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Dimerization of Immunoglobulin G

Dimerization of Immunoglobulin G



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I would like to know the specific determinants for formation of IgG dimers. My understanding is the stem of the antibody is a homodimer of two heavy chains, covalently bonded through two disulfide bridges called the hinge region. Is there any association between the constant region? My reading would suggest no but I have not found a specific reference as such.


For human IgG1 and 4, the only inter-heavy chain disulfide bonds are the two in the hinge region. IgG2 has 4 disulfides in the hinge, and IgG3 has 11:

There are intra-heavy chain disulfides in the Fc region, and of course there are non-covalent bonds between the two heavy chains (as well as the light chains) to maintain structure, but aside from the hinge region the only other inter-chain disulfides are between heavy and light.

Here is an interesting review of classical (determined in the 1960s) and non-classical (more recently discovered) disulfide bond formation and structure in human IgGs.


Fragments of Bacterial Endoglycosidase S and Immunoglobulin G Reveal Subdomains of Each That Contribute to Deglycosylation *

Endoglycosidase S (EndoS) is a glycoside-hydrolase secreted by the bacterium Streptococcus pyogenes. EndoS preferentially hydrolyzes the N-linked glycans from the Fc region of IgG during infection. This hydrolysis impedes Fc functionality and contributes to the immune evasion strategy of S. pyogenes. Here, we investigate the mechanism of human serum IgG deactivation by EndoS. We expressed fragments of IgG1 and demonstrated that EndoS was catalytically active against all of them including the isolated CH2 domain of the Fc domain. Similarly, we sought to investigate which domains within EndoS could contribute to activity. Bioinformatics analysis of the domain organization of EndoS confirmed the previous predictions of a chitinase domain and leucine-rich repeat but also revealed a putative carbohydrate binding module (CBM) followed by a C-terminal region. Using expressed fragments of EndoS, circular dichroism of the isolated CBM, and a CBM-C-terminal region fusion revealed folded domains dominated by β sheet and α helical structure, respectively. Nuclear magnetic resonance analysis of the CBM with monosaccharides was suggestive of carbohydrate binding functionality. Functional analysis of truncations of EndoS revealed that, whereas the C-terminal of EndoS is dispensable for activity, its deletion impedes the hydrolysis of IgG glycans.


Classes of immunoglobulins

The five primary classes of immunoglobulins are IgG, IgM, IgA, IgD and IgE. These are distinguished by the type of heavy chain found in the molecule. IgG molecules have heavy chains known as gamma-chains IgMs have mu-chains IgAs have alpha-chains IgEs have epsilon-chains and IgDs have delta-chains.

Differences in heavy chain polypeptides allow these immunoglobulins to function in different types of immune responses and at particular stages of the immune response. The polypeptide protein sequences responsible for these differences are found primarily in the Fc fragment. While there are five different types of heavy chains, there are only two main types of light chains: kappa (κ) and lambda (λ).

Antibody classes differ in valency as a result of different numbers of Y-like units (monomers) that join to form the complete protein. For example, in humans, functioning IgM antibodies have five Y-shaped units (pentamer) containing a total of 10 light chains, 10 heavy chains and 10 antigen-binding.

IgG class

  • Molecular weight: 150,000
  • H-chain type (MW): gamma (53,000)
  • Serum concentration: 10 to 16 mg/mL
  • Percent of total immunoglobulin: 75%
  • Glycosylation (by weight): 3%
  • Distribution: intra- and extravascular
  • Function: secondary response

IgM class

  • Molecular weight: 900,000
  • H-chain type (MW): mu (65,000)
  • Serum concentration: 0.5 to 2 mg/mL
  • Percent of total immunoglobulin: 10%
  • Glycosylation (by weight): 12%
  • Distribution: mostly intravascular
  • Function: primary response

IgA class

  • Molecular weight: 320,000 (secretory)
  • H-chain type (MW): alpha (55,000)
  • Serum concentration: 1 to 4 mg/mL
  • Percent of total immunoglobulin: 15%
  • Glycosylation (by weight): 10%
  • Distribution: intravascular and secretions
  • Function: protect mucus membranes

IgD and IgE class

  • Molecular weight: 180,000
  • H-chain type (MW): delta (70,000)
  • Serum concentration: 0 to 0.4 mg/mL
  • Percent of total immunoglobulin: 0.2%
  • Glycosylation (by weight): 13%
  • Distribution: lymphocyte surface
  • Function: unknown
  • Molecular weight: 200,000
  • H-chain type (MW): epsilon (73,000)
  • Serum concentration: 10 to 400 ng/mL
  • Percent of total immunoglobulin: 0.002%
  • Glycosylation (by weight): 12%
  • Distribution: basophils and mast cells in saliva and nasal secretions
  • Function: protect against parasites

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Abstract

The terminal carbohydrate residues of the N-glycan on the immunoglobulin G (IgG) fragment crystallizable (Fc) determine whether IgG activates pro- or anti-inflammatory receptors. The IgG Fc alone becomes potently anti-inflammatory upon addition of α2–6-linked N-acetylneuraminic acid residues to the N-glycan, stimulating interest in use of this entity in novel therapies for autoimmune disease [Kaneko et al. (2006) Science313, 670–3]. Complete Fc sialylation has, however, been deemed challenging due to a combination of branch specificity and perceived protection by glycan–protein interactions. Here we report the preparation of high levels of disialylated Fc by using sufficient amounts of a highly active α2–6 sialyltransferase (ST6Gal1) preparation expressed in a transiently transformed human cell culture. Surprisingly, ST6Gal1 sialylated the two termini of the complex-type binantennary glycan in a manner remarkably similar to that observed for the free N-glycan, suggesting the Fc polypeptide does not greatly influence ST6Gal1 specificity. In addition, sialylation of either branch terminus does not appear to dramatically alter the motional behavior of the N-glycan as judged by solution NMR spectroscopy. Together these, data suggest the N-glycan occupies two distinct states: one with both glycan termini sequestered from enzymatic modification by an α1–6Man–branch interaction with the polypeptide surface and the other with both glycan termini exposed to the bulk solvent and free from glycan–polypeptide interactions. The results suggest new modes by which disialylated Fc can act as an anti-inflammatory effector.


