Name: targeting cysteine thiols for in vitro site-specific glycosylation of recombinant proteins
Uploaded: 2017-10-04T12:00:00
Description: Watch this Scientific Journal Video about Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins at JoVE.com

This research was supported by the Natural Sciences and Engineering Research Council of Canada (05239 to P.B.S.), Canadian Foundation for Innovation/Ontario Research Fund (to P.B.S.), Prostate Cancer Fight Foundation - Telus Ride for Dad (to P.B.S.) and Ontario Graduate Scholarship (to Y.J.C. and N.S.).

1. Polymerase chain reaction (PCR)-mediated site-directed mutagenesis for the incorporation of Cys into a bacterial pET-28a expression vector.
<ol>
	<li>Determine the concentration of the pET-28a vector (i.e. double stranded DNA) using a ultraviolet (UV) extinction coefficient of 0.020 (&#956;g/mL) cm-1 at 260 nm.</li>
	<li>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...

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...

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-&#947; (PLC&#947;) 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 &#945;-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.

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

targeting cysteine thiols for in vitro site-specific glycosylation of recombinant proteins

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.

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...

Watch this Scientific Journal Video about Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins at JoVE.com

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

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.

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

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

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