Bump-and-hole engineering of human polypeptide N-acetylgalactosamine transferases to dissect their protein substrates and glycosylation sites in cells

. 2023 Jan 11;4(1):101974. doi: 10.1016/j.xpro.2022.101974

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

IRDye® 800CW Donkey anti-rabbit IgG (used at 1:10000)	LI-COR Biosciences (Lincoln, USA)	LI-COR Biosciences Cat# 926-32213; RRID AB_621848
Rabbit anti-FLAG (used at 1:1000)	Thermo Fisher Scientific (Waltham, USA)	Thermo Fischer Scientific Cat# PA1-984B; RRID: AB_347227
Rabbit anti-VSV-G (used at 1:500)	Thermo Fisher Scientific	Thermo Fischer Scientific Cat# PA1-29903; RRID: AB_1961363

Chemicals, peptides, and recombinant proteins

Ac₄GalN6yne (compound 3)	Cioce et al.³	N/A
Ac₄ManNAl	Click Chemistry Tools (Scottsdale, USA)	Click Chemistry Tools Cat# 1154-100; CAS No. 935658-93-8
Benzonase® Nuclease	Merck (Darmstadt, Germany)	Merck Cat# E1014-5KU; CAS No. 9025-65-4
Biotin-DADPS-picolyl azide	Schumann et al.¹; subsequently custom synthesized from Sussex Research Laboratories (Ottawa, CA)	N/A
BTTAA	Jena Bioscience (Jena, Germany)	Jena Bioscience Cat# CLK-067-25; CAS No. 1334179-85-9
Bis(S-acetyl-2-thioethyl) 3,4,6-tri-O-acetyl-2-deoxy-2-(5-hexynoyl)amido-a-Dgalactopyranosyl phosphate (compound 2)	Schumann et al.¹	N/A
CF680 picolyl azide	Biotium (Fremont, USA)	Biotium Cat# 96003
CloneAmp™ HiFi PCR Premix	Takara Bio (Kusatsu, Japan)	Takara Cat# 639298
EA2 peptide PTTDSTTPAPTTK	AnaSpec (Fremont, USA)	AnaSpec Cat# AS-63841
Halt™ Protease Inhibitor Cocktail (100×)	Thermo Fisher Scientific	Thermo Fisher Scientific Cat# 1861279
Intercept (TBS) Blocking Buffer	LI-COR Biosciences	LI-COR Biosciences Cat# 927-60001
IRDye® 800CW Streptavidin (used at 1:5000)	LI-COR Biosciences	LI-COR Biosciences Cat# 926-32230
Lipofectamine™ LTX Reagent with PLUS™ Reagent	Thermo Fisher Scientific	Thermo Fisher Scientific Cat# 15338100
Lys-C, Mass Spec Grade	Promega (Madison, USA)	Promega Cat# VA1170
Ni-NTA® Agarose beads	Qiagen (Hilden, Germany)	Qiagen Cat# 30210
Nuclease free water	Thermo Fisher Scientific	Thermo Fischer Scientific Cat# AM9937
Opti-MEM® I Reduced Serum Medium	Thermo Fisher Scientific	Thermo Fischer Scientific Cat# 31985062
PNGase F	Promega	Promega Cat# V4831A
PUGNAc	Sigma-Aldrich (St. Louis, USA)	Sigma Cat# A7229; CAS No. 132489-69-1
RapiGest SF	Waters (Milford, USA)	Waters Cat# 186002122
SafeBlue Protein Stain	NBS Biologicals (Huntingdon, UK)	NBS Biologicals Cat# NBS-SB1L
Sera-Mag SpeedBeads Neutravidin-Coated Magnetic Beads	Cytiva (Marlborough, USA)	Cytiva Cat# 78152104011150
SfiI restriction enzyme	New England Biolabs (Ipswich, USA)	New England Biolabs Cat# R0123S
Soluble His₆-tagged BH-T2 protein	Schumann et al.¹	N/A
Trypsin Gold, mass spectrometry grade	Promega	Promega Cat# V528A
Uridine 5′-diphospho-N-acetylgalactosamine disodium salt (UDP-GalNAc)	Sigma-Aldrich	Sigma Aldrich Cat# U5252; CAS No. 108320-87-2
Uridine 5′-diphospho-2-deoxy-2-(5-hexynoyl)amido-α-D-galactopyranoside disodium salt (compound 1)	Choi et al.¹²	N/A

Critical commercial assays

ExpiFectamine™ 293 Transfection Kit	Thermo Fischer Scientific	Thermo Fischer Scientific Cat# A14525
In-Fusion HD Cloning Kit	Takara Bio	Takara Bio Cat# 102518
NucleoSpin® Gel and PCR Clean-up Kit	Macherey-Nagel (Düren, Germany)	Macherey-Nagel Cat# 740609.50
Pierce™ BCA Protein Assay Kit	Thermo Fischer Scientific	Thermo Fischer Scientific Cat# 23227
Q5® Site-Directed Mutagenesis Kit	New England Biolabs	New England Biolabs Cat# E0554S
QIAprep Spin Miniprep Kit	Qiagen	Qiagen Cat# 27106
Revert™ 700 Total Protein Stain Kit	LI-COR Bioscience	LI-COR Bioscience Cat# 926-11016
Trans-Blot Turbo RTA Midi 0.2 μm Nitrocellulose Transfer Kit	Bio-Rad Laboratories (Hercules, USA)	Bio-Rad Laboratories Cat# 1704271
ZymoPURE™ II Plasmid Maxiprep Kit	Zymo Research (Irvine, USA)	Zymo Research Cat# D4203

