Activity Detection of GalNAc Transferases by Protein-Based Fluorescence Sensors In Vivo

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2016 Sep 16

PMID 27632006

Authors

Lina Song

Collin Bachert

Adam D Linstedt

Affiliations

Soon will be listed here.

Abstract

Mucin-type O-glycosylation occurring in the Golgi apparatus is an important protein posttranslational modification initiated by up to 20 GalNAc-transferase isozymes with largely distinct substrate specificities. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and misregulation causes human diseases. Here we describe the use of protein-based fluorescence sensors that traffic in the secretory pathway to monitor GalNAc-transferase activity in living cells. The sensors can either be "pan" or isozyme specific.

References

Kathiresan S, Melander O, Guiducci C, Surti A, Burtt N, Rieder M . Six new loci associated with blood low-density lipoprotein cholesterol, high-density lipoprotein cholesterol or triglycerides in humans. Nat Genet. 2008; 40(2):189-97. PMC: 2682493. DOI: 10.1038/ng.75. View

Schjoldager K, Vester-Christensen M, Goth C, Petersen T, Brunak S, Bennett E . A systematic study of site-specific GalNAc-type O-glycosylation modulating proprotein convertase processing. J Biol Chem. 2011; 286(46):40122-32. PMC: 3220544. DOI: 10.1074/jbc.M111.287912. View

Song L, Bachert C, Schjoldager K, Clausen H, Linstedt A . Development of isoform-specific sensors of polypeptide GalNAc-transferase activity. J Biol Chem. 2014; 289(44):30556-30566. PMC: 4215235. DOI: 10.1074/jbc.M114.599563. View

Bachert C, Linstedt A . A sensor of protein O-glycosylation based on sequential processing in the Golgi apparatus. Traffic. 2012; 14(1):47-56. PMC: 3548014. DOI: 10.1111/tra.12019. View

Kato K, Jeanneau C, Tarp M, Benet-Pages A, Lorenz-Depiereux B, Bennett E . Polypeptide GalNAc-transferase T3 and familial tumoral calcinosis. Secretion of fibroblast growth factor 23 requires O-glycosylation. J Biol Chem. 2006; 281(27):18370-7. DOI: 10.1074/jbc.M602469200. View

Kong Y, Joshi H, Schjoldager K, Madsen T, Gerken T, Vester-Christensen M . Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis. Glycobiology. 2014; 25(1):55-65. PMC: 4245906. DOI: 10.1093/glycob/cwu089. View

Bennett E, Mandel U, Clausen H, Gerken T, Fritz T, Tabak L . Control of mucin-type O-glycosylation: a classification of the polypeptide GalNAc-transferase gene family. Glycobiology. 2011; 22(6):736-56. PMC: 3409716. DOI: 10.1093/glycob/cwr182. View

Yoshida A, Suzuki M, Ikenaga H, Takeuchi M . Discovery of the shortest sequence motif for high level mucin-type O-glycosylation. J Biol Chem. 1997; 272(27):16884-8. DOI: 10.1074/jbc.272.27.16884. View

Szent-Gyorgyi C, Stanfield R, Andreko S, Dempsey A, Ahmed M, Capek S . Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces. J Mol Biol. 2013; 425(22):4595-613. PMC: 3919518. DOI: 10.1016/j.jmb.2013.08.014. View

10.

Gerken T, Jamison O, Perrine C, Collette J, Moinova H, Ravi L . Emerging paradigms for the initiation of mucin-type protein O-glycosylation by the polypeptide GalNAc transferase family of glycosyltransferases. J Biol Chem. 2011; 286(16):14493-507. PMC: 3077648. DOI: 10.1074/jbc.M111.218701. View

11.

Schjoldager K, Vester-Christensen M, Bennett E, Levery S, Schwientek T, Yin W . O-glycosylation modulates proprotein convertase activation of angiopoietin-like protein 3: possible role of polypeptide GalNAc-transferase-2 in regulation of concentrations of plasma lipids. J Biol Chem. 2010; 285(47):36293-303. PMC: 2978557. DOI: 10.1074/jbc.M110.156950. View

12.

Boskovski M, Yuan S, Pedersen N, Goth C, Makova S, Clausen H . The heterotaxy gene GALNT11 glycosylates Notch to orchestrate cilia type and laterality. Nature. 2013; 504(7480):456-9. PMC: 3869867. DOI: 10.1038/nature12723. View

13.

Gill D, Tham K, Chia J, Wang S, Steentoft C, Clausen H . Initiation of GalNAc-type O-glycosylation in the endoplasmic reticulum promotes cancer cell invasiveness. Proc Natl Acad Sci U S A. 2013; 110(34):E3152-61. PMC: 3752262. DOI: 10.1073/pnas.1305269110. View

14.

van Der Post S, Subramani D, Backstrom M, Johansson M, Vester-Christensen M, Mandel U . Site-specific O-glycosylation on the MUC2 mucin protein inhibits cleavage by the Porphyromonas gingivalis secreted cysteine protease (RgpB). J Biol Chem. 2013; 288(20):14636-14646. PMC: 3656315. DOI: 10.1074/jbc.M113.459479. View

15.

Schjoldager K, Clausen H . Site-specific protein O-glycosylation modulates proprotein processing - deciphering specific functions of the large polypeptide GalNAc-transferase gene family. Biochim Biophys Acta. 2012; 1820(12):2079-94. DOI: 10.1016/j.bbagen.2012.09.014. View

16.

Moremen K, Tiemeyer M, Nairn A . Vertebrate protein glycosylation: diversity, synthesis and function. Nat Rev Mol Cell Biol. 2012; 13(7):448-62. PMC: 3934011. DOI: 10.1038/nrm3383. View

17.

Szent-Gyorgyi C, Schmidt B, Schmidt B, Creeger Y, Fisher G, Zakel K . Fluorogen-activating single-chain antibodies for imaging cell surface proteins. Nat Biotechnol. 2007; 26(2):235-40. DOI: 10.1038/nbt1368. View