Abstract

Antibody therapeutics are now in widespread use and provide a new approach for treating serious diseases such as rheumatic diseases and cancer. Monoclonal antibodies used as therapeutic agents must be of high quality, and their safety must be guaranteed. Aggregated antibody is a degradation product that may be generated during the manufacturing process. To maintain the high quality and safety of antibody therapeutics, it is necessary to understand the mechanism of aggregation and to develop technologies to strictly control aggregate formation. Here, we extensively investigated the conformational and colloidal characteristics of isolated antibody constant domains, and provided insights into the molecular mechanism of antibody aggregation. Isolated domains (CH2, CH3, CL, and CH1-CL dimer) of human immunoglobulin G were synthesized, solubilized using 49 sets of solution conditions (pH 2–8 and 0–300 mM NaCl), and characterized using circular dichroism, intrinsic tryptophan fluorescence, and dynamic light scattering. Salt-induced conformational changes and oligomer formation were kinetically analyzed by NaCl-jump measurements (from 0 to 300 mM at pH 3). Phase diagrams revealed that the domains have different conformational and colloidal stabilities. The unfolded fractions of CH3 and CH2 at pH 3 were larger than that of CL and CH1-CL dimer. The secondary and tertiary structures and particle sizes of CH3 and CH2 showed that, in non-native states, these domains were sensitive to salt concentration. Kinetic analyses suggest that oligomer formation by CH3 and CH2 proceeds through partially refolded conformations. The colloidal stability of CH3 in non-native states is the lowest of the four domains under the conditions tested. We propose that the impact of IgG constant domains on aggregation follows the order CH3 > CH2 > CH1-CL dimer > CL furthermore, we suggest that CH3 plays the most critical role in driving intact antibody aggregation under acidic conditions.


Antibody Functions

Antibodies, part of the humoral immune response, are involved in pathogen detection and neutralization.

Learning Objectives

Differentiate among affinity, avidity, and cross-reactivity in antibodies

Key Takeaways

Key Points

  • Antibodies are produced by plasma cells, but, once secreted, can act independently against extracellular pathogen and toxins.
  • Antibodies bind to specific antigens on pathogens this binding can inhibit pathogen infectivity by blocking key extracellular sites, such as receptors involved in host cell entry.
  • Antibodies can also induce the innate immune response to destroy a pathogen, by activating phagocytes such as macrophages or neutrophils, which are attracted to antibody-bound cells.
  • Affinity describes how strongly a single antibody binds a given antigen, while avidity describes the binding of a multimeric antibody to multiple antigens.
  • A multimeric antibody may have individual arms with low affinity, but have high overall avidity due to synergistic effects between binding sites.
  • Cross reactivity occurs when an antibody binds to a different-but-similar antigen than the one for which it was raised this can increase pathogen resistance or result in an autoimmune reaction.

Key Terms

  • avidity: the measure of the synergism of the strength individual interactions between proteins
  • affinity: the attraction between an antibody and an antigen

Antibody Functions

Differentiated plasma cells are crucial players in the humoral immunity response. The antibodies they secrete are particularly significant against extracellular pathogens and toxins. Once secreted, antibodies circulate freely and act independently of plasma cells. Sometimes, antibodies can be transferred from one individual to another. For instance, a person who has recently produced a successful immune response against a particular disease agent can donate blood to a non-immune recipient, confering temporary immunity through antibodies in the donor’s blood serum. This phenomenon, called passive immunity, also occurs naturally during breastfeeding, which makes breastfed infants highly resistant to infections during the first few months of life.

Antibodies coat extracellular pathogens and neutralize them by blocking key sites on the pathogen that enhance their infectivity, such as receptors that “dock” pathogens on host cells. Antibody neutralization can prevent pathogens from entering and infecting host cells, as opposed to the cytotoxic T-cell-mediated approach of killing cells that are already infected to prevent progression of an established infection. The neutralized antibody-coated pathogens can then be filtered by the spleen and eliminated in urine or feces.

Mechanisms of antibody action: Antibodies may inhibit infection by (a) preventing the antigen from binding to its target, (b) tagging a pathogen for destruction by macrophages or neutrophils, or (c) activating the complement cascade.

Antibodies also mark pathogens for destruction by phagocytic cells, such as macrophages or neutrophils, because they are highly attracted to macromolecules complexed with antibodies. Phagocytic enhancement by antibodies is called opsonization. In another process, complement fixation, IgM and IgG in serum bind to antigens, providing docking sites onto which sequential complement proteins can bind. The combination of antibodies and complement enhances opsonization even further, promoting rapid clearing of pathogens.

