We conclude from observations in our model system that polyglutamine-expanded huntingtin protein not seen in large, visible aggregates is the toxic species in HD and that the site of toxicity is within the nucleus. During the course of our previous studies attempting to find a huntingtin nuclear localization signal 14 , we noted the high conservation of the first 18 amino acids of huntingtin in all vertebrate species. By molecular modeling, it appeared that huntingtin 1—18 may form an amphipathic alpha helical structure, with the hydrophobic face potentially involved in membrane interactions Fig.
This led us to hypothesize that this conserved domain may be involved in huntingtin sub-cellular localization. Immunofluorescence with mAB monoclonal antibody 20 in striatal-derived ST Hdh cells demonstrated that most of huntingtin protein was localized to the cytoplasm and perinucleus in a reticular structure Fig.
This signal had a high degree of overlap with the immunofluorescence signal from the ER lumen chaperone protein, calreticulin 21 Fig. Given this ER-localization of huntingtin, we then fused this 1—18 sequence to the amino-terminus of eYFP, keeping the huntingtin start methionine intact, and assayed localization of the fusion proteins in live striatal-derived ST Hdh cells by both confocal and wide-field deconvolution microscopy under minimal expression conditions.
The same localization was seen for huntingtin 1—, 1— fragments and full-length huntingtin as carboxyl-terminal eYFP fusions Fig. This localization appeared similar to what has been reported for huntingtin by immunofluorescence in ST Hdh cells 19 , 22 and human neurons Huntingtin 1—18 is highly conserved in all diverse vertebrate species.
B Huntingtin 1—18 modeled as an alpha-helix, on a helical wheel model. C Same model in space-filling representation. D Predicted hydrophobic face of huntingtin 1—18 with hydrophobic residues in yellow. E Predicted charged face with aspartic acids 5 and 12 highlighted in blue.
F Color key for D and E. A 3D deconvolution of huntingtin immunofluorescence with mAB anti-huntingtin monoclonal antibody panel b and anti-calreticulin antibody panel a. Nuclear DNA is stained with Hoechst dye in panel c, and the merged image with white merge of magenta-green is in panel d. B Three-dimensional image restoration of huntingtin 1—18, 1—, 1— expressed in STHdh cells, and huntingtin 1— full-length expressed in HEK cells for 24 h as carboxyl-terminal fusions to eYFP.
All huntingtin constructs are in Q15 wild-type context. Lanes show the nuclear fraction Nuclear , the post nuclear supernatant PNS and soluble and pellet fractions from 10 g S10, P10 , and g S, P centrifugation. Most of endogenous huntingtin co-fractionates with calreticulin in the high-speed fraction pellet see arrows. To test for huntingtin ER localization in vitro , we lysed and subjected ST Hdh cells to fractionation following nuclear isolation by low-speed and high-speed centrifugation Fig.
Unlike the ER lumen located calreticulin, huntingtin's 1—18 sequence suggested us that it may behave more like the hepatitis non-structural protein 5A NS5A ER membrane association signal, which is an amphipathic alpha helix We hypothesized that correct huntingtin ER localization may require an optimal ER, as several membrane resident proteins can mis-localize when stress is induced by the purification procedure.
When the cells were cooled prior to fixation, huntingtin signal was skewed towards the nucleus Fig. Huntingtin 1—eYFP was then tested for temperature-dependent localization to the ER by cooling and warming live cells in a heated tissue culture dish Fig. Huntingtin 1—eYFP was seen to reversibly target the ER depending on temperature, and could be cycled on and off repeatedly data not shown. Huntingtin 1—18 -eYFP was small enough to diffuse into and out of the nucleus when it was not targeted to the ER.
The same effect was seen with full-length endogenous huntingtin Fig. Huntingtin 1—18 ER-targeting is sensitive to ER temperature stress. See also Supplementary Material, Video S1. Taking advantage of our live cell system, we then tested whether huntingtin localization to the ER required energy.
