Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Summary

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Abstract

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca²⁺) entry which is the principal Ca²⁺ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca²⁺ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Introduction

Store operated calcium (Ca²⁺) entry (SOCE) is the major pathway by which immune cells take up Ca²⁺ from the extracellular space into the cytosol. In T lymphocytes, T cell receptors located on the plasma membrane (PM) bind antigens which activate protein tyrosine kinases (reviewed in ^1,2,3). A phosphorylation cascade leads to the activation of phospholipase-γ (PLCγ) which subsequently mediates the hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate (PIP₂) into diacylglycerol and inositol 1,4,5-trisphosphate (IP₃). ....

Protocol

1. Polymerase chain reaction (PCR)-mediated site-directed mutagenesis for the incorporation of Cys into a bacterial pET-28a expression vector.

1. Determine the concentration of the pET-28a vector (i.e. double stranded DNA) using a ultraviolet (UV) extinction coefficient of 0.020 (μg/mL) cm^-1 at 260 nm.
2. Synthesize a pair of complementary mutagenic primers for each Cys mutation such that i) there are a minimum of 15 nucleotides complementary to the template prior to the first base mismatch and 15 nucleotides complementary to the template after the final base mismatch, ii) total primer length does not exceed 45 nucleotides,....

Results

The first step of this approach requires the mutagenesis of the candidate glycosylation residues to Cys residues which can be modifiable using the glucose-5-MTS. EFSAM has no endogenous Cys residues, so no special considerations need to be made prior to the mutagenesis. However, native Cys residues must be mutated to non-modifiable residues prior to performing the described chemistry. To minimally effect the native structure, we suggest performing a global sequence alignment of the protei.......

Discussion

Protein glycosylation is a post-translational modification where sugars are covalently attached to polypeptides primarily through linkages to amino acid side chains. As many as 50% of mammalian proteins are glycosylated ⁵⁴, where the glycosylated proteins can subsequently have a diverse range of effects from altering biomolecular binding affinity, influencing protein folding, altering channel activity, targeting molecules for degradation and cellular trafficking, to name a few (reviewed in

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phusion DNA Polymerase	Thermo Fisher Scientific	F530S	Use in step 1.3.
Generuler 1kb DNA Ladder	Thermo Fisher Scientific	FERSM1163	Use in step 1.6.
DpnI Restriction Enzyme	New England Biolabs, Inc.	R0176	Use in step 1.8.
Presto Mini Plasmid Kit	GeneAid, Inc.	PDH300	Use in step 1.16.
BL21 DE3 codon (+) E. coli	Agilent Technologies, Inc.	230280	Use in step 2.1.
DH5a E. coli	Invitrogen, Inc.	18265017	Use in step 1.9.
0.22 mm Syringe Filter	Millipore, Inc.	SLGV033RS	Use in step 2.3.
HisPur Ni2+-NTA Agarose Resin	Thermo Fisher Scientific	88221	Use in step 3.3.
3,500 Da MWCO Dialysis Tubing	BioDesign, Inc.	D306	Use in step 3.8, 3.16, 4.2, 4.5 and 4.6.
Bovine Thrombin	BioPharm Laboratories, Inc.	SKU91-055	Use in step 3.9.
5 mL HiTrap Q FF Anion Exchange Column	GE Healthcare, Inc.	17-5156-01	Use in step 3.11.
Glucose-5-MTS	Toronto Research Chemicals, Inc.	G441000	Use in step 4.1.
Vivaspin 20 Ultrafiltration Centrifugal Concentrators	Sartorius, Inc.	VS2001	Use in step 3.11, 4.2, 4.5 and 4.6.
PageRuler Unstained Broad Protein Ladder	Thermo Fisher Scientific	26630	Use in step 3.7, 3.10 and 3.15
HiTrap Q FF Anion Exchange Column	GE Healthcare, Inc.	17-5053-01	Use in step 3.12.
AKTA Pure Fast Protein Liquid Chromatrography System	GE Healthcare, Inc.	29018224	Use in step 3.14.
600 MHz Varian Inova NMR Spectrometer	Agilent Technologies, Inc.		Use in step 5.2 and 5.5.