Affinity, avidity, and cross reactivity

Not all antibodies bind with the same strength, specificity, and stability. In fact, antibodies exhibit different affinities (attraction) depending on the molecular complementarity between antigen and antibody molecules. An antibody with a higher affinity for a particular antigen would bind more strongly and stably. It would be expected to present a more challenging defense against the pathogen corresponding to the specific antigen.

Antibody affinity, avidity, and cross reactivity: (a) Affinity refers to the strength of single interactions between antigen and antibody, while avidity refers to the strength of all interactions combined. (b) An antibody may cross-react with different epitopes.

The term avidity describes binding by antibody classes that are secreted as joined, multivalent structures (such as IgM and IgA). Although avidity measures the strength of binding, just as affinity does, the avidity is not simply the sum of the affinities of the antibodies in a multimeric structure. The avidity depends on the number of identical binding sites on the antigen being detected, as well as other physical and chemical factors. Typically, multimeric antibodies, such as pentameric IgM, are classified as having lower affinity than monomeric antibodies, but high avidity. Essentially, the fact that multimeric antibodies can bind many antigens simultaneously balances their slightly-lower-binding strength for each antibody/antigen interaction.

Antibodies secreted after binding to one epitope on an antigen may exhibit cross reactivity for the same or similar epitopes on different antigens. Cross reactivity occurs when an antibody binds not to the antigen that elicited its synthesis and secretion, but to a different antigen. Because an epitope corresponds to such a small region (the surface area of about four to six amino acids), it is possible for different macromolecules to exhibit the same molecular identities and orientations over short regions.

Cross reactivity can be beneficial if an individual develops immunity to several related pathogens despite having been exposed to or vaccinated against only one of them. For instance, antibody cross reactivity may occur against the similar surface structures of various Gram-negative bacteria. Conversely, antibodies raised against pathogenic molecular components that resemble self molecules may incorrectly mark host cells for destruction, causing autoimmune damage. Patients who develop systemic lupus erythematosus (SLE) commonly exhibit antibodies that react with their own DNA. These antibodies may have been initially raised against the nucleic acid of microorganisms, but later cross-reacted with self-antigens. This phenomenon is also called molecular mimicry.


References

Lynch, I. & Dawson, K. A. Protein-nanoparticle interactions. Nano Today 3, 40–47 (2008).

Luker, K. E. et al. Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. Proc. Natl. Acad. Sci. 101, 12288–12293 (2004).

Tiwary, P., Limongelli, V., Salvalaglio, M. & Parrinello, M. Kinetics of protein–ligand unbinding: Predicting pathways, rates, and rate-limiting steps. Proc. Natl. Acad. Sci. 112, E386–E391 (2015).

Boccaletti, S., Latora, V., Moreno, Y., Chavez, M. & Hwang, D. U. Complex networks: Structure and dynamics. Phys. Rep. 424, 175–308 (2006).

Scott, D. E., Bayly, A. R., Abell, C. & Skidmore, J. Small molecules, big targets: Drug discovery faces the protein-protein interaction challenge. Nat. Rev. Drug Discov. 15, 533–550 (2016).

Abu-Salah, K. et al. DNA-Based Nanobiosensors as an Emerging Platform for Detection of Disease. Sensors 15, 14539–14568 (2015).

Vo-Dinh, T. et al. SERS Nanosensors and Nanoreporters: Golden Opportunities in Biomedical Applications. Wiley Interdiscip. Rev. Nanomedicine Nanobiotechnology 7, 17–33 (2015).

Cooper, M. A. Optical biosensors in drug discovery. Nat. Rev. Drug Discov. 1, 515–528 (2002).

Nguyen, H. H., Park, J., Kang, S., Kim, M. & Cooper, M. A. Surface plasmon resonance: A versatile technique for biosensor applications. Sensors (Switzerland) 15, 10481–10510 (2015).

Yan, Y. & Marriott, G. Analysis of protein interactions using fluorescence technologies. Curr. Opin. Chem. Biol. 7, 635–640 (2003).

Charmet, J., Arosio, P. & Knowles, T. P. J. Microfluidics for Protein Biophysics. J. Mol. Biol. 430, 565–580 (2018).

Homola, J. & Piliarik, M. Surface Plasmon Resonance (SPR) Sensors. In 45–67 (2006).

Patching, S. G. Surface plasmon resonance spectroscopy for characterisation of membrane protein-ligand interactions and its potential for drug discovery. Biochim. Biophys. Acta - Biomembr. 1838, 43–55 (2014).

Olaru, A., Bala, C., Jaffrezic-Renault, N. & Aboul-Enein, H. Y. Surface Plasmon Resonance (SPR) Biosensors in Pharmaceutical Analysis. Crit. Rev. Anal. Chem. 45, 97–105 (2015).

Then, W. L., Aguilar, M. I. & Garnier, G. Quantitative blood group typing using surface plasmon resonance. Biosens. Bioelectron. 73, 79–84 (2015).

Kim, S., Wark, A. W. & Lee, H. J. Femtomolar Detection of Tau Proteins in Undiluted Plasma Using Surface Plasmon Resonance. Anal. Chem. 88, 7793–7799 (2016).

Šípová, H. & Homola, J. Surface plasmon resonance sensing of nucleic acids: A review. Anal. Chim. Acta 773, 9–23 (2013).

Michaelis, S., Wegener, J. & Robelek, R. Label-free monitoring of cell-based assays: Combining impedance analysis with SPR for multiparametric cell profiling. Biosens. Bioelectron. 49, 63–70 (2013).