Addition of an ATP-inhibition cocktail resulted in the release of huntingtin 1—18 from ER and diffusion into the nucleus of all cells observed representative cells in Fig. Loss of ER-targeting was also noted in response to cell treatments with UPR inducers tunicamycin or dithiothreitol Fig. Thus, huntingtin 1—18 targeting to the ER is reversible, requires energy and can be inhibited by inducers of ER stress. Huntingtin ER-targeting is ATP-dependent and sensitive to inducers of the unfolded protein response.
Transfected cells were identified by red fluorescence without observation of the green fluorescent channel, thus blinding the investigator to the data at the time of acquisition, a method described by others Cells were imaged capturing three fluorescent signal channels: the red channel to define mRFP and diffusion across the cell without any localization, the blue channel to define the area of the nucleus and the green channel to define the localization of eYFP fusions.
From this, percent cytoplasmic localization was determined for each construct over cells. As seen in Figure 5 , alanine point mutants on the predicted hydrophobic face of the helix Fig. However, a hydrophobic residue predicted to be on the opposite face Fig. These point-mutant data indicate that the sequence was behaving with an amphipathic nature. To test for the presence of structure, we substituted methionine 8 with a proline residue to induce a structure-breaking proline turn.
Mutations in the charged residues on the charged face of the helix at positions E5A and E12A resulted in increased vesicle targeting Fig. As with full-length huntingtin, huntingtin 1—eYFP was seen to co-fractionate with calreticulin, however, not in the nuclear fraction Fig. This suggested that while huntingtin 1—18 could target ER alone, specificity of this ER targeting was enhanced by additional sequences in huntingtin.
Huntingtin behaves as an amphipathic alpha helix in vivo. Quantitative cytoplasmic targeting assay with huntingtin 1—18 and mutant moieties expressed along with mRFP control and stained with Hoechst DNA dye. Panels A—D, wild-type huntingtin 1— Panels E—H, huntingtin methionine 8 to proline mutation. Merge of magenta and green images are shown as white in panels D versus H. R values of nuclear intensity correlation are shown in the top right corners of the merged images in D and H. Vesicles are highlighted by white arrows.
See also supplementary video 2. No affect of mutating basic residues K6, K9 or both to alanines on cytoplasmic targeting shown in M,N,O. Finally, to directly analyze and confirm the structure of huntingtin 1—18, we performed circular dichroism spectroscopy studies of the 1—18 sequence produced as a synthetic peptide Genescript. This spectrum did not change upon dilution, indicating that this was not likely a dimerization domain.
When incubated with synthetic unilamellar vesicles SUVs , the helical content was seen to change in the presence of the vesicles, indicating a conformational change of huntingtin 1—18 in direct response to the presence of membranes Fig. To test for the phenotype of the M8P mutation on huntingtin 1—18 structure, we performed CD spectroscopy on a 1—18 M8P synthetic peptide Genescript.
CD spectrum showed no helical content in the peptide Fig. These results by mutational studies in vivo and spectroscopy in vitro correlate to show that huntingtin 1—18 is an amphipathic alpha helix, and that this structure is required to recognize membranes. Huntingtin 1—18 is an amphipathic alpha helix in vitro.
A Circular dichroism spectroscopy of huntingtin 1—18 synthetic peptide at two concentrations, showing typical alpha-helical content, with no effect of dilution. To test for the physiological relevance of our described huntingtin 1—18 point mutants in the context of a larger fragment of huntingtin protein, we either deleted amino acids 2—13 or 5—13 in the context of huntingtin 1—[Q15] Fig. This fragment of huntingtin contains most of the known modifications and interaction domains of huntingtin interacting proteins, as well as the first three HEAT-repeats within huntingtin As with our previous assays, transfections were observed at minimal time points 12—14 h in live ST Hdh cells.
Deletion of huntingtin 2—13, 5—13 or the M8P mutation resulted in reduced ER targeting, and constitutive huntingtin nuclear entry, with M8P phenotype equal to deletion of residues 2—13 Fig. Therefore, it appeared that in the absence of ER targeting, huntingtin was actively entering the nucleus by sequences distal to the amino-terminal first 81 amino acids exon 1.
