X-ray diffraction experiments described in this paper were performed using beamlines 08-ID and 08-BM at the Canadian Light Source, which is supported by the Canada Foundation for Innovation, Natural Sciences and Engineering Research Council of Canada, the University of Saskatchewan, the Government of Saskatchewan, Western Economic Diversification Canada, the National Research Council Canada, and the Canadian Institutes of Health Research. We would like to acknowledge the Structural &#38; Biophysical Core Facility, The Hospital for Sick Children, for access to the ITC and BLI instruments. J.E.O. was supported by Banting Postdoctoral Fellowship BPF-144483 from the Canadian Institutes of Health Research. T.S. is a recipient of a Canada Graduate Scholarship Master&#39;s Award and a Vanier Canada Graduate Scholarship&#160;from the Canadian Institutes of Health Research. This work was supported by operating grant PJT-148811 (J.-P.J.) from the Canadian Institutes of Health Research. This research was undertaken, in part, thanks to funding from the Canada Research Chairs program (J.-P.J.).

The authors declare no competing interests.

Membrane-anchored glycoproteins are critical for cell function and are attractive therapeutic targets. Here, we present a protocol for the structural and biophysical characterization of the ECD of membrane glycoproteins, both alone and in complex with small molecule ligands and Fab fragments. We have successfully used this protocol to determine the crystal structure of the three N-terminal-most Ig domains of the extracellular portion of human CD2228, a critical co-receptor on B cells involved in keeping humoral immunity in check79. We have also characterized the binding site of CD22 with its natural ligand &#945;2-6 sialyllactose, and defined the mode of recognition of a therapeutic antibody towards human CD22. These results provide insights into the structure-function relationship of a key member of the Siglecs family that has restricted expression on B cells, and a molecular roadmap towards the development of new CD22 targeted small molecule and antibody-based therapeutics. While this protocol was used successfully for an Ig-containing B cell receptor, we propose that our approach can be applied for the structural and biophysical characterization of any membrane glycoprotein with a distinct domain organization. In such cases, construct design and combinatorial N-linked glycan mutations (either to Gln or Ala) can be evaluated to find a construct suitable for crystal growth and high-resolution diffraction.
Obtaining a homogeneous and pure glycoprotein sample is of critical importance for crystal growth and X-ray diffraction, as well as for downstream biophysical characterization. N-linked glycans present on glycoproteins are inherently heterogeneous, and can cause conformational and chemical heterogeneity within the glycoprotein that can deter crystal formation. To reduce this micro-heterogeneity, strategies that introduce point mutations to remove Asn residues predicted to harbor N-linked glycans, or using mutant cell lines (such as HEK293S) followed by treatment with endoglycosidases (such as EndoH) can considerably improve crystallization success15,21,22. In this protocol, we discuss the purification of soluble glycoproteins and Fabs that are secreted into the cell supernatant. Glycoprotein secretion provides a relatively simple route towards purity, without the need for cell lysis or the addition of harsh chemicals or detergents. The cell supernatant, obtained following cell harvesting is then run directly over a column that has affinity for the protein of interest (e.g., Ni-NTA for His-tagged glycoproteins, or LC affinity for Fab fragments). However, depending on the column of use and the conditions of the cell supernatant (e.g., pH), the binding capability of the protein of interest to the column may be affected. If this is the case, it may be necessary to concentrate and buffer exchange the cell supernatant to improve binding to the column. Furthermore, it is strongly recommended that quality control steps during purification be employed to help assess protein purity. Running an SDS-PAGE gel or Western blot of all samples (before, during and after purification steps) can yield insights into whether the proposed purification scheme is suitable for the protein of interest. If contaminating bands are visible on SDS-PAGE, or if several species are obtained during purification (e.g., several peaks on size exclusion), additional purification steps should be considered, e.g., ion exchange chromatography, to gain in purity and increase chances of downstream crystallization80.
For macromolecular crystallization, it is often critical to obtain high yields of the protein of interest to allow for the screening of a large number of potential crystallization conditions at high protein concentrations to find suitable crystal hits. Generally, the HEK293 cell lines discussed here (HEK293F and HEK293S) are robust expression systems, and can be easily scaled up to produce more sample as necessary. However, it is possible that the protein of interest may not express sufficiently within these cell lines. In these cases, other cell lines, such as Expi293 cells81,82, have been found to show superior levels of protein expression and should be considered as an alternative.
If well-ordered, diffracting crystals are not obtained following testing of several constructs of the protein of interest despite high purity, it may be necessary to expand crystallization techniques to promote crystal formation. It has been shown that Fab fragments of antibodies and nanobodies can be&#160;excellent crystallization enhancers, and promote well-ordered crystal packing83,84,85. These fragments can be expressed and purified to homogeneity, and used in a complex with the protein of interest to promote crystallization. Importantly, Fab fragments produced as described in Section 10 can have a tendency to form non-functional LC dimers86. These dimers are contaminants and should be removed during purification. In our experience, LC dimers often have a different retention volume on size exclusion, or elute as a distinct peak on ion exchange chromatography, and thus can be removed from the Fab purification - however this is not always the case. If these techniques are insufficient to remove LC dimers from the Fab purification, additional purification methods, such as Protein G affinity purification, can be employed to improve purity.
Alternative to co-complexation with Fab fragments, well-documented techniques such as random matrix microseeding can improve chances of obtaining well-ordered crystals63,70. This method involves the addition of small amounts of crushed, suboptimal crystals into the crystallization condition, providing a crystal nucleate to promote crystal growth. This can be performed using crystals of the protein of interest, or ones with similar domain architecture and tertiary structure. Furthermore, random matrix microseeding can&#160;be performed in attempts to crystallize the protein alone, or in complex with a Fab fragment or small molecule of interest. Recent advances in cryo-electron microscopy also make this technique an attractive alternative to X-ray crystallography for obtaining high-resolution structural information for molecules with appropriate features87,88,89,90,91.
When phasing of X-ray diffraction datasets fails by MR, HA soaking may be needed to solve the phase problem by anomalous dispersion or isomorphous replacement. Inspection of the amino acid sequence of the protein can provide clues about the strategy for HA derivatization, including optimum pH for binding. In particular, unpaired cysteines within the protein can specifically bind HA compounds that contain mercury. Soaking native crystals with HA compounds is an iterative process to determine the identity of the optimal HA compound, its concentration, and the required incubation time. If initial soaking attempts do not yield well-diffracting crystals containing a HA suitable for phasing, it may be necessary to introduce amino acid substitutions to improve the probability of HA binding and improve anomalous signal. Examples include mutations to include a free cysteine residue to efficiently bind Hg, Au, Pt or Pb. Expression of proteins for anomalous phasing in a seleno-methionine supplemented media in E. coli is extensively used for anomalous phasing, however an equivalent system that reliably incorporates seleno-methionine is not readily available for mammalian cells in suspension92,93, and is an area of future development.
Once the unliganded structure of the glycoprotein of interest is obtained, soaking the crystals with small molecule ligands can be performed to obtain a structure of the immune receptor-ligand complex. These data provide a blueprint for the rational design of more specific and high-affinity ligands that can be used as small-molecule therapeutics as well as provides high resolution insights into the biological function of the glycoprotein. When attempting to soak glycoprotein crystals with small molecule ligands of interest, inspection of the unliganded crystal structure can indicate whether soaking should be possible. If close crystal-packing contacts are found around the ligand-binding site or around regions expected to undergo conformational changes upon ligand binding, soaking will likely be problematic. In this case, other methods such as co-crystallization of the protein-ligand complex should be performed.