Raz, S. R., Bremer, M. G. E. G., Haasnoot, W. & Norde, W. Label-free and multiplex detection of antibiotic residues in milk using imaging surface plasmon resonance-based immunosensor. Anal. Chem. 81, 7743–7749 (2009).

Duan, X. et al. Quantification of the affinities and kinetics of protein interactions using silicon nanowire biosensors. Nat. Nanotechnol. 7, 401–407 (2012).

Wang, W. U., Chen, C., Lin, K.-H., Fang, Y. & Lieber, C. M. Label-free detection of small-molecule-protein interactions by using nanowire nanosensors. Proc. Natl. Acad. Sci. 102, 3208–3212 (2005).

Chen, K. I., Li, B. R. & Chen, Y. T. Silicon nanowire field-effect transistor-based biosensors for biomedical diagnosis and cellular recording investigation. Nano Today 6, 131–154 (2011).

Zheng, G., Patolsky, F., Cui, Y., Wang, W. U. & Lieber, C. M. Multiplexed electrical detection of cancer markers with nanowire sensor arrays. Nat. Biotechnol. 23, 1294–1301 (2005).

Im, H., Huang, X. J., Gu, B. & Choi, Y. K. A dielectric-modulated field-effect transistor for biosensing. Nat. Nanotechnol. 2, 430–434 (2007).

Esadze, A. & Iwahara, J. Stopped-flow fluorescence kinetic study of protein sliding and intersegment transfer in the target DNA search process. J. Mol. Biol. 426, 230–244 (2014).

Berlow, R. B., Dyson, H. J. & Wright, P. E. Hypersensitive termination of the hypoxic response by a disordered protein switch. Nature 543, 447–451 (2017).

Guinn, E. J., Jagannathan, B. & Marqusee, S. Single-molecule chemo-mechanical unfolding reveals multiple transition state barriers in a small single-domain protein. Nat. Commun. 6, 1–8 (2015).

Hessel, V., Löwe, H. & Schönfeld, F. Micromixers - A review on passive and active mixing principles. Chem. Eng. Sci. 60, 2479–2501 (2005).

Lin, X. et al. Determination of cell metabolite VEGF165 and dynamic analysis of protein-DNA interactions by combination of microfluidic technique and luminescent switch-on probe. Biosens. Bioelectron. 79, 41–47 (2016).

Li, Y. et al. A novel microfluidic mixer based on dual-hydrodynamic focusing for interrogating the kinetics of DNA-protein interaction. Analyst 138, 4475–4482 (2013).

Duncombe, T. A., Tentori, A. M. & Herr, A. E. Microfluidics: Reframing biological enquiry. Nat. Rev. Mol. Cell Biol. 16, 554–567 (2015).

Srisa-Art, M., Dyson, E. C., DeMello, A. J. & Edel, J. B. Monitoring of real-time streptavidin-biotin binding kinetics using droplet microfluidics. Anal. Chem. 80, 7063–7067 (2008).

Shang, L., Cheng, Y. & Zhao, Y. Emerging Droplet Microfluidics. Chemical Reviews 117, 7964–8040 (2017).

Song, H. & Ismagilov, R. F. Millisecond Kinetics on a Microfluidic Chip Using Nanoliters of Reagents. J. Am. Chem. Soc. 125, 14613–14619 (2003).

Mercadal, P. A., Fraire, J. C., Motrich, R. D. & Coronado, E. A. Enzyme-Free Immunoassay Using Silver Nanoparticles for Detection of Gliadin at Ultralow Concentrations. ACS Omega 3, 2340–2350 (2018).

Fraire, J. C., Motrich, R. D. & Coronado, E. A. Design of a novel plasmonic nanoconjugated analytical tool for ultrasensitive antigen quantification. Nanoscale 8, 17169–17180 (2016).

Mercadal, P. A., Fraire, J. C., Motrich, R. D. & Coronado, E. A. Plasmonic sensing through bioconjugation of Ag nanoparticles: Towards the development of immunoassays for ultralow quantification of antigens in colloidal dispersions. Adv. Mater. Lett. 9(6), 456–461 (2018).

Ianni, J. C. A comparison of the Bader-Deuflhard and the Cash-Karp Runge-Kutta integrators for the GRI-MECH 3.0 model based on the chemical kinetics code Kintecus. In Computational Fluid and Solid Mechanics 2003 1368–1372, https://doi.org/10.1016/B978-008044046-0.50335-3 (Elsevier, 2003).

Ianni, J. C. Kintecus. Windows Version 4.55. (2014).

Mandl, A., Filbrun, S. L. & Driskell, J. D. Asymmetrically Functionalized Antibody–Gold Nanoparticle Conjugates to Form Stable Antigen-Assembled Dimers. Bioconjug. Chem. 28, 38–42 (2017).

Kang, J.-H. et al. Gold nanoparticle-based colorimetric assay for cancer diagnosis. Biosens. Bioelectron. 25, 1869–1874 (2010).

Medley, C. D. et al. Gold Nanoparticle-Based Colorimetric Assay for the Direct Detection of Cancerous Cells. Anal. Chem. 80, 1067–1072 (2008).

Rosi, N. L. & Mirkin, C. A. Nanostructures in Biodiagnostics. Chem. Rev. 105, 1547–1562 (2005).

Zhao, W., Brook, M. A. & Li, Y. Design of Gold Nanoparticle-Based Colorimetric Biosensing Assays. Chem. Bio. Chem. 9, 2363–2371 (2008).

Kimling, J. et al. Turkevich Method for Gold Nanoparticle Synthesis Revisited. J. Phys. Chem. B 110, 15700–15707 (2006).

