Native Multimers Research Articles

Sendai virus particles carrying target virus glycoproteins for antibody induction

Induction of antibodies targeting viral glycoproteins is a key for the development of a vaccine against enveloped virus infection. Glycoproteins on the virion exhibiting native multimer structure may be a good immunogen to present antibody epitopes, but it is often difficult to prepare immunogenic inactivated virions. Preparation of soluble glycoprotein multimers has been attempted, while virus-like particles carrying target glycoproteins can be a more immunogenic antigen. In the present study, a target glycoprotein-embedded Sendai virus (SeV) particle was developed for induction of anti-virus antibodies. We constructed a chimeric antigen, HIV-1 EnvF, consisting of HIV-1 Env ectodomain and SeV F transmembrane-cytoplasmic domain, which was shown to be efficiently incorporated into the SeV virion. EnvF was recognized by anti-HIV-1 broadly-neutralizing monoclonal antibodies (bnAbs) including 35O22 that targets an Env trimer-dependent epitope. Analysis revealed that HIV-1AD8 EnvF can mediate viral entry into the cells, which is inhibited by anti-HIV-1 bnAbs and HIV-1 entry inhibitors, suggesting that the EnvF exhibits an HIV-1 Env native-like functional structure to present bnAb epitopes. Immunization of mice with replication-defective SeVs expressing HIV EnvF and non-infectious SeV particles (NVP) carrying HIV EnvF efficiently induced anti-HIV Env antibodies. HTLV-1 EnvF also showed the potential to efficiently induce anti-HTLV-1 Env antibodies. These results indicate that SeV particles carrying EnvF can be a promising vaccine platform for induction of antibodies targeting enveloped virus glycoproteins.

Crowded organelles, lipid accumulation, and abnormal membrane tubulation in cellular models of enhanced α-synuclein membrane interaction

Previous work from our group showed that certain engineered missense mutations to the α-synuclein (αS) KTKEGV repeat motifs abrogate the protein’s ability to form native multimers. The resultant excess monomers accumulate in lipid-membrane-rich inclusions associated with neurotoxicity exceeding that of natural familial Parkinson’s disease mutants such as E46K. We presented an initial characterization of the lipid-rich inclusions and found similarities to the αS- and vesicle-rich inclusions that form in baker’s yeast when αS is expressed. We also discussed, with some caution, a possible role of membrane-rich inclusions as precursors to filamentous Lewy bodies, the widely accepted hallmark pathology of Parkinson’s disease and other synucleinopathies. In the meantime, advances in the microscopic characterization of Lewy bodies have highlighted the presence of crowded organelles and lipid membranes in addition to αS accumulation. This prompted us to revisit the αS inclusions caused by our repeat motif variants in neuroblastoma cells. In addition to our previous characterization, we found that these inclusions can often be seen by brightfield microscopy, overlap with endogenous vesicle markers in immunofluorescence experiments, stain positive for lipid dyes, and can be found to be closely associated with mitochondria. We also observed abnormal tubulation of membranes, which was subtle in inducible lines and pronounced in cells that transiently expressed high amounts of the highly disruptive KTKEGV motif mutant “KLKEGV”. Membrane tubulation had been reported before as an αS activity in reductionist systems. Our in-cellulo demonstration now suggests that this mechanism could possibly be a relevant aspect of aberrant αS behavior in cells.

Metal ions and redox balance regulate distinct amyloid-like aggregation pathways of GAPR-1

Members of the CAP superfamily (Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-Related 1 proteins) are characterized by the presence of a structurally conserved CAP domain. The common structure-function relationship of this domain is still poorly understood. In this study, we unravel specific molecular mechanisms modulating the quaternary structure of the mammalian CAP protein GAPR-1 (Golgi-Associated plant Pathogenesis-Related protein 1). Copper ions are shown to induce a distinct amyloid-like aggregation pathway of GAPR-1 in the presence of heparin. This involves an immediate shift from native multimers to monomers which are prone to form amyloid-like fibrils. The Cu2+-induced aggregation pathway is independent of a conserved metal-binding site and involves the formation of disulfide bonds during the nucleation process. The elongation process occurs independently of the presence of Cu2+ ions, and amyloid-like aggregation can proceed under oxidative conditions. In contrast, the Zn2+-dependent aggregation pathway was found to be independent of cysteines and was reversible upon removal of Zn2+ ions. Together, our results provide insight into the regulation of the quaternary structure of GAPR-1 by metal ions and redox homeostasis with potential implications for regulatory mechanisms of other CAP proteins.

Advancing multimer analysis of von Willebrand factor by single-molecule AFM imaging.