Surface proteins play critical roles in cellular function. Through their extracellular domains, these membrane proteins can modulate cell-cell interactions, adhesion, transport and signalling1,2. The extracellular localization of these proteins makes them attractive targets for the development of therapeutics for the treatment of a wide range of diseases, including cancer and autoimmune diseases3,4,5,6,7. One of the most common folds of human membrane protein ectodomains is the immunoglobulin-like (Ig) fold, which is formed by seven or more &#946;-strands arranged into two &#946;-sheets8,9. Typically, Ig-containing glycoproteins are multi-domain structures with Ig domains sequentially arranged on the extracellular portion of the membrane protein10. Post-translational modifications of these cell-surface proteins, particularly N- and O-linked glycosylation, have been shown to play essential roles in their regulation, folding, secretion and function11. To improve our understanding of their function and to better design therapeutics that can target them, techniques are required that allow for their detailed molecular characterization. Here, we present a combination of techniques that allow for the biophysical (biolayer interferometry (BLI) and isothermal titration calorimetry (ITC)) and structural (X-ray crystallography) characterization of the extracellular domain of Ig-containing membrane glycoproteins, alone and&#160; in complex with their biologically relevant ligands and therapeutic molecules (Figure 1).
N-linked glycosylation is one of the most common post-translation modifications on mammalian proteins, and occurs during protein maturation within the endoplasmic reticulum and Golgi12,13. Cell lines, such as human embryonic kidney (HEK) 293 cells, have been developed for the recombinant expression of large quantities of glycosylated mammalian proteins14,15. This cell line has been developed in a suspension format, which allows for the ease of scaling up protein production to larger quantities&#160;in comparison to adherent cell lines. Here, we utilize two HEK293 cell lines: HEK293F and HEK293 Gnt I-/- (HEK293S), which differ by the absence of N-acetylglucosaminyl transferase I (Gnt I) in the latter. In turn, production of complex glycans (as seen in HEK293F) is not possible and instead high mannose-type glycans (predominantly Man5GlcNAc2) reside at&#160;N-linked glycan sites18,19,20. Using these two cell lines in parallel allows studying the effect of glycan size and complexity on biological function and therapeutic targeting. Indeed, glycoproteins produced in HEK293F cells will have larger, more complex glycans compared to the same glycoprotein produced in HEK293S cells. Glycoproteins produced in HEK293S cells are more amenable to crystallization, because of the reduced chemical and conformational heterogeneity of their N-linked glycans. To further improve crystallizability, glycoproteins produced in HEK293S (but not HEK293F) cells can be treated with the enzyme endoglycosidase H (Endo H), which results in the cleavage of high mannose glycans such that only a single N-acetylglucosamine (GlcNAc) moiety remains at each N-linked glycosylation site21,22. Other methods can also be used to limit N-glycan processing within the cells, such as the addition of glycosyltransferase inhibitors during glycoprotein expression, including kifunensine23. Alternative approaches involve the expression of native glycoproteins (in HEK293F cells) followed by enzymatic deglycosylation using peptide N-glycosidase F (PNGaseF). However, deglycosylation with PNGaseF has been shown to be less effective under native conditions and increases aggregation in some proteins; in cases when the protein remains soluble after treatment, it acquires negative charges on its surface due to the deamidation of the asparagine residue to aspartic acid24, which might be detrimental for its crystallization. Predicted N-glycosylation sites can also be mutated, most often to alanine or glutamine residues, to prevent N-linked glycosylation at these sites and to generate glycoprotein samples of high homogeneity. Alternatively, glycoproteins can be produced in other eukaryotic cell cultures, including yeast, insect, and plant systems, or other mammalian cell lines such as Chinese hamster ovarian (CHO) cells16,17.
Many mammalian expression vectors, including pHLsec, allow for the secretion of recombinant glycoprotein ectodomains into the cell medium25. Secretion of glycoproteins from HEK293 cells allows for rapid and easy purification without the need for cell lysis. Addition of purification tags (e.g., His-tag, Strep-tag, Flag-tag, Myc-tag, HA-tag) to the N or C terminus of the target glycoprotein allows purification by a single-step affinity chromatography. Subsequently, size exclusion chromatography can be used to yield a monodisperse sample for biophysical and structural characterization.