Fraire, J. C., Perez, L. A. & Coronado, E. A. Rational design of plasmonic nanostructures for biomolecular detection: Interplay between theory and experiments. ACS Nano 6, 3441–3452 (2012).

Palik, E. D. Handbook of Optical Constant of Solids (1985).


Materials and Methods

Materials

PECAM-1.3, a murine anti–human PECAM-1 mAb specific for Ig homology domain 1 (Sun et al. 1996b), was fluorescently labeled with Alexa 488 using a protein labeling kit (Molecular Probes). A mouse anti–human mAb, specific for FKBP-12, MAR4 (mouse anti–human α5 integrin), and IIA1 (mouse anti–human β1 integrin) were purchased from PharMingen. P1D6 (mouse anti–human α5 integrin) and P4C10 (mouse anti–human β1 integrin) were purchased from GIBCO BRL. AP1510 and the pCF1E vector encoding FKBP cDNA were provided by ARIAD Pharmaceuticals, Inc. (Cambridge, MA). PMSF was purchased from Sigma-Aldrich. Chemical cross-linking agents, bis(sulfosuccinikidyl) suberate (BS 3 ), and dithiobis(sulfosuccinimidylpropionate) (DTSSP) were purchased from Pierce Chemical Co. DTSSP is thiol cleavable, whereas BS 3 is noncleavable.

Construction of cDNAs Encoding PECAM-1/FKBP-1 and PECAM-1/FKBP-2 Fusion Proteins

One or two copies of the FK506-binding protein (FKBP), FKBP-12, were fused, to the COOH terminus of the cytoplasmic domain of PECAM-1, using standard overlapping PCR techniques (Fig. 1). PECAM-1/FKBP-1, containing one FKBP motif, was produced by PCR. A segment of PECAM-1 was amplified from amino acid residue 375 to the COOH terminus of PECAM-1 using PECAM-1 primer 1 (5′-CCTGTCAAGTAAGGTGGTGGAGTCT-3′) and PECAM-1 primer 2 (5′-CACCTGCA-CGCCTCTAGAAGTTCCATCAAGGGAGCC-3′), and was joined to a full-length FKBP motif using FKBP primer 3 (5′-GGCTCCCTTGATGGAACTTCTAGAGGCGTGCAGGTG-3′) and FKBP primer 4 (5′-GTCCTGAATGCTCTTCCAGGCGGCCGCTTATGCGTAGTCTGGTAC-3′). The two segments were subsequently mixed together with primers 1 and 4, and secondary overlap PCR was performed. The final amplified product was digested with Nhe I and Not I and inserted into similarly cut wild-type PECAM-1 cDNA that had been cloned into the mammalian expression vector pcDNA3. PECAM-1/FKBP-2, containing two FKBP domains fused to the COOH terminus of PECAM-1, was constructed by addition of a second FKBP domain to PECAM-1/FKBP-1. The integrity and authenticity of both constructs were confirmed by nucleotide sequencing.

Development of Stable PECAM-1/FKBP-1– and PECAM-1/FKBP-2–expressing HEK-293 Cell Lines

HEK (human embryonic kidney)-293 cells and human erythroleukemia (HEL) cells were obtained from American Type Culture Collection and cultured in MEM and RPMI-1640 medium (Sigma-Aldrich), respectively, with 10% heat-inactivated fetal calf serum at 37°C in a humidified atmosphere of 5% CO2. HEK-293 cells were grown to 80–90% confluence in 100-mm dishes, incubated with 10 μg of pcDNA3.0 (Invitrogen) containing PECAM-1/FKBP-1 or PECAM-1/FKBP-2 in a lipofectamine mixture (GIBCO BRL) for 4 h in serum-free medium (Sigma-Aldrich). Cells were cultured for an additional 48 h in serum-containing MEM before adding 0.6 mg/ml G418 (geneticin GIBCO BRL). G418-resistant cell lines were analyzed, using flow cytometry, for cell surface expression of PECAM-1/FKBP-1 and PECAM-1/FKBK2. Cell clones were obtained by cell sorting and limited dilution subcloning. The properties of at least three different clonal lines were examined so that the influence of clonal variation on the observed phenotype could be determined.

Western Blot and Immunoprecipitation Analysis

HEL and HEK-293 cells were lifted using 0.01% trypsin and 10 mM EDTA, washed in sterile HPBS, and resuspended at a final concentration of 10 6 cells/ml. Chemical cross-linking was initiated by addition of 2 mM BS 3 or DTSSP for 1 h at 4°C, and then 50 mM Tris-HCl was added to stop the reaction. Cell lysates were prepared in lysis buffer (2% Triton X-100, 50 mM Tris-HCl, 2 mM PMSF), and the 15,000 g Triton-soluble fraction was analyzed under both reducing and nonreducing conditions on a 7% SDS–polyacrylamide gel. After transfer to an Immobilion-P membrane (Millipore), proteins were visualized using antibodies to PECAM-1 (PECAM-1.3) or FKBP-12 in conjunction with enhanced chemiluminescence immunodetection (Amersham Pharmacia Biotech) using peroxidase-conjugated goat anti–mouse IgG (Jackson ImmunoResearch Laboratories). For immunoprecipitation analysis, detergent cell lysates were prepared as above, and the Triton-soluble fractions were incubated with 10 μg/ml PECAM-1.3 IgG overnight at 4°C. Immune complexes were captured with 50 μl of 50% slurry of protein G–Sepharose for 1 h at 4°C, and then washed five times with immunoprecipitation wash buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, and 2% Triton X-100). Bound proteins were eluted from the beads by boiling for 10 min in 30 μl of SDS-reducing buffer and analyzed by SDS-PAGE, as described above.