The formation of hemostatic plugs at sites of vascular injury crucially involves the multimeric glycoprotein von Willebrand factor (VWF). VWF multimers are linear chains of N-terminally linked dimers. The latter are formed from monomers via formation of the C-terminal disulfide bonds Cys2771-Cys2773’, Cys2773-Cys2771’, and Cys2811-Cys2811’. Mutations in VWF that impair multimerization can lead to subtype 2A of the bleeding disorder von Willebrand Disease (VWD). Commonly, the multimer size distribution of VWF is assessed by electrophoretic multimer analysis. Here, we present atomic force microscopy (AFM) imaging as a method to determine the size distribution of VWF variants by direct visualization at the single-molecule level. We first validated our approach by investigating recombinant wildtype VWF and a previously studied mutant (p.Cys1099Tyr) that impairs N-terminal multimerization. We obtained excellent quantitative agreement with results from earlier studies and with electrophoretic multimer analysis. We then imaged specific mutants that are known to exhibit disturbed C-terminal dimerization. For the mutants p.Cys2771Arg and p.Cys2773Arg, we found the majority of monomers (87 ± 5% and 73 ± 4%, respectively) not to be C-terminally dimerized. While these results confirm that Cys2771 and Cys2773 are crucial for dimerization, they additionally provide quantitative information on the mutants’ different abilities to form alternative C-terminal disulfides for residual dimerization. We further mutated Cys2811 to Ala and found that only 23 ± 3% of monomers are not C-terminally dimerized, indicating that Cys2811 is structurally less important for dimerization. Furthermore, for mutants p.Cys2771Arg, p.Cys2773Arg, and p.Cys2811Ala we found ‘even-numbered’ non-native multimers, i.e. multimers with monomers attached on both termini; a multimer species that cannot be distinguished from native multimers by conventional multimer analysis. Summarizing, we demonstrate that AFM imaging can provide unique insights into VWF processing defects at the single-molecule level that cannot be gained from established methods of multimer analysis.

Characterization of a human UT‐B urea transporter antibody (897.3)

Urea entry into the gastrointestinal tract is facilitated by the urea transporter UT‐B1 (Stewart 2011). This process is crucial to our intestinal wellbeing as it is the first step in the urea nitrogen salvaging mechanism, which is necessary for maintaining healthy gut bacterial populations. In a previous study using a bovine UT‐B antibody, a 35kDa human UT‐B1 protein was found to be highly expressed in the ascending colon (Collins et al., 2010). The aim of this study was to produce a reliable human UT‐B antibody to be used for further investigations of human UT‐B expression. Initial PCR analysis using primers designed along the UT‐B gene confirmed UT‐B1 as the major isoform expressed at the RNA level in all human gastrointestinal tissues. Three polyclonal human UT‐B antibodies ‐ one designed against the N‐terminal (UT‐B#N) and two against the C‐terminal (UT‐B#C and UT‐Bc19) ‐ were made according to the UT‐B1 amino acid sequence and extensive characterization was carried out using Western blot analysis. While both C‐terminal antibodies detected UT‐B1 in the membrane of erythrocytes as a large glycosylated signal at 45‐60kDa as expected, the UT‐B#N antibody failed to do so and was rejected. As UT‐B transporters are glycoproteins, deglycosylation experiments were performed on UT‐B1 protein using PNGaseF enzyme. While UT‐Bc19 successfully detected 36 kDa deglycosylated UT‐B1, UT‐B#C failed to do so and was also rejected. Next, β‐mercaptoethanol was used to reduce UT‐B1 from its native multimer to a monomer and UT‐Bc19 successfully detected a big shift from a smeared 40‐100kDa signal to a neat 40kDa signal. Finally, UT‐Bc19 also detected a strong 40‐70kDa glycosylated UT‐B1 signal in human ileum protein. In conclusion, these data have shown that UT‐Bc19 is the only consistent human UT‐B antibody. Future investigations of UT‐B1 expression will therefore utilise UT‐Bc19 with confidence and help establish the physiological significance of UT‐B1 in the human gastrointestinal tract.Grant Funding Source: Supported by The Wellcome Trust and Science Foundation Ireland.

Trapping of Human Hemoglobin by Haptoglobin: Molecular Mechanisms and Clinical Applications

Haptoglobin (Hp) is an abundant plasma protein controlling the fate of hemoglobin (Hb) released from red blood cells after intravascular hemolysis. The complex formed between Hp and Hb is extraordinary strong, and once formed, this protein-protein association can be considered irreversible. A model of the Hp-Hb complex has been generated and the first steps toward understanding the mechanism behind the shielding effects of Hp have been taken. The clinical potential of the complex for modulating inflammatory reactions and for functioning as an Hb-based oxygen carrier have been described. The three-dimensional structure of the Hp-Hb complex is unknown. Moreover, Hp is not a homogeneous protein. There are two common alleles at the Hp genetic locus denoted Hp1 and Hp2, which when analyzed on the protein levels result in differences between their physiological behavior, particularly in their shielding against Hb-driven oxidative stress. Additional cysteine residues on the α-subunit allow Hp2 to form a variety of native multimers, which influence the biophysical and biological properties of Hp. The multimeric conformations, in turn, also modulate the glycosylation patterns of Hp by steric hindrance. A detailed analysis of the influence of Hp glycosylation will be instrumental to generate a deeper understanding of its biological function. Several pathological conditions also modify the glycan compositions allowing Hp to be potentially used as a marker protein for these disorders.