A highly pure and homogeneous glycoprotein sample under appropriate conditions can result in well-diffracting crystals. Once a full X-ray diffraction dataset has been obtained from such crystals, initial phases need to be determined to calculate the electron density of the glycoprotein. Thanks to an ever increasing number of structures in the Protein Data Bank (PDB), the most commonly used method for phasing has by far become molecular replacement (MR), which uses a related protein structure to obtain initial phases26. However, when MR fails to solve the phase problem, as has occasionally been the case for multi-Ig domain glycoproteins27,28,29, alternative methods are required. In this article, we detail a method to soak crystals with heavy atoms (HA) for phasing, which was required for solving the structure of the CD22 ectodomain28. Identifying the right HA for phasing is an iterative process that depends on HA reactivity, available atoms in the glycoprotein in a given crystal lattice, and the crystallization solution30,31. Alternatively, natural sulfur atoms in cysteine and methionine residues can be used for phasing if present at a high enough ratio to other atoms in the glycoprotein, and if X-ray diffraction data can be collected with high enough redundancy32,33.
The biological function of membrane glycoproteins is often mediated by protein-protein interactions or protein-ligand interactions, such as with carbohydrates. When the ligand is small enough to diffuse from the solution to the glycoprotein binding site in the crystal lattice, soaking experiments can be successful to obtain a glycoprotein-ligand co-crystal structure to better understand ligand recognition.
The protocols presented here are also relevant for understanding the interactions of surface glycoproteins with synthetic therapeutic ligands34,35 and antibody therapeutics36,37. When combined with structural information, binding kinetics and thermodynamics can be powerful to understand and improve their mechanisms of action. One technique that allows for the kinetic analysis of therapeutic antibodies binding to a glycoprotein is BLI38,39. BLI uses biosensors with an immobilized ligand to measure the association and dissociation kinetics with a binding partner, ultimately determining an equilibrium dissociation constant (KD). BLI is an attractive approach because small amounts of glycoproteins are required (&#60;100 &#181;g), experiment time is fast (~10-15 min per run), and it can be automated. ITC is also useful for studying affinities between glycoproteins and binding partners40,41,42,43. While ITC is more time and reagent intensive, valuable information can be obtained regarding the thermodynamics of the interaction (&#916;G, &#916;H, &#916;S, and stoichiometry). ITC is also very useful for studying weak interactions that are often associated with the transient binding of surface glycoproteins to ligands. Furthermore, these techniques can be used in conjunction to evaluate the binding of various constructs and assess the effect of different N-linked glycoforms obtained from expressing the glycoprotein in different cell lines. Performing BLI and ITC with glycoproteins produced in HEK293F, HEK293S and treated with Endo H can provide an in-depth view of the role of glycans in biological activity and therapeutic engagement.
We successfully applied these protocols to characterize the extracellular domain (ECD) of human CD2228, a glycoprotein member of the sialic acid-binding Ig-like lectins (Siglecs) family that is essential for maintaining B-cell homeostasis44. We performed in-depth construct design to facilitate crystallization and phased the X-ray dataset by HA soaking with Hg. We also soaked CD22 crystals with its ligand sialic acid (&#945;2-6 sialyllactose) to obtain a structure of the immune receptor-ligand complex and thus provided the blueprints for the structure-guided design of glycan mimetics45,46. In addition, we generated the fragment antigen binding (Fab) of the anti-CD22 therapeutic antibody epratuzumab - a therapeutic candidate currently in phase III clinical trials for non-Hodgkin&#39;s lymphoma47- to determine its binding affinity by BLI and ITC to differentially glycosylated CD22 ECD constructs. These studies revealed a critical role for N-linked glycosylation in epratuzumab engagement, with potential implications for CD22 recognition on dysfunctional B cells.