Inactivation of huntingtin ER targeting results in huntingtin nuclear entry, inhibition of polyglutamine-dependent aggregation and increased toxicity. D Scoring of percent cells with nuclear huntingtin present above threshold, comparing the huntingtin 1—18 deletions with just the M8P mutation and huntingtin in [Q15] and [Q] contexts, as carboxyl-terminal fusion to mRFP or amino-terminal fusions to eGFP, cells for each construct were counted.
E—F Effect of M8P mutation inhibiting polyglutamine-dependent aggregation of huntingtin 1— [Q] fragment. G—H Effect of M8P mutation in full-length [Q15] huntingtin context resulting in increased nuclear levels.
I Quantification of toxicity in SThdh striatal-derived cell line. In the context of toxic polyglutamine-expanded huntingtin 1— fragments, polyglutamine-expanded huntingtin Q will typically induce the formation of multiple large aggregates of protein within 18 h of expression, and cell death is then seen to occur Fig. This same fragment is also pathogenic in a HD mouse model With the M8P mutation in 1— huntingtin context, we observed two striking phenotypes: the complete absence of polyglutamine-mediated aggregation at Q or even at Q lengths data not shown , and increased huntingtin nuclear localization Fig.
However, despite the absence of any visible aggregates, the toxicity of huntingtin 1— M8P increased dramatically, but this toxicity was still polyglutamine-dependent Fig. Similar phenotypes were seen with M8P mutation in the context of full-length huntingtin, with increased nuclear localization Fig. The full-length huntingtin M8P also established that huntingtin 1—18 is the only signal in huntingtin required to associate with the ER in huntingtin. Therefore, it appeared that the amino-terminal membrane association activity in huntingtin was both important to trigger the onset of polyglutamine-dependent protein aggregation and modulate polyglutamine-mediated huntingtin toxicity.
This was to prevent any artifactual nuclear localization due to diffusion. Thus, the reversible huntingtin ER localization by 1—18 could result in huntingtin nuclear entry when huntingtin is released from the ER, through an active nuclear import ability within residues 81— in huntingtin. Huntingtin nuclear entry upon 1—80 deletion is mediated by distal sequences in 81— Nuclear localization at pre-bleach indicates an active nuclear localization.
Active transport was assayed by nuclear FRAP. Panel M, quantification of fluorescence recovery. Having established that huntingtin can target the ER and that disruption of this targeting could lead to nuclear entry of huntingtin and increased mutant huntingtin toxicity, we then sought to determine the specific vesicular population targeted by huntingtin 1— By live cell video analysis, we could see that the puncta being targeted by huntingtin 1—18 moved in a manner consistent with vesicles, with a concentration of signal near the microtubule organizing center Supplementary Material, Video S2.
Huntingtin localized to vesicular populations and ER, but did not co-localize with the early endosomal marker RhoB 28 , 29 Fig. Thus, huntingtin localization to vesicles via 1—18 is specific to late endosomes and autophagic vesicles, with vacuolar membrane localization evident Fig. Huntingtin 1—18 vesicular targeting is to late endosomes and autophagic vesicles.
Live cell co-localization analysis of huntingtin 1—[Q15]-mRFP magenta in ST Hdh cells after 14 hours expression with various endosomal markers green , and merged signal white. A—F Poor-co-localization with early endosomal marker RhoB. Vacuolar membrane localization is highlighted with white arrows in G —I. Results indicative of cells each observed in three replicate experiments.
Knowing the normal biological function of huntingtin is critical in understanding the role of polyglutamine-expansion in huntingtin during HD. Several groups have seen huntingtin localized to the ER, vesicles and the nucleus by a variety of methods, including immunofluorescence in fixed neurons and in tissue culture model cells 19 , 22 ; biochemical cell fractionation 12 , 15 ; and live cell imaging in tissue culture cells 14 , neurons 35 and transgenic Drosophila models 7 , 8.