Flow Cytometry

4 μg of PECAM-1.3 or PECAM-1/IgG in 100 μl of HPBS was added to 5 × 10 5 wild-type or transfected HEK-293 cells in 100 μl of HPBS and allowed to incubate for 60 min at room temperature in the presence or absence of various concentrations of AP1510. The cells were then washed by centrifugation with 1 ml of HPBS and then resuspended in 100 μl of HPBS containing 1 μg of FITC-labeled goat anti–human or goat anti–mouse IgG γ chain (Jackson ImmunoResearch Laboratories). After a 30 min incubation at 4°C, the cells were washed once more in HPBS, and then subjected to flow cytometric analysis.

Fluorescence Microscopy

Wild-type or stably transfected 293 cells were incubated in the presence or absence of 1 μM AP1510 for 30 min at room temperature, and then by 5 μg/ml Alexa 488–conjugated PECAM-1.3 for 30 min at 4°C. The cells were then washed, fixed with 1% formaldehyde, and mounted onto glass slides using a cytospin preparation. The cell surface distribution of PECAM-1 was analyzed using a Zeiss fluorescent microscope (ZEISS).

Quantitative Evaluation of the Binding of 293 Cells To Immobilized Fibronectin

Adherent 293 cells were lifted using 0.01% trypsin and 10 mM EDTA, washed in sterile HPBS, and resuspended at 10 6 cells/ml in prewarmed (37°C) serum-free medium. After incubation with 2 μg/ml calcein AM (Molecular Probes) for 30 min at 37°C, cells were washed and resuspended at 10 6 cells/ml in prewarmed serum-free medium, and then incubated again, this time with varying concentrations of AP1510 in the presence of selected antibodies for 30 min at 22°C. 200-μl cell aliquots were then added to wells of 96-well microtiter tissue culture plates (Dynatech Laboratories) that had been coated with either 10 μg/ml fibronectin (Sigma-Aldrich) or 10 mg/ml BSA (ICN Biomedicals). Antibody competition experiments were performed by preincubating cells with varying concentrations of selected integrin-specific mAbs for 30 min at 22°C, before addition of cells to the wells. The reaction was stopped by addition of 70 μl of 4% formaldehyde for 15 min at 37°C, and total well fluorescence was determined in a CytoFluor fluorescent plate reader (PerSeptive Biosystem). Loosely adherent and unbound cells were then removed by washing the wells with prewarmed serum-free medium, and fluorescence was measured once more to determine percent cell adhesion.


DISCUSSION

As a member of the Atlastin GTPase family functioning in mediating the fusion of different ER tubules in plant cells, the mechanistic basis of the action of RHD3 in the ER membrane fusion is still not well studied. Previous studies showed that RHD3 undergoes a GTP-dependent homotypic interaction, which can be enhanced by the phosphorylation on the divergent sequences of the CT of RHD3 ( Chen et al., 2011 Ueda et al., 2015). Although the structure of RHD3 has not been resolved, based on the high structure similarity between RHD3 and Sey1p, a yeast member of the Atlastin GTPases, we simulated the structure of the cytosolic N terminus of RHD3. The simulated cytosolic structure of RHD3 suggests that the GDs and the first two 3HBs of two RHD3 monomers could twist together to form a dimer. In consistence with this simulation, we find that a full-length RHD3 can form homotypic interactions with its GD as well as its middle domain. When the potential polar linkage between D185 of one RHD3 monomer and R101 of another monomer in the interface of the dimer is disrupted, the interaction between a full-length RHD3 and the GD of RHD3 is weakened. Similarly, mutations in the first two 3HBs in the middle domain could also reduce the interaction between a full-length RHD3 and its middle domain. We suggest that the dimerization of RHD3 mediated by its GD and 3HBs is highly likely. Interestingly, when we expressed the GD or cytosolic N terminus of RHD3 in plant cells, the expression has a negative effect on the formation of the polygonal ER network, suggesting that the function of endogenous RHD3 is affected. This influence is possibly through the interaction between the GD or cytosolic N terminus of RHD3 and full-length, endogenous RHD3. In animal cells, it is believed that, to facilitate different ER membrane fusion, the cytosolic N terminus of Atlastin-1 in two different ER membranes first undergo a dimerization between GDs to tether different ER membrane together. This dimerization is then stabilized by 3HBs in the middle domain, after which a GTP-hydrolysis dependent conformational change occurs to fuse the ER membranes. Because the interaction between a full-length RHD3 and its GD or first two 3HBs was required for the efficient ER fusion, we think that a similar mechanism may exist for RHD3.

A major difference between RHD3 and Atlastin-1 is that the predicted helical bundle enriched middle domain of RHD3 is much longer than that of Atlastin-1 ( Stefano and Brandizzi, 2014). Like RHD3, Sey1p also has a middle domain longer than Atlastin-1. It has been shown that the deletion of 3HB-3 and 3HB-4 inactivates Sey1p ( Yan et al., 2015). Here we find that in RHD3, 3HB-3 and 3HB-4 play a vital role in the protein stability of RHD3. Although both 3HB-1 and 3HB-2 are required for possible dimerization of RHD3, it is interesting to note that 3HB-1 mutants RHD3(A285P) and RHD3(L355P) localized to thick bundled ER tubules, while 3HB-2 mutants RHD3(L379P) and RHD3(A434P) form punctates and aggregations on the ER tubules. This suggests that the mutations in 3HB-2 (L379P and A434P) may also influence the distribution of RHD3 on ER tubules.