<table><tbody><tr><td>0.22 &amp;mu;m Steritop filter</td><td>EMD Millipore</td><td>SCGPS02RE</td><td></td></tr><tr><td>10 well 4-15% gradient SDS-PAGE gel</td><td>Bio-Rad</td><td>4561084</td><td></td></tr><tr><td>10x glycobuffer 3</td><td>New England Biolabs</td><td>P0702S</td><td>Comes with Endo H reagent</td></tr><tr><td>10x Kinetics Buffer</td><td>PALL Fort&amp;eacute;Bio</td><td>18-1092</td><td></td></tr><tr><td>10x Tris/Glycine/SDS Buffer</td><td>Bio-Rad</td><td>1610732</td><td></td></tr><tr><td>1 mL round bottom 96 well block</td><td>ThermoFisher</td><td>260251</td><td></td></tr><tr><td>22 mm cover slip</td><td>Hampton research</td><td>HR3-231</td><td></td></tr><tr><td>4x Laemmli Sample Buffer</td><td>Bio-Rad</td><td>1610747</td><td></td></tr><tr><td>96-3 well INTELLIPLATE&amp;nbsp; low volume reservior</td><td>Art Robbins Instruments</td><td>102-0001-03</td><td></td></tr><tr><td>AgeI</td><td>New England Biolabs</td><td>R0552S</td><td></td></tr><tr><td>&amp;Auml;KTA Pure</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>&amp;Auml;KTA Start&amp;nbsp;</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Amicon Ultra 15 centrifugal filtration device 10KDa MWCO</td><td>Millipore</td><td>UFC901008</td><td></td></tr><tr><td>Amicon Ultra 4 centrifugal filtration device 10KDa MWCO</td><td>Millipore</td><td>UFC801008</td><td></td></tr><tr><td>Auto-iTC200</td><td>Malvern</td><td></td><td></td></tr><tr><td>Axygen MaxyClear Snaplock 1.5 mL microtubes</td><td>Fisher Scientific</td><td>MCT150C</td><td></td></tr><tr><td>Countess Cell Counting Chamber Slides</td><td>Thermo Fisher Scientific</td><td>C10228</td><td></td></tr><tr><td>CryoLoop 18 x 0.05-0.1 mm</td><td>Hampton research</td><td>HR4-945</td><td></td></tr><tr><td>CryoLoop 18 x 0.1-0.2 mm</td><td>Hampton research</td><td>HR4-947</td><td></td></tr><tr><td>CryoLoop 18 x 0.2-0.3 mm</td><td>Hampton research</td><td>HR4-970</td><td></td></tr><tr><td>Digital Dry Bath</td><td>Bio-Rad</td><td>1660562EDU</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; DH5&amp;alpha;</td><td>Invitrogen</td><td>18258012</td><td></td></tr><tr><td>Endo H</td><td>New England Biolabs</td><td>P0702S</td><td></td></tr><tr><td>Erlenmeyer flask (baffled base), polycarbonate, sterile, 500 mL, DuoCAP</td><td>TriForest Labware</td><td>FBC05000S</td><td></td></tr><tr><td>Erlenmeyer flask 125 mL (baffled base), polycarbonate, sterile, 125 mL with vented cap</td><td>VWR</td><td>89095-258</td><td></td></tr><tr><td>Falcon Disposable sterile serological pipet, non-pyrogenic, 10 mL</td><td>Greiner Bio-One</td><td>607180</td><td></td></tr><tr><td>Falcon Disposable sterile serological pipet, non-pyrogenic, 25 mL</td><td>Greiner Bio-One</td><td>760180</td><td></td></tr><tr><td>Falcon Disposable sterile serological pipet, non-pyrogenic, 5 mL</td><td>Greiner Bio-One</td><td>606180</td><td></td></tr><tr><td>Falcon Disposable sterile serological pipet, non-pyrogenic, 50 mL</td><td>Greiner Bio-One</td><td>768180</td><td></td></tr><tr><td>FectoPRO DNA Transfection Reagent, Polyplus</td><td>VWR</td><td>10118-842</td><td></td></tr><tr><td>Freestyle 293F cells</td><td>Thermo Fisher Scientific</td><td>R79007</td><td></td></tr><tr><td>Freestyle Expression medium</td><td>Thermo Fisher Scientific</td><td>12338001</td><td></td></tr><tr><td>Heavy