases. Alignments with characterized mammalian isoforms revealed strong sequence similarities between certain fly and mammalian isoforms, highlighting putative orthologues between the species. In vitro activity assays demonstrated biochemical transferase activity for each gene, with three isoforms requiring glycosylated substrates. Comparison of the activities of Drosophila and mammalian orthologues revealed conservation of substrate preferences against a panel of peptide and glycopeptide substrates. Furthermore, Edman degradation analysis demonstrated that preferred sites of GalNac addition were also conserved between certain fly and mammalian orthologues. Semiquantitative PCR amplification of Drosophila cDNA revealed expression of most isoforms at each developmental stage, with some isoforms being less abundant at certain stages relative to others. In situ hybridization to Drosophila embryos revealed specific staining of pgant5 and pgant6 in the salivary glands and pgant5 in the developing hindgut. Additionally, pgant5 and pgant6 expression within the egg chamber was restricted to the follicle cells, cells known to be involved in egg formation and subsequent embryonic patterning. The characterization reported here provides additional insight into the use of this model system to dissect the biological role of this enzyme family in vivo during both fly and mammalian development.

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“…The ppGalNAc-T3-specific synthetic peptide HIV gp120 (RGPGRAFVTIGKIGNMR) (22) and the control substrate EA2 (PTTDSTTPAPTTK, corresponding to the tandem repeat sequence of rat submandibular gland apomucin, detailed in Albone and colleagues (23) ) were synthesized by the Facility for Biotechnology Resources, Center for Biologics Evaluation and Research (Bethesda, MD, USA). Reactions were carried out at 378C in 25-ml mixture containing 500 mM gp 120 or EA2 peptides and 51 mM UDP-[ 14 C]GalNAc (7.8 mCi/mmol, PerkinElmer Life Sciences, Waltham, MA, USA) in 10 mM MnCl 2 , 40 mM sodium cacodylate (pH 6.6), 40 mM b-mercaptoethanol, and 0.1% Triton X-100 as described.…”

Section: Cytoimmunochemistry and Laser Confocal Microscopymentioning

confidence: 99%

Mechanism of FGF23 processing in fibrous dysplasia

Bhattacharyya

Wiench

Dumitrescu

et al. 2012

Journal of Bone and Mineral Research

107

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Fibroblast growth factor-23 (FGF23) is a phosphate-and vitamin D-regulating hormone derived from osteoblasts/osteocytes that circulates in both active (intact, iFGF23) and inactive (C-terminal, cFGF23) forms. O-glycosylation by O-glycosyl transferase Nacetylgalactosaminyltransferase 3 (ppGalNAcT3) and differential cleavage by furin have been shown to be involved in regulating the ratio of active to inactive FGF23. Elevated iFGF23 levels are observed in a number of hypophosphatemic disorders, such as X-linked, autosomal recessive, and autosomal dominant hypophosphatemic rickets, whereas low iFGF23 levels are found in the hyperphosphatemic disorder familial tumoral calcinosis/hyperphosphatemic hyperostosis syndrome. Fibrous dysplasia of bone (FD) is associated with increased total FGF23 levels (cFGF23 þ iFGF23); however, classic hypophosphatemic rickets is uncommon. Our results suggest that it can be explained by increased FGF23 cleavage leading to an increase in inactive cFGF23 relative to active iFGF23. Given the fact that FD is caused by activating mutations in the small G-protein G s a that results in increased cyclic adenosine monophosphate (cAMP) levels, we postulated that there may be altered FGF23 cleavage in FD and that the mechanism may involve alterations in cAMP levels and ppGalNacT3 and furin activities. Analysis of blood specimens from patients with FD confirmed that the elevated total FGF23 levels are the result of proportionally increased cFGF23 levels, consistent with less glycosylation and enhanced cleavage by furin. Analysis of primary cell lines of normal and mutation-harboring bone marrow stromal cells (BMSCs) from patients with FD demonstrated that BMSCs harboring the causative G s a mutation had higher cAMP levels, lower ppGalNAcT3, and higher furin activity. These data support the model wherein glycosylation by ppGalNAcT3 inhibits FGF23 cleavage by furin and suggest that FGF23 processing is a regulated process that controls overall FGF23 activity in FD patients. ß

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Cloning and Expression of Mouse UDP-GalNAc:PolypeptideN-Acetylgalactosaminyltransferase-T3

Cited by 60 publications

References 0 publications

Functional Conservation of Subfamilies of Putative UDP-N-acetylgalactosamine:Polypeptide N-Acetylgalactosaminyltransferases inDrosophila, Caenorhabditis elegans, and Mammals

Functional Conservation of Subfamilies of Putative UDP-N-acetylgalactosamine:Polypeptide N-Acetylgalactosaminyltransferases inDrosophila, Caenorhabditis elegans, and Mammals

Functional Characterization and Expression Analysis of Members of the UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferase Family from Drosophila melanogaster

Mechanism of FGF23 processing in fibrous dysplasia

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