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). IP₃ is a small diffusible messenger which binds to IP₃ receptors (IP₃R) on the endoplasmic reticulum (ER) thereby opening this receptor channel and permitting Ca²⁺ to flow down the concentration gradient from the ER lumen to the cytosol (reviewed in ⁴). Receptor signaling from G protein coupled and tyrosine kinase receptors in a variety of other excitable and non-excitable cell types lead to the same production of IP₃ and activation of IP₃Rs.

Due to the finite Ca²⁺ storage capacity of the ER, the IP₃-mediated release and resultant increase in cytosolic Ca²⁺ is only transient; however, this depletion of the ER luminal Ca²⁺ profoundly effects stromal interaction molecule-1 (STIM1), a type-I transmembrane (TM) protein mostly found on the ER membrane ^5,6,7. STIM1 contains a lumen-oriented Ca²⁺ sensing domain made up of an EF-hand pair and sterile α-motif (EFSAM). Three cytosolic-oriented coiled-coil domains are separated from EFSAM by the single TM domain (reviewed in ⁸). Upon ER luminal Ca²⁺ depletion, EFSAM undergoes a destabilization-coupled oligomerization ^7,9 which causes structural rearrangements of the TM and coiled-coil domains ¹⁰. These structural changes culminate in a trapping of STIM1 at ER-PM junctions ^11,12,13,14 through interactions with PM phosphoinositides ^15,16 and Orai1 subunits ^17,18. Orai1 proteins are the PM subunits which assemble to form Ca²⁺ channels ^19,20,21,22. The STIM1-Orai1 interactions at ER-PM junctions facilitate an open Ca²⁺ release activated Ca²⁺ (CRAC) channel conformation which enables the movement of Ca²⁺ into the cytosol from the high concentrations of the extracellular space. In immune cells, the sustained cytosolic Ca²⁺ elevations via CRAC channels induce the Ca²⁺-calmodulin/calcineurin dependent dephosphorylation of the nuclear factor of activated T-cells which subsequently enters the nucleus and begins transcriptional regulation of genes promoting T-cell activation ^1,3. The process of CRAC channel activation by STIM1 ^23,24 via agonist-induced ER luminal Ca²⁺ depletion and the resulting sustained cytosolic Ca²⁺ elevation is collectively termed SOCE ²⁵. The vital role of SOCE in T-cells is evident by studies demonstrating that heritable mutations in both STIM1 and Orai1 can cause severe combined immunodeficiency syndromes ^3,19,26,27. EFSAM initiates SOCE after sensing ER-luminal Ca²⁺ depletion via the loss of Ca²⁺ coordination at the canonical EF-hand, ultimately leading to the destabilization-coupled self-association ^7,28,29.

Glycosylation is the covalent attachment and processing of oligosaccharide structures, also known as glycans, through various biosynthetic steps in the ER and Golgi (reviewed in ^30,32,33). There are two predominant types of glycosylation in eukaryotes: N-linked and O-linked, depending on the specific amino acid and atom bridging the linkage. In N-glycosylation, glycans are attached to the side chain amide of Asn, and in most cases, the initiation step occurs in the ER as the polypeptide chain moves into the lumen ³⁴. The first step of N-glycosylation is the transfer of a fourteen-sugar core structure made up of glucose (Glc), mannose (Man), and N-acetylglucosamine (GlcNAc) (i.e. Glc₃Man₉GlcNAc₂) from an ER membrane lipid by an oligosaccharyltransferase ^35,36. Further steps, such as cleavage or transfer of glucose residues, are catalyzed in the ER by specific glycosidases and glycosyltransferases. Some proteins that leave the ER and move into the Golgi can be further processed ³⁷. O-glycosylation refers to the addition of glycans, usually to the side chain hydroxyl group of Ser or Thr residues, and this modification occurs entirely in the Golgi complex ^33,34. There are several O-glycan structures which can be made up of N-acetylglucosamine, fucose, galactose, and sialic acid with each monosaccharide added sequentially ³³.

While no specific sequence has been identified as prerequisite for many types of O-glycosylation, a common consensus sequence has been associated with the N-linked modification: Asn-X-Ser/Thr/Cys, where X can be any amino acid except Pro ³³. STIM1 EFSAM contains two of these consensus N-glycosylation sites: Asn131-Trp132-Thr133 and Asn171-Thr172-Thr173. Indeed, previous studies have shown that EFSAM can be N-glycosylated in mammalian cells at Asn131 and Asn171 ^38,39,40,41. However, previous studies of the consequences of N-glycosylation on SOCE have been incongruent, suggesting suppressed, potentiated or no effect by this post-translational modification on SOCE activation ^38,39,40,41. Thus, research on the underlying biophysical, biochemical, and structural consequences of EFSAM N-glycosylation is vital to comprehending the regulatory effects of this modification. Due to the requirement for high levels of homogeneous proteins in these in vitro experiments, a site-selective approach to covalently attach glucose moieties to EFSAM was applied. Interestingly, Asn131 and Asn171 glycosylation caused structural changes that converge within the EFSAM core and enhance the biophysical properties which promote STIM1-mediated SOCE ⁴².

The chemical attachment of glycosyl groups to Cys thiols has been well-established by a seminal work which first demonstrated the utility of this enzyme-free approach to understanding the site-specific effects of glycosylation on protein function ^43,44. More recently and with respect to STIM1, the Asn131 and Asn171 residues were mutated to Cys and glucose-5-(methanethiosulfonate) [glucose-5-(MTS)] was used to covalently link glucose to the free thiols ⁴². Here, we describe this approach which not only uses mutagenesis to incorporate site specific Cys residues for modification, but also applies solution nuclear magnetic resonance (NMR) spectroscopy to rapidly assess both modification efficiency and structural perturbations as a result of the glycosylation. Notably, this general methodology is easily adaptable to study the effects of either O– or N-glycosylation of any recombinantly produced protein.

Protocol

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

Representative 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<sup cla…

Materials

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.