Atom Screens Au</td><td>Hampton research</td><td>HR2-444</td><td></td></tr><tr><td>Heavy Atom Screens Hg</td><td>Hampton research</td><td>HR2-446</td><td></td></tr><tr><td>Heavy Atom Screens M1</td><td>Hampton research</td><td>HR2-448</td><td></td></tr><tr><td>Heavy Atom Screens M2</td><td>Hampton research</td><td>HR2-450</td><td></td></tr><tr><td>Heavy Atom Screens Pt</td><td>Hampton research</td><td>HR2-442</td><td></td></tr><tr><td>HEK 293S</td><td>ATCC</td><td>ATCC CRL-3022</td><td></td></tr><tr><td>HisTrap Affinity Column</td><td>GE Healthcare</td><td>17525501</td><td></td></tr><tr><td>HiTrap KappaSelect Affinity Columns</td><td>GE Healthcare</td><td>17545811</td><td></td></tr><tr><td>HiTrap LambdaSelect Affinity Columns</td><td>GE Healthcare</td><td>17548211</td><td></td></tr><tr><td>KpnI</td><td>New England Biolabs</td><td>R0142S</td><td></td></tr><tr><td>MCSG-1 Crystal Screen 1.7 mL block</td><td>Anatrace</td><td>MCSG-1</td><td></td></tr><tr><td>MCSG-2 Crystal Screen 1.7 mL block</td><td>Anatrace</td><td>MCSG-2</td><td></td></tr><tr><td>MCSG-3 Crystal Screen 1.7 mL block</td><td>Anatrace</td><td>MCSG-3</td><td></td></tr><tr><td>MCSG-4 Crystal Screen 1.7 mL block</td><td>Anatrace</td><td>MCSG-4</td><td></td></tr><tr><td>Mercuric chloride</td><td>Sigma</td><td>1044170100</td><td></td></tr><tr><td>Microplate, 96 well, polypropelene, flat bottom, black&amp;nbsp;</td><td>Greiner Bio-One</td><td>655209</td><td></td></tr><tr><td>Minstrel DT UV</td><td>Formulatrix</td><td></td><td></td></tr><tr><td>Multitron Pro shaker</td><td>Infors HT</td><td>MP25-TA-CO2HB</td><td></td></tr><tr><td>Nanodrop 2000/2000c Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>ND-2000</td><td></td></tr><tr><td>Nanosep 3K Omega centrifugal device</td><td>PALL Life Science</td><td>OD003C33</td><td></td></tr><tr><td>Ni-NTA biosensors</td><td>PALL Fort&amp;eacute;Bio</td><td>18-5102</td><td></td></tr><tr><td>Octet RED96</td><td>PALL ForteBio</td><td></td><td></td></tr><tr><td>Oryx 4 crystallizaiton robot</td><td>Douglas Instrument</td><td>ORY-4/1</td><td></td></tr><tr><td>Platinum chloride</td><td>Sigma</td><td>520632-1g</td><td></td></tr><tr><td>Precision Plus Protein Standard</td><td>Bio-Rad</td><td>161-0374</td><td></td></tr><tr><td>PureLink HiPure Plasmid Maxiprep Kit</td><td>Invitrogen</td><td>K210006</td><td></td></tr><tr><td>Quick Coomassie Stain</td><td>Protein Ark</td><td>GEN-QC-STAIN-1L</td><td></td></tr><tr><td>Steriflip Sterile 50 mL Disposable Vacuum Filtration System 0.22 &amp;micro;m Millipore Express</td><td>EMD Millipore</td><td>SCGP00525</td><td></td></tr><tr><td>Superdex 200 Increase 10/300 GL</td><td>GE Healthcare</td><td>28990944</td><td></td></tr><tr><td>Superose 6 10/300 GL</td><td>GE Healthcare</td><td>17517201</td><td></td></tr><tr><td>Tantalum bromide cluster</td><td>Jena bioscience</td><td>PK-103</td><td></td></tr><tr><td>Top96 Crystallization Screen</td><td>Rigaku Reagents</td><td>1009846</td><td></td></tr><tr><td>Tryphan Blue</td><td>Thermo Fisher Scientific</td><td>T10282</td><td></td></tr><tr><td>VDX 24-well with sealant</td><td>Hampton research</td><td>HR3-172</td><td></td></tr><tr><td>&amp;alpha;2-6 sialyllactose</td><td>Sigma Aldrich</td><td>A8556-1mg</td><td></td></tr></tbody></table>