Here, we describe the first 18 amino acids of huntingtin as a membrane-targeting domain that can mediate the association of huntingtin with the ER and late endosomes. This functional domain in huntingtin now directly ties huntingtin function to the ER. ER stress is becoming increasingly important in understanding the pathology of several protein-misfolding neurodegenerative diseases, including Parkinson's and Alzheimer's diseases Huntingtin over-expression has been seen to stimulate endosomal-lysosomal activity, endosomal tubulation and autophagy Reduction of huntingtin levels in cells by siRNA-mediated methods results in perturbation of the ER 40 , and genetic knockouts of huntingtin in embryonic stem cells demonstrated that huntingtin was essential for normal ER structure The huntingtin 1—18 sequence has the structure of an amphipathic alpha helix.
These structures are also seen in other ER and vesicle-associated proteins: the vesicle-associated membrane proteins, or VAMPs 16 amino acid signal 41 ; the ER-associated hepatitis C 5a protein 30 amino acid signal 23 ; and the yeast ER-associated nuclear receptor activator, AF2 We noted that huntingtin 1—18 ER-targeting ability appears to be saturable, by observing increasing live cell expression over time in excess of 48 h.
At very high levels of over-expression, excess huntingtin 1—18 appears soluble, but does not cause any toxicity data not shown. Also, using in vitro pure vesicle targeting assays with purified huntingtin 1—eYFP, we did not observe any direct targeting, nor could we see huntingtin 1—18 targeting other membranous structures like the plasma membrane, even when highly over-expressed data not shown.
This suggests that huntingtin 1—18's ability to target membranes may be limited by another factor, and is likely not due to direct membrane insertion. This is consistent with our observations of reversible huntingtin association upon temperature stress. This suggests that reversible association of huntingtin with ER and vesicles may be an important activity of huntingtin. The alpha helical structure is critical to huntingtin's ability to target the ER, as the M8P point-mutant or the hydrophobic residue point-mutants phenotype of disrupted membrane targeting is similar to deletion of the entire membrane association domain.
The mutations on the charged face of the helix indicate the importance of acidic residues, but not basic residues. The mutation of all basic lysines to arginines without effect indicates that lysine modifications such as acetylation and SUMOylation 18 are not required for huntingtin membrane association, but these data do not exclude the possibility that post-translational modification of 1—18 may be important for huntingtin release from the ER.
This function is likely important for huntingtin's biological role, as the 1—18 sequence is completely conserved in all vertebrate species of huntingtin. Upon sensing misfolded protein, Ire1p protein is modified and cleaved, resulting in nuclear entry of an Ire1p fragment and allowing its function as a transcription activator for a series of genes, including those that encode chaperones and cysteine isomerases.
ER stress induces the release of huntingtin from the ER, where it is then seen to actively enter the nucleus by distal sequences. Small fragment mouse transgenic models of HD have typically more severe phenotypes than full-length huntingtin models 26 , 44 , The ability to simply diffuse into the nucleus may be partially responsible for the accelerated phenotype in these models.
Others have shown that addition of exogenous nuclear export or import signals to mutant huntingtin can modulate its toxicity, with NES addition resulting in decreased toxicity Like Ire1p, huntingtin has been shown to be involved in transcription regulation for a series of genes containing neuronal restrictive silencing elements, or NRSEs 16 , But unlike Ire1p, huntingtin is not a lumenal protein, but at the cytoplasmic ER membrane. The concept of an ER membrane-bound protein translocating to the nucleus in response to stress is not novel.
The antioxidant response element ARE binding transcription factor, Nrf1, associates with the ER membrane via an amino-terminal helical transmembrane domain. In response to cellular oxidative stress, Nrf1's ER-targeting domain is cleaved, and Nrf1 enters the nucleus to act as a transcription activator for a series of genes involved in anti-oxidant activity The fact that we can observe reversible huntingtin targeting to ER via 1—18 in live cells indicates that the huntingtin membrane-association signal is likely not cleaved, and again suggests that it is not likely to be a direct membrane insertion signal.