RHD3 is an ER localized protein. Our results indicate that the TMs of RHD3 are important for the targeting and retention of RHD3 in the ER. The TMs not only serve as an ER membrane anchor, they are also able to interact with each other. Atlastin-1 in animal cells undergo a GTP-independent association through its TMs. This TM-mediated association is proposed to increase the density of Atlastin-1 at the site of membrane tethering ( Liu et al., 2012 Yan et al., 2015), so multiple cycles of dimerization of Atlastin-1 in the different membranes can occur for the efficient ER membrane fusion. Likely, RHD3 may also undergo a TM-mediated association for the efficient ER membrane fusion in plant cells.

Although the TMs are important for the targeting and retention of RHD3 in the ER, we also found the amphipathic helix located in the CT of RHD3 has an ability for the ER and Golgi targeting. Given the fact that a large portion of RHD3(ƊTM) or the CT of RHD3 is in the cytosol, we think a portion of RHD3(ƊTM) or the CT of RHD3 is anchored to the membrane of the ER and Golgi. When the hydrophobic residues on the same hydrophobic face in the amphipathic helix are replaced by the acidic residues, all the mutations abolished the membrane attachment ability of the CT of RHD3 however, different hydrophobic residue replacement did not compromise the membrane association of the CT of RHD3. Thus, it is highly likely that the membrane anchoring of RHD3(ƊTM) or the CT of RHD3 is due to a hydrophobic interaction between the amphipathic helix and membrane lipids. The amphipathic helix of Atlastin-1 has been proposed to facilitate the ER membrane fusion by providing the driving force for outer leaflet mixing of two different membranes through interacting with and destabilizing the lipid bilayer ( Liu et al., 2012). The mutations in the hydrophobic residues on the same hydrophobic face in the helix not only abolish the membrane attachment ability of the CT of RHD3, the full-length RHD3 with such mutations in the amphipathic helix also has reduced ability to rescue ƊSey1p and rhd3-8 mutants. This indicates that membrane anchoring mediated by the amphipathic helix of RHD3 also play a role in the efficient ER membrane fusion, possibly through its interaction with lipid bilayers like the amphipathic helix of Atlastin-1.

It is interesting to note that a portion of RHD3(ƊTM) or the CT of RHD3 is not only anchored to the ER membrane, but also the Golgi membrane. This is probably a reflection of the specific organization of the ER-Golgi interface in plant cells that Golgi is physically linked to the ER ( Sparkes et al., 2009). However, when the CT of yeast Sey1p was expressed in plant cells, no Golgi targeting is revealed ( Supplemental Fig. S5A ), but replacing the amphipathic helix in the CT of yeast Sey1p with the amphipathic helix of RHD3 will target YFP to the Golgi or ER membrane ( Supplemental Fig. S5B ), so the Golgi targeting of RHD3(ƊTM) or the CT of RHD3 is RHD3 specific and is determined by the amphipathic helix of the CT of RHD3. However, the full-length RHD3 is ER localized ( Chen et al., 2011), so the physiological relevance of this Golgi membrane preference of the CT of RHD3 is not clear.

CONCLUSIONS

Based on our systemic structure-function analysis of RHD3, we hypothesize that, to mediate different ER membrane fusion, different RHD3 molecules in different ER membranes need to undergo homotypic interaction through their GDs and middle domains. Likely, RHD3 may also undergo a TM-mediated association to increase the density of RHD3 in the same ER membrane for efficient ER membrane fusion. Meanwhile, the amphipathic helix of the C terminus of RHD3 would attach to the ER membrane to disturb the lipid bilayer to facilitate the membrane fusion.


1. Edelman GM, Cunningham BA, Gall WE, Gottlieb PD, Rutishauser U, Waxdal MY. The covalent structure of an entire γG immunoglobulin molecule. The Rockefeller University. Communicated by Theodore Shedlovsky. 1969

2. Germenis A. Medical Immunology. Papazisis Publications. 2000

3. Branden C, Tooze J. Introduction to protein structure. 2nd Edition, Garland Publishing, Inc, New York. 1999

4. Owen JA, Punt J, Stanford SA, Jones PP. KUBY Immunology. Seventh Edition, W. H. Freeman and Company, New York. 2013

5. Abbas AK, Lichtman AH. Basic Immunology. Functions and Disorders of the Immune System. Saunders, second edition. 2004

6. Schroeder HW Jr, Cavacini L. Structure and Function of Immunoglobulins. J Allergy Clin Immunol. 2009125:41-52

7. Bruhns P. Properties of mouse and human IgG receptors and their contribution to disease models. Blood Journal. 2012199:5640-5649

8. Biermann MHC, Griffante G, Podolska MJ, Boeltz S, Strurmer J, Munoz LE, Bilyy R, Herrmann M. Sweet but dangerous - the role of immunoglobulin G glycosylation in autoimmunity and imflammation. Lupus. 201625:934-942

9. Hmiel LK, Brorson KA, Boyne MT. Post-translational structural modifications of immunoglobulin G and their effect on biological activity. Anal Bioanal Chem. 2014 Springer

10. Derry C, Rooperanian DC, Akilesh S. FcRn: The neonatal Fc receptor comes of age. Nature Reviews Immunology. 20077:715-725