characterization of glycoproteins with the immunoglobulin fold by x-ray crystallography and biophysical techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

Several constructs of CD22 ECD were successfully cloned into the pHLsec expression vector and overexpressed in mammalian HEK293F and HEK293S cell lines (Figure&#160;2 and 3A). All constructs were purified to size homogeneity by size exclusion chromatography and yielded a highly pure sample for crystallization studies (Figure&#160;3B and 3C). The CD22 construct that led to well-diffracting crystals was the d1-d3 truncation (residues 20-330), with&#160;five&#160;of the six predicted N-linked glycosylation sites mutated from Asn to Ala (N67A, N112A, N135A, N164A and N231A), produced in HEK293S cells, such that only the glycosylation site at position N101 was retained (this construct is named CD2220-330, 5A). Crystals were obtained in several conditions of the MCSG-1 sparse-matrix screen, but the best crystals were from a condition containing 30% (w/v) polyethylene glycol 4000, 0.2 M lithium chloride and 0.1 M Tris, pH 8.5. These native crystals diffracted to 2.1 &#197; resolution; using known structures of Ig domains of related Siglec proteins did not yield any solutions in MR searches.
To acquire phasing information, we soaked native crystals with a panel of HA compounds that included Hg, Pt, Os, Ta, and Br at concentrations ranging from 1-20 mM of HA compound for an incubation time from 5 min to 1 d (Figure 4). We monitored crystals for changes in morphology, and found that crystals soaked with HA compound at 20 mM resulted in the rapid cracking and dissolving of the crystal. We froze a total of 63 crystals that retained their shape following set incubation times that were soaked with tantalum bromide cluster, platinum chloride, mercuric acetate and mercuric chloride. Crystals soaked with 7 mM of mercuric chloride for 30 min showed anomalous signal on a fluorescence scan at the Canadian Light Source (CLS) 08-BM beamline (Saskatoon, Canada) and allowed for multi-wavelength anomalous dispersion X-ray data collection on a single crystal. These datasets allowed us to solve the mercury substructure of CD2220-330, 5A, which revealed a single mercury atom bound to a free cysteine at position C308 and ultimately allowed us to build the structure of CD2220-330, 5A into the phased electron density map using AutoBuild78.
Once the unliganded structure was solved, we were interested in solving the structure of CD22 bound to its ligand, &#945;2-6 siallylactose. We first calculated the affinity of CD22 towards &#945;2-6 sialyllactose using ITC to characterize the binding thermodynamics of the interaction. We observed an affinity of ~280 &#181;M and used this information to identify an initial concentration (~ 100 x KD) of ligand to use for soaking of our native CD2220-330,5A crystals. We soaked the CD2220-330, 5A crystals with 25 mM siallylactose for 5 min, 2 h, 14 h, 40 h and 5 d and monitored for changes in crystal morphology. A total of ~75 crystals were frozen from various time points and sent to the CLS synchrotron beamline 08-ID (Saskatoon, Canada) for remote data collection. A total of six X-ray datasets were collected from well-diffracting crystals. The structure from each X-ray dataset was solved by MR&#160;using the unliganded CD2220-330, 5A structure as an initial search model. The resulting electron density for all datasets was then inspected for positive density in the Fo-Fc map that would correspond to bound &#945;2-6 sialyllactose within the binding site of CD22. Remarkably, all datasets collected, even those from crystals soaked after only 5 min of incubation time, contained positive density corresponding to the ligand in the binding site. Overall structures of the unliganded and liganded CD22 were highly similar with minimal conformational changes, which might explain the success of soaking experiments with &#945;2-6 sialyllactose.
We next characterized the antigenic surface of CD22 recognized by therapeutic antibody epratuzumab in BLI and ITC experiments (Figure 5). Kinetics and thermodynamics profiles of epratuzumab Fab binding to CD22 constructs with different glycoforms revealed an increasing affinity to CD22 with reduced N-linked glycan size, with up to a 14-fold improvement in affinity for smaller glycans (327 nM vs 24 nM in BLI; 188 nM vs 58 nM in ITC). The CD22 N-linked glycan restricting access of the antibody to its epitope was identified by BLI using single-point mutants of CD22 and by solving the epratuzumab Fab-CD22 d1-d3 co-crystal structure28.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57750/57750fig1.jpg" /> 
Figure 1. Overview of glycoprotein characterization from construct design to biophysical and structural characterization. (1) Primary sequence analysis of representative glycoprotein. In grey, the extracellular domain (ECD); in green, the transmembrane (TM) segment; and in blue, the cytosolic domain of the glycoprotein. Predicted N-linked glycans are labeled. (2) Cloning of ECD constructs. (3) Expression of ECD constructs in mammalian cells. (4) Glycoprotein purification. While proteins expressed in HEK293F will contain complex glycans, proteins expressed in HEK293S will have high mannose glycans. Enzymatic treatment of glycoproteins produced in HEK293S cells with Endo H results in glycoproteins with only a GlcNAc moiety at N-linked glycosylation sites. (5a) Glycoproteins are tested for their binding to antibodies by biolayer interferometry (BLI) and isothermal titration calorimetry (ITC). Affinity to small ligands can also be measured by ITC. (5b) Crystallization trials of glycoproteins with homogeneous N-linked glycans, such as those expressed in HEK293S and deglycosylated with Endo H. (6) In some cases, mutation of N-linked glycosylation sites is necessary to obtain crystals. <a href="https://www.jove.com/files/ftp_upload/57750/57750fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57750/57750fig2.jpg" /> 