Our data on huntingtin 1—18 activity mediating polyglutamine-dependent aggregation is consistent with data from yeast models that demonstrated that sequences flanking the polyglutamine tract can effect polyglutamine-mediated aggregation and toxicity However, in those studies, different amino-terminal fusions as well as proline-rich regions could modulate huntingtin exon1 fragment aggregation and toxicity, indicating that regions of huntingtin protein on either side of the polyglutamine tract control huntingtin toxicity, and may even work in concert for normal huntingtin function.
The proline-rich region in huntingtin carboxyl to the polyglutamine tract has been shown to mediate interaction with several vesicular proteins This suggests that the huntingtin proline-rich region interacting proteins may communicate with proteins bound to 1—18 across the polyglutamine tract to mediate specificity. Thus, huntingtin may act as a molecular scaffold with the polyglutamine tract as an essential component.
All species of vertebrate huntingtin contain at least four glutamines in this tract, and a glutamine-tract deletion mutant knockin HD mouse displays a neurologic phenotype The complete disruption of polyglutamine-dependent visible aggregates of huntingtin by the M8P mutation led to greatly increased toxicity, consistent to what has been seen by others with huntingtin in neurons 50 , and in another polyglutamine-expansion disease, SCA7 The polyglutamine expansion in M8P context may be in another, more toxic structural conformation than what is seen in classic large, visible aggregates.
This is a subject of further study, but indicates that there may be targets for therapeutic development in huntingtin protein in addition to the polyglutamine tract. One of the classic pathological observations in HD is the accumulation of the mutant huntingtin protein in neuronal nuclei Our data with huntingtin 1—18 function controlling huntingtin nuclear entry suggest that polyglutamine-expansion may subtly inhibit proper reversible ER-targeting or nuclear shuttling of huntingtin, thus resulting in increased nuclear levels over time.
In M8P[Q] huntingtin context, the protein was unable to form visible aggregates, yet was far more toxic than [Q] huntingtin. M8P[Q] huntingtin was also notably more nuclear than [Q] huntingtin. These data demonstrated that the polyglutamine-expanded huntingtin protein not seen in visible aggregates was the toxic species, and that the site of toxicity was within the nucleus.
The increased toxicity of M8P[Q] huntingtin in the absence of any visible aggregates also suggests that in our cell culture model, physical blocking of vesicular trafficking is not a trigger of cell toxicity, contrary to what has been suggested by others in Drosophila HD models 7.
The activities of vesicular interaction, trafficking and nuclear entry as a transcription factor for huntingtin are strikingly similar to that recently proposed for the amino-terminal Huntingtin-Interacting Protein 1, or Hip1 Our observations conclude that while huntingtin 1—18 is sufficient to target membranes, the specificity of that interaction to late endosomes is mediated by additional sequences in huntingtin.
Others have seen that huntingtin can interact with early endosomes via an interaction with the huntingtin-associated protein 40, or Hap40, which interacts with huntingtin via the carboxyl-terminus 9. This indicates that full-length huntingtin has the ability to interact with both early and late endosomes through different regions of the protein as a mechanism to traffic from synapses down long axons to the cell body. This also stresses the importance of analysis of huntingtin function in a full-length context.
However, the early endosome interaction may not be relevant to HD, as many mouse models show HD-like pathology in the absence of the carboxyl-terminus of huntingtin 26 , With wild-type huntingtin, localization of the amino-terminus is specific to the ER, late endosomes and autophagic vesicles, which are precursors to autophagic vacuoles. In HD patient lymphoblasts, the presence of autophagic vacuoles is greatly enhanced in a polyglutamine length-dependent manner Thus, huntingtin may have a normal role in the trafficking of vesicles and the formation of autophagic vacuoles.
Nuclear export may also be the mediator of this response back in the cytoplasm via exported factors Fig. This suggests that the normal role of huntingtin, present in all cells, is in nuclear-ER communication in response to ER stress. This stress response may be more critical to the health of neuronal cell populations such as those in the striatum and the cortex.