Deposited data

Crystal structure BH-T2/EA2/UDP/Mn2+	Schumann et al.¹	PDB: 6E7I
Crystal structure BH-T2/compound 1/Mn2+	Schumann et al.¹	PDB: 6NQT
Gel and blot full images	Schumann et al.¹	https://doi.org/10.17632/nh4vww6hxj.2
Glycoproteomics raw data	Schumann et al.¹	PRIDE accession ID: PXD018048
Proteomics raw data	Schumann et al.¹	PRIDE accession ID: PXD017989

Experimental models: Cell lines

Expi293F™ cells	Thermo Fischer Scientific	Thermo Fischer Scientific Cat# A14527
K-562	Laboratory of Jonathan Weissman, UCSF	N/A
K-562 pSBbi-WT-hAGX1-BH-T1	Schumann et al.¹	N/A
K-562 pSBbi-mut-hAGX1-WT-T1	Schumann et al.¹	N/A
K-562 pSBbi-mut-hAGX1-BH-T1	Schumann et al.¹	N/A
K-562 pSBbi-WT-hAGX1-BH-T2	Schumann et al.¹	N/A
K-562 pSBbi-mut-hAGX1-WT-T2	Schumann et al.¹	N/A
K-562 pSBbi-mut-hAGX1-BH-T2	Schumann et al.¹	N/A
MCF7 cells	ATCC (Manassas, USA)	ATCC Cat# HTB-22; RRID: CVCL_0031
MCF7 pSBbi-mut-hAGX1-NahK-BH T2	Cioce et al.³	N/A
Stellar™ Competent Cells	Takara Bio	Takara Bio Cat# 636763

Oligonucleotides

T1 and T2 site-directed mutagenesis primers	Choi et al.¹²	N/A
T1 and T2 In-Fusion primers	Schumann et al.¹	N/A

Recombinant DNA

pCMV(CAT)T7-SB100	Addgene (Watertown, USA)	Addgene Cat# 34879; RRID: Addgene_34879
pDONR221	Thermo Fischer Scientific	Thermo Fischer Scientific Cat# 12536017
pGEN2-DEST	Moremen et al.³⁰	N/A
pOPING-trunc-BH-T2	Schumann et al.¹	N/A
pSBbi-WT-hAGX1-BH-T1	Schumann et al.¹	N/A
pSBbi-mut-hAGX1-WT-T1	Schumann et al.¹	N/A
pSBbi-mut-hAGX1-BH-T1	Schumann et al.¹	N/A
pSBbi-WT-hAGX1-BH-T2	Schumann et al.¹	N/A
pSBbi-mut-hAGX1-WT-T2	Schumann et al.¹	N/A
pSBbi-mut-hAGX1-BH-T2	Schumann et al.¹	N/A

Software and algorithms

Byonic	Protein Metrics (Cupertino, USA)	N/A
GraphPad Prism (non-linear Michaelis-Menten and kcat fitting programs)	Dotmatics (Boston, USA)	N/A
Image Studio	LI-COR Bioscience	N/A
Perseus	Tyanova et al.³²	N/A
PyMOL	Schrödinger, Inc. (New York, USA)	N/A
MaxQuant	Cox et al.³³	N/A
SnapGene	Dotmatics	N/A

Other

4%–20% Criterion™ TGX™ Precast Midi Protein Gel, 12+2 well, 45 μL	Bio-Rad Laboratories	Bio-Rad Laboratories Cat# 5671093
4%–20% Criterion™ TGX™ Precast Midi Protein Gel, 18 well, 30 μL	Bio-Rad Laboratories	Bio-Rad Laboratories Cat# 5671094
4%–20% Criterion™ TGX™ Precast Midi Protein Gel, 26 well, 15 μL	Bio-Rad Laboratories	Bio-Rad Laboratories Cat# 5671095
Steritop Threaded Bottle Top Filter	Sigma-Aldrich	Sigma Aldrich Cat# S2GPT01RE
Amicon Ultra-15 Centrifugal Filters (3 kDa MWCO)	Merck	Merck Cat# UFC900308
Protein LoBind® Tubes, 1.5 mL	Eppendorf (Hamburg, Germany)	Eppendorf Cat# 0030108116
Nalgene™ 0.2 μM PES syringe filter	Thermo Fisher Scientific	Thermo Fisher Scientific Cat# 720-1320
Odyssey CLx	LI-COR Bioscience	LI-COR Bioscience Model # 9140
SnakeSkin™ Dialysis Tubing, 10K MWCO, 16 mm	Thermo Fisher Scientific	Thermo Fisher Scientific Cat# 88243
UltraMicroSpin™ columns	The Nest group, Inc. (Ipswich, USA)	The Nest group, Inc. Cat# SUM SS18V