Figure 2. Design of CD22 ectodomain DNA constructs for expression in mammalian cells.&#160;A) Representation of the pHLsec plasmid used for transient transfection of CD22 ECD constructs. AgeI and KpnI sites used for cloning are indicated with red boxes. B) The CD22 ECD contains seven Ig domains (d1-d7) and 12 predicted N-linked glycosylation sites (in blue). Four constructs were designed from the CD22 ECD. C) 1% agarose gel showing PCR amplicons of CD22 ECD constructs for cloning into the pHLsec mammalian expression vector. First lane contains 1 kb DNA marker. <a href="https://www.jove.com/files/ftp_upload/57750/57750fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57750/57750fig3.jpg" /> 
Figure 3. Expression and purification of glycoproteins.&#160;A) Effect of cell density on expression yields. Glycoprotein expression in small-scale 25 mL culture of HEK293F suspension cells transfected using three different starting densities of cells (0.5 x 106 cells mL-1, 1.0 x 106 cells mL-1, and 1.5 x 106 cells mL-1). Quantification performed by densitometry from SDS-PAGE in left panel and by quantitative BLI in right panel. Values are representative of one glycoprotein preparation. B) Chromatogram of the first purification step for construct CD2220-330, 5A from 600 mL of supernatant using a Ni-NTA affinity column. The glycoprotein was eluted using a gradient of imidazole (grey line), where 100% corresponds to the elution buffer, which contains 500 mM imidazole. Pooled fractions are depicted with vertical lines. C) Size-exclusion chromatogram for construct CD2220-330,5A using a high-performance gel filtration column. Pooled fractions from the elution peak are depicted with vertical lines. Inset: Coomassie-stained SDS-PAGE gel showing the purity of the glycoprotein. <a href="https://www.jove.com/files/ftp_upload/57750/57750fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57750/57750fig4.jpg" /> 
Figure 4. Crystal soaking with heavy atoms. A) Sample workstation for soaking native crystals with HA compounds. All required tools are labeled. B) Steps followed to soak crystals of construct CD2220-330, 5A with HA compounds. Step 1, Open well containing crystals, and transfer crystals using a loop to a 0.2 &#181;L drop on a cover slip containing HA solution diluted in the crystallization condition such that the final concentration of HA ranges from 1-10 mM. Step 2, seal the drop in the crystallization plate and incubate crystals with the HA compound for different periods of time. Step 3, mount the soaked crystal in the loop and back-soak for 30 s in three consecutive 0.2 &#181;l drops containing the mother liquor solution supplemented with 20% (v/v) glycerol dispensed on a cover slip. Step 4, flash freeze the crystal mounted on a loop with liquid nitrogen and place it in a puck for shipment to the synchrotron beamline. <a href="https://www.jove.com/files/ftp_upload/57750/57750fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57750/57750fig5.jpg" /> 
Figure 5. Biolayer interferometry and isothermal titration calorimetry measurements. A) Representative BLI experiment. Top panel: example of plate setup for a kinetics experiment, where the following are labeled: 1x kinetics buffer (B), His6x-tagged glycoprotein loading (L), representative Fab concentrations (500, 250, 125,&#160; 62.5&#160;nM), PBS + 500 mM regeneration buffer (R), and 1x kinetics neutralization buffer (B). Each well contains 200 &#181;L of solution. Step number for the kinetics experiment is indicated at the top of the plate. Middle panel: Representative raw data of BLI experiment performed using Ni-NTA biosensors and the plate described in the top panel. Step numbers correspond to baseline (1), His6x glycoprotein loading (2), baseline (3), association in serial dilution of Fab (4) and dissociation (5). Regeneration steps are not represented (Steps 6-7). Bottom panel: Representative analyzed data showing raw association and dissociation (blue line) with the corresponding 1:1 fit (red line). B)&#160;Top panel: Representative plate setup for a single ITC run on an automated ITC instrument with seven experiments in a 96-well round bottom block. Each experiment is comprised of three wells. The first well (red) corresponds to sample for the cell (400 &#181;L), the second well (green) corresponds to sample for the syringe (120 &#181;L). The third well is left empty, and the mixed samples will be returned to this well following experiment completion. Experiments 1, 2, and 7 are buffer into buffer controls. Experiments 3-5 represent triplicate experiments with glycoprotein (P) in the cell and Fab or ligand (L) in the syringe. Experiment 6 represents a ligand heat of dilution control and should be subtracted from experiments 3-5 during data analysis. Bottom panel: Representative raw (top) and processed (bottom) ITC data showing Fab (epratuzumab) binding to CD22 ECD produced in HEK293F cells. <a href="https://www.jove.com/files/ftp_upload/57750/57750fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques at JoVE.com