Purification and characterization of a heteromultimeric glycoprotein from Artocarpus heterophyllus latex with an inhibitory effect on human blood coagulation.

Plant latex has many health benefits and has been used in folk medicine. In this study, the biological effect of Artocarpus heterophyllus (jackfruit) latex on human blood coagulation was investigated. By a combination of heat precipitation and ion-exchange chromatography, a heat stable heteromultimeric glycoprotein (HSGPL1) was purified from jackfruit milky latex. The apparent molecular masses of the monomeric proteins on SDS/PAGE were 33, 31 and 29 kDa. The isoelectric points (pIs) of the monomers were 6.63, 6.63 and 6.93, respectively. Glycosylation and deglycosylation tests confirmed that each subunit of HSGPL1 formed the native multimer by sugar-based interaction. Moreover, the multimer of HSGPL1 also resisted 2-mercaptoethanol action. Peptide mass fingerprint analysis indicated that HSGPL1 was a complex protein related to Hsps/chaperones. HSGPL1 has an effect on intrinsic pathways of the human blood coagulation system by significantly prolonging the activated partial thrombin time (APTT). In contrast, it has no effect on the human extrinsic blood coagulation system using the prothrombin time (PT) test. The prolonged APTT resulted from the serine protease inhibitor property of HSGPL1, since it reduced activity of human blood coagulation factors XI(a) and α-XII(a).

Glycoproteomic analysis and molecular modeling of haptoglobin multimers

Extra-thiol groups on the α-subunit allow haptoglobin (Hp) to form a variety of native multimers which influence the biophysical and biological properties of Hp. In this work, we demonstrated how differences of multimeric conformation alter the glycosylation of Hp. The isoform distributions of different multimers were examined by an alternative approach, i.e. 3-D-(Native/IEF/SDS)-PAGE, which revealed differences in N-glycosylation among individual multimers of the same Hp sample. Glycomic mapping of permethylated N-glycan indicated that the assembled monomer and multimeric conformation modulate the degree of glycosylation, especially the reduction in terminal sialic acid residues on the bi-antennary glycan. Loss of the terminal sialic acid in the higher order multimers increases the number of terminal galactose residues, which may contribute to conformation of Hp. A molecular model of the glycosylated Hp multimer was constructed, suggesting that the effect of steric hindrance on multimeric formation is critical for the enlargement of the glycan moieties on either side of the monomer. In addition, N241 of Hp was partially glycosylated, even though this site is unaffected by steric consideration. Thus, the present study provides evidence for the alteration of glycan structures on different multimeric conformations of Hp, improving our knowledge of conformation-dependent function of this glycoprotein.

Native multimer analysis of plasma and platelet von Willebrand factor compared to denaturing separation: Implication for the interpretation of satellite bands

Blue native electrophoresis (BNE) was applied to analyze the von Willebrand factor (vWF) multimers in their native state and to present a methodology to perform blue native electrophoresis on human plasma proteins, which has not been done before. The major difference between this method and the commonly used SDS-agarose gel electrophoresis is the lack of satellite bands in the high-resolution native gel. To further analyze this phenomenon, a second dimension was performed under denaturing conditions. Thereby, we obtained a pattern in which each protein sub-unit from the first dimension dissociates into three distinct sub-bands. These bands confirm the triplet structure, which consists of an intermediate band and two satellite bands. By introducing the second dimension, our novel method separates the triplet structure into a higher resolution than the commonly used SDS-agarose gel electrophoresis does. This helps considerably in the classification of ambiguous von Willebrand's disease subtypes. In addition, our method has the additional advantage of being able to resolve the triplet structure of platelet vWF multimers, which has not been identified previously through conventional SDS-agarose electrophoresis multimer analysis. This potential enables us to compare the triplet structure from platelet and plasmatic vWF, and may help to find out whether structural abnormalities concern the vWF molecule in the platelet itself, or whether they are due to the physiological processing of vWF shed into circulation. Owing to its resolution and sensitivity, this native separation technique offers a promising tool for the analysis and detection of von Willebrand disorder, and for the classification of von Willebrand's disease subtypes.