While we do not see any binary difference between wild type and mutant huntingtin for ER or vesicle targeting, this is consistent with the late age-onset, progressive and subtle nature of HD. Any effects of the polyglutamine expansion on huntingtin 1—18 activity will likely be understood better by the detailed analysis of huntingtin 1—18 direct interacting proteins, which are the subjects of future study.
Hypothetical model of huntingtin function in relation to sub-cellular localization signals. Huntingtin is an ER-localized protein, tethered to membranes by the amphipathic alpha helix in 1—18, allowing huntingtin to bind ER, late endosomes and autophagic vesicles. Early endosome localization is via Hap40 interaction at the carboxyl-terminus 9. Effects of huntingtin in the nucleus have been described by others, and the off switch of this activity is huntingtin nuclear export via its NES near the carboxyl terminus.
Inactivation of huntingtin 1—18 targeting by M8P mutation results in increased nuclear huntingtin, inhibition of aggregation and greatly increased toxicity, but only when expanded polyglutamine is present. The membrane association domain of huntingtin is therefore an important modulator of huntingtin function and mutant huntingtin nuclear entry and toxicity. This membrane-association domain may therefore be a good application of targeted drug design against HD.
The role of post-translational modification and signaling affecting huntingtin 1—18 function, as well as the nuclear import signal in huntingtin 81— will additionally be the subjects of further study.
The K D were displayed using line charts Figure 4 B. The combination and dissociation sensorgrams of the modified VHHs at the same concentration were presented in Figure S1. Compared with the corresponding samples without any treatment, the K D of NB, NB, NB and NB about increased to two-fold, and both on-rate and off-rate were influenced, indicating that extra processing steps including incubation and ultrafiltration slightly impacted the affinity of VHHs.
A Double logarithm coordinate was used; the diagonal line was equal affinity line K D. B Single logarithm coordinate was used to show the affinity variation directly. Different VHHs were distinguished by colors. Five samples were measured for each VHH. Briefly, the K D of NB-1 was limitedly affected, increasing from 1. The K D of NB-2 increased fold, from 7. The K D of NB-3 increased fold, from 1. By analyzing the kinetic properties Table S1 , it could be found that, the increase of K D were mainly caused by the increase of on-rate.
This result suggests that fluorescein induced VHH denaturation even when no covalent binding occurred. To identify the secondary structural changes resulted from amino modifications, the original and modified proteins were evaluated by far-UV CD spectra Figure 5 A. For the spectra of NB-1, there is no significant difference between the five groups, showing no substantial changes in the secondary structure among original or modified Nb When modified by increased molar ratios of NHS-Fluo, the spectra of NB-2 were accompanied by a faint red shift 2 nm around nm, and a simultaneous flattening of the spectra from nm to nm.
The spectra of modified NB-3 did not change obviously and has no significant shift or peak, indicating NB-3 is more stable than NB On one hand, like NB-2, the peak at nm shifted slightly and the spectra flattened out from nm to nm. On the other hand, the positive peaks around nm were dissimilar. It was clear from the drastic changes in the spectra that the amino modification affected the structure of NB-4 significantly. It is important to note that these results are consistent with the decrease of stability and bioactivity resulting from amino modification Figure 3 and Figure 4.
Five samples were taken for each VHH. The spectrum signals at nm were exported for thermal denaturation analysis. The effect of amino modification on the thermostability of four VHHs was evaluated by thermal denaturation analysis. The midpoints of the denaturation profiles were estimated to characterize the thermostability changes of four VHHs Table 3.
As modified by increased molar ratios of NHS-Fluo, NB-2 showed the biggest drop in denaturation temperature, followed by NB-3, and NB-1 that maintained good thermostability after modification. As observed from these results, the amino group modified products of NB-1 exhibited better resistant secondary structure against changes in temperature than NB-2 and NB Fluorescence imaging was performed to verify the application potential of fluorescein labeled VHH. Labeled NB-1 stained beads visualized by laser scanning confocal microscope.