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

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12.6K Views.  The Hospital for Sick Children Research Institute. Benaderingen voor de biofysische en structurele karakterisatie van glycoproteïnen presenteren wij met de immunoglobuline vouw door biolayer interferometrie, isothermische titratie calorimetrie en röntgendiffractie.

Video: Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

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Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are found throughout nature, however, because of their physical properties and tendency to aggregate, they are traditionally viewed as being especially difficult to crystallize. Here, we utilize a variety of quick and simple techniques designed to identify a series of possible domain boundaries for a given coiled-coil protein, and then quickly characterize the behavior of these proteins in solution. With the addition of a strongly fluorescent tag (mRuby2), protein characterization is simple and straightforward. The target protein can be readily visualized under normal lighting and can be quantified with the use of an appropriate imager. The goal is to quickly identify candidates that can be removed from the crystallization pipeline because they are unlikely to succeed, affording more time for the best candidates and fewer funds expended on proteins that do not produce crystals. This process can be iterated to incorporate information gained from initial screening efforts, can be adapted for high-throughput expression and purification procedures, and is augmented by robotic screening for crystallization.

We describe a framework incorporating straightforward biochemical and computational analysis to guide the characterization and crystallization of large coiled-coil domains. This framework can be adapted for globular proteins or extended to incorporate a variety of high-throughput techniques.

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are ...

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

&#946;-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of &#946;-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach

Single-chain fragment variable (scFv) antibodies were previously constructed of variable light and heavy chains joined by a (Gly4-Ser) 3 linker. The linker was created using molecular modeling software as a loop structure. Here, we introduce a protocol forin silico analysis of a complete scFv antibody that interacts with the epidermal growth factor receptor (EGFR). The homology modeling, with Pyrx of protein-protein docking and molecular dynamic simulation of the interacting scFv antibody and EGFR First, the authors used a protein structure modeling program and Python for homology modeling, and the antibody scFv structure was modeled for homology. The investigators downloaded Pyrx software as a platform in the docking study. The Molecular dynamic simulation was run using modeling software. Results show that when the MD simulation was subjected to energy minimization, the protein model had the lowest binding energy (-5.4 kcal/M). In addition, the MD simulation in this study showed that the docked EGFR-scFv antibody was stable for 20-75 ns when the movement of the structure increased sharply to 7.2 &#197;. In conclusion, in silicoanalysiswas performed, and the molecular docking and molecular dynamics simulations of the scFv antibody proved the effectiveness of the designed immune-therapeutic drug scFv as a specific drug therapy for EGFR.

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach

This antibody homology modeling prediction protocol is followed by antibody-receptor Pyrx docking and molecular dynamic simulation. These three primary methods are used to visualize the accurate antibody-receptor binding areas and the binding stability of the final structure.

Single-chain fragment variable (scFv) antibodies were previously constructed of variable light and heavy chains joined by a (Gly4-Ser) 3 linker. The ...

Genetics

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

Immunology and Infection

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Biology

Bioinformatics Resources for the Study of Glycan-Mediated Protein Interactions

The Glyco@Expasy initiative was launched as a collection of interdependent databases and tools spanning several aspects of knowledge in glycobiology. In particular, it aims at highlighting interactions between glycoproteins (such as cell surface receptors) and carbohydrate-binding proteins mediated by glycans. Here, major resources of the collection are introduced through two illustrative examples centered on the N-glycome of the human Prostate Specific Antigen (PSA) and the O-glycome of human serum proteins. Through different database queries and with the help of visualization tools, this article shows how to explore and compare content in a continuum to gather and correlate otherwise scattered pieces of information. Collected data are destined to feed more elaborate scenarios of glycan function. Glycoinformatics introduced here is, therefore, proposed as a means to either strengthen, shape, or refute assumptions on the specificity of a protein glycome in a given context.

This protocol illustrates how to explore, compare, and interpret human protein glycomes with online resources.

The Glyco@Expasy initiative was launched as a collection of interdependent databases and tools spanning several aspects of knowledge in glycobiology. In ...

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

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