11. Hirschberg C. Topography of glycosylation in the Rough Endoplasmic Protein Reticulum and Golgi apparatus. Ann. Rev. Biochem. 198756:63-87

12. Maverakis E, Kim K, Shimoda M, Gershwin ME, Patel F, Wilken R, Raychaudhuri S, Renee Ruhaak L, Lebrilla CB. Glycans in the immune system and the altered glycan theory of Autoimmunity: A critical review. J Autoiummun. 20150:1-13

13. Walsh G. Post-translational Modification of Protein Biopharmaceuticals. Part One, Glycosylation. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. 2009

14. Walsh T. Christopher. Posttranslational Modification of Proteins. Expanding Nature Inventory. Romberts and Company Pumplshers, Englegood, Colorado. 2006

15. Shinkawa T, Nakamura K, Yamane N, Shoji-Hosaka E, Kanda Y, Sakurada M, Uchida K, Anazawa H, Satoh M, Yamasaki M, Hanai N, Shitara K. The absence of fucose but not the presence of Galactose or Bisecting N-Acetylglucosamine of Human IgG1 Complex-type Oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. The journal of biological chemistry. 2003278:3466-3473

16. Arnold JN, Wormald MR, Sim RB, Rudd PM, Dwek RA. Structure of Human Immunoglobulins. Annu. Rev. Immunol. 200725:21-50

17. Krapp S, Mimura Y, Jefferis R, Huber R, Sondermann P. Structural Analysis of Human IgG-Fc Glycoforms Reveals a Correlation Between Glycosylation and Structural Integrity. J Mol Biol. 2003325:979-989

18. Ravetch JM, Bolland S. IgG Fc Receptors. Annu. Rev. Immunol. 200119:275-90

19. Mimura Y. et al. Contrasting glycosylation profiles between Fab and Fc of a human IgG protein studied by electrospray ionization mass spectrometry. Journal of Immunological Methods. 2007326:116-126

20. Masuda K, Yamaguchi Y. et al. Pairing of oligosaccharides in the Fc region of immunoglobulin G. FEBS Letters. 2000473:349-357

21. Nimmerjahn F, Anthony RM, Ravetch JV. Agalactosylated IgG antibodies depend on cellular Fc receptors for in vivo activity. Proceedings of the National Academy on Sciences (PNAS). 2007104:20

22. De Haan N, Reiding KR, Driessen G, Van Der Burg M, Wuhrer M. Changes in healthy human IgG Fc-glycosylation after birth and during early childhood. Journal of Proteome Research. 2016 http://pubs.acs.org

23. Williams PJ, Arkwright PD, Rudd P, Scragg IG, Edge CJ, Wormald MR, Rademacher TW. Short Communication: Selective Placental Transport of Maternal IgG to the Fetus. Placenta. 199516:749-756

24. Sazinsky SL, Ott RG, Silver NW, Tidor B, Ravetch JM, Wittrup KD. Aglycosylated immunoglobulin G1 variants productively engage activating Fc receptors. Proceedings of the National Academy on Sciences (PNAS). 2008105:20167-20172

25. Ferrara C, Stuart F, Sondermann P, Brunker P, Umana P. The Carbohydrate at FcγRIIIa Asn-162. An element required for high affinity binding to non-fucosylated IgG glycoforms. The Journal of Biological Chemistry. 2006281:5032-5036

26. Shields RL, Lai J, Keck R, O'Conneli LY, Hong K, Meng YG, Weikert SHA, Presta L. Lack of Fucose on human IgG1 N-Linked Oligosaccharide improves binding to human FcγRIII and Antibody-dependent Cellular Toxicity. Journal of Biological Chemistry. 2002277:26733-26740

27. Warner TG, deKremer RD, Sjoberg ER, Mock AK. Characterization and analysis of Branched-chain N-Acetylglucosaminyl Oligosaccharides Accumulating in Sandhoff Disease Tissue. The Journal of Biological Chemistry. 1985260:6194-6199

28. Kibe T, Fujimoto S, Ishida C, Togari H, Wada Y, Okada S, Nakagawa H, Tsukamoto Y, Takahashi N. Glycosylation and Placental Transport of Immunoglobulin G. J Clin Biochem Nutr. 199621:57-63

29. Nimmerjahn F, Ravetch JV. Fcγ Receptors: Old Friends and New Family Members. Immunity. 200624:19-28

30. Kouings A. et al. Site-specific glycosylation of human immunoglobulin G is altered in four rheymatoid arthritis patients. The Biochemical Journal. 1996314:621-630

31. Malhotra R, Wormald MR, Rudd PM, Fischer PB, Dwek RA, Sim RB. Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein. Nature Publishing Group. 19951:237-243

32. Malhotra R. et al. Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein. Nature Medicine. 19951:237-243

33. Theodoratou E, Thaci K, Agakov F, Timofeeva MN, Stambuk J, Pucic-Bakovic M, Vuckovic F, Orchard P. et al. Glycosylation of plasma IgG in colorectal cancer prognosis. Scientific Reports 6. 2016 article number 28098

34. Vuckovic F, Theodoratou E, Thaci K, Timofeeva M, Vojta A, Stambuk J, Pucic-Bakovic M, Rudd PM. et al. IgG glycome in colorectal cancer. Biology of Human Tumors, Clinical Cancer Research. 2016 American Association for Cancer Research

35. Ruhaak LR, Miyamoto S, Lebrilla C. Developments in the identification of Glycan Biomarkers for the detection of cancer. Mol Cell Proteomics. 201312:846-855