Disulfide-bonded multimers of proteoglycan 4 (PRG4) are present in normal synovial fluids

Background The proteoglycan 4 ( PRG4) gene encodes for a mucin-like O-linked glycosylated protein with several names, including lubricin and superficial zone protein. The objective of this study was to analyze PRG4 in normal bovine calf and steer synovial fluids for evidence of native multimers formed by intermolecular disulfide bonds. Methods A combination of mucin biochemical techniques, with antibodies to both terminal domains and the mucin-like domain of PRG4, were used for analyses. Results Multimers were present in both calf and steer fluids, and reduction and alkylation converts the multimeric complex (likely dimeric) into monomeric subunits. Tandem mass spectrometry analyses supported the Western blot data and identified PRG4 in the reduced ∼ 345 kDa monomeric form. Interestingly, ∼ 70 kDa fragments released upon reduction contained peptides from both the N and C terminal regions, which most likely represent fragments of a sparsely glycosylated PRG4 population that are disulfide-linked to extensively glycosylated, intact monomers. Conclusions The analyses described here have demonstrated the presence of native disulfide-bonded multimers of PRG4 in normal bovine synovial fluids. General significance These structures are similar to those described for multimerization of mucins in general. Such multimerization and proteolytic cleavage of PRG4 may have functional significance in joint health and disease.

Novel molecular architecture of the multimeric archaeal PEP-synthase homologue (MAPS) from Staphylothermus marinus

The phosphoenolpyruvate (PEP)-synthases belong to the family of structurally and functionally related PEP-utilizing enzymes. The only archaeal member of this family characterized thus far is the Multimeric Archaeal PEP-Synthase homologue from Staphylothermus marinus (MAPS). This protein complex differs from the bacterial and eukaryotic representatives characterized to date in its homomultimeric, as opposed to dimeric or tetrameric, structure. We have probed the molecular architecture of MAPS using limited proteolytic digestion in conjunction with electron microscopic, biochemical, and biophysical techniques. The 2.2 MDa particle was found to be organized in a concentric fashion. The 93.7 kDa monomers possess a pronounced tripartite domain structure and are arranged such that the N-terminal domains form an outer shell, the intermediate domains form an inner shell, and the C-terminal domains form a core structure responsible for the assembly into a multimeric complex. The core domain was shown to be capable of assembling into the native multimer by recombinant expression in Escherichia coli. Deletion mutants as well as a synthetic peptide were investigated for their state of oligomerization using native polyacrylamide gel electrophoresis, molecular sieve chromatography, analytical ultracentrifugation, circular dichroism (CD) spectroscopy, and chemical cross-linking. Our data confirmed the existence of a short C-terminal, α-helical oligomerization motif that had been suggested by multiple sequence alignments and secondary structure predictions. We propose that this motif bundles the monomers into six groups of four. An additional formation of 12 dimers between globular domains from different bundles leads to the multimeric assembly. According to our model, each of the six bundles of globular domains is positioned at the corners of an imaginary octahedron, and the helical C-terminal segments are oriented towards the centre of the particle. The edges of the octahedron represent the dimeric contacts. Phylogenetic analysis suggests that the ancient predecessor of this family of enzymes contained the C-terminal oligomerization motif as a feature that was preserved in some hyperthermophiles.

In vitro antigen challenge of human antibody libraries for vaccine evaluation: the human immunodeficiency virus type 1 envelope.

Human antibody responses, or versions thereof, can be cloned as phage display libraries. In vaccine evaluation, the possibility therefore exists of challenging the human response in vitro, rather than in vivo, in order to assist in establishing the most promising vaccine leads. The characteristics of the antibodies retrieved directly indicate the strengths and weaknesses of the vaccine at the molecular level. We applied this approach to compare recombinant and native human immunodeficiency virus type 1 envelope preparations. We conclude that recombinant gp160, gp140, and, to a lesser extent, gp120 present epitopes around the CD4 binding site in a conformation different from that of the native multimer and contrary to expected vaccine requirements. Antibodies to the potently neutralizing b12 epitope were selected preferentially from an immune library by purified human immunodeficiency virus type 1 virions. This suggests that b12 is a major epitope on the virions, in contrast to recombinant envelope preparations, in which related, weakly neutralizing epitopes predominate. Although the majority of virions in the preparation used are expected to be noninfective, it appears that they predominantly express a native envelope configuration and would be able to elicit potent neutralizing antibodies.

Reconstruction of symmetry-related molecules from protein data bank (PDB) files

Many natural proteins are active as multimers. Crystallographic protein databases, however, generally store only part of the native multimer, the asymmetric unit, along with symmetry information. As a result of inaccuracies in the data, it is not always possible to reconstruct the native multimer. Here, a set of methods is presented that are designed to cope with inconsistencies in symmetry information. Applications include the validation of Protein Data Bank entries and the automatic generation of symmetry contacts for inspection and analysis.