In biotechnology applications like fluorescence immunostaining and immunodetection, labeled antibodies are required to diffuse rapidly to tissue, and bind to antigens robustly and specifically. Beijing, China. The structure with highest global model quantity estimation GMQE was employed as template for homology-modelling. After that, figures were prepared using Yasara [ 32 ]. The amino acid was numbered in Kabat scheme.
The residual NHS-Fluo was removed by ultrafiltration. After, the gel was stained with Coomassie brilliant blue and visualized. The blank channel used as a subtraction was blocked by ethanolamine. Before and after modifications, samples were analyzed in general approach. Samples were diluted by 20 mM phosphate buffer pH 8.
Far-UV CD spectra were obtained in a 10 mm path-length cell at room temperature. The curve was smoothed in BioKine, using Savitzky-Golay mathematics, 15 window pointsf, polynomial order 3. MOS and TCU Bio-Logic with a 10 mm path-length cell were used in combination for denature protein analysis and calculation of denaturation temperature. Before each measurement, wait for 1 min to equalize the temperature of samples. After that, the curve of ellipticity change with temperatures rise in nm was exported, and smoothed in Origin , using Savitzky-Golay arithmetic, with 15 points of window, polynomial order 2.
The fraction of native protein was calculated by formula F1 :. In the formula, F N represents the fraction of native protein, y t represents the optical rotation value in temperature t, y u represents the optical rotation value of completely denatured protein, and y N represents the optical rotation value of native protein.
Briefly, the protein was dissolved into a coupling buffer, composed of 0. In this study, we characterized the influence of four selected VHHs modified by fluorescein on their amino groups. The results fully demonstrated the individual variations of VHHs after amino modification, however, our analysis did not explain precisely which characteristics made VHHs robust during amino modification due to the complexity of VHHs. In particular, we found at last one VHH that retained its structural stability, antigen binding ability and thermostability after modification by NHS-Fluo, and a high titer of labels with good homogeneity was achieved.
The labeled VHHs presented good performance in fluorescence imaging. Accordingly, amino modification could be a good option for VHH functionalization. The combination and dissociation sensor grams of the different molar ratio modified VHHs in the same concentration. Table S1. The kinetic properties of modified VHHs.
Conceptualization J. The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Sample Availability: Not available. Published online Jul Author information Article notes Copyright and License information Disclaimer.
Received Jun 11; Accepted Jul This article has been cited by other articles in PMC. Associated Data Supplementary Materials moleculess Abstract The functionalization of VHHs enables their application in almost every aspect of biomedical inquiry. Keywords: VHHs, single domain antibodies, amino modification, protein structures, fluorescent.
Introduction Heavy-chain-only antibodies HcAbs are a unique type of antibody devoid of light chains, originally found in the serum of camelids [ 1 ]. Results and Discussion 2. Amino Group Distribution in Four VHHs Homology-modeling visualized the structure character and lysine residues distribution in three-dimensional space Table 1 and Figure 1.
Open in a separate window. Figure 1. Isomers Composition of the Modified VHHs Amino modification has been criticized for its inevitable generation of heterogeneous products. Figure 2. Figure 3. Figure 4. Figure 5. Table 3 Midpoints of the denature profiles of the modified VHHs. Figure 6. Materials and Methods 3. Conclusions In this study, we characterized the influence of four selected VHHs modified by fluorescein on their amino groups.
Click here for additional data file. Author Contributions Conceptualization J. Conflicts of Interest The authors declare no conflict of interest. Footnotes Sample Availability: Not available. References 1. Hamers-Casterman C. Naturally occurring antibodies devoid of light chains. Fang T. Nanobody immunostaining for correlated light and electron microscopy with preservation of ultrastructure. Giorgino T. Nanobody interaction unveils structure, dynamics and proteotoxicity of the Finnish-type amyloidogenic gelsolin variant.
Acta Mol. Basis Dis. Kabatas S. Costa S. Nano Lett. Fernandes J. Therapeutic application of antibody fragments in autoimmune diseases: Current state and prospects. Drug Discov. Platonova E. Van Lith S. Bioconjugate Chem.
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