Heterobifunctional Cross-linking Reagent Research Articles

Enzymatic Protein Immobilization for Nanobody Array.

Antibody arrays play a pivotal role in the detection and quantification of biomolecules, with their effectiveness largely dependent on efficient protein immobilization. Traditional methods often use heterobifunctional cross-linking reagents for attaching functional residues in proteins to corresponding chemical groups on the substrate surface. However, this method does not control the antibody's anchoring point and orientation, potentially leading to reduced binding efficiency and overall performance. Another method using anti-antibodies as intermediate molecules to control the orientation can be used but it demonstrates lower efficiency. Here, we demonstrate a site-specific protein immobilization strategy utilizing OaAEP1 (asparaginyl endopeptidase) for building a nanobody array. Moreover, we used a nanobody-targeting enhanced green fluorescent protein (eGFP) as the model system to validate the protein immobilization method for building a nanobody array. Finally, by rapidly enriching eGFP, this method further highlights its potential for rapid diagnostic applications. This approach, characterized by its simplicity, high efficiency, and specificity, offers an advancement in the development of surface-modified protein arrays. It promises to enhance the sensitivity and accuracy of biomolecule detection, paving the way for broader applications in various research and diagnostic fields.

Serum Albumin Domain Structures in Human Blood Serum by Mass Spectrometry and Computational Biology

Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low density of cross-link data has so far precluded its use in determining structures de novo. Cross-linking density has been typically limited by the chemical selectivity of the standard cross-linking reagents that are commonly used for protein cross-linking. We have implemented the use of a heterobifunctional cross-linking reagent, sulfosuccinimidyl 4,4′-azipentanoate (sulfo-SDA), combining a traditional sulfo-N-hydroxysuccinimide (sulfo-NHS) ester and a UV photoactivatable diazirine group. This diazirine yields a highly reactive and promiscuous carbene species, the net result being a greatly increased number of cross-links compared with homobifunctional, NHS-based cross-linkers. We present a novel methodology that combines the use of this high density photo-cross-linking data with conformational space search to investigate the structure of human serum albumin domains, from purified samples, and in its native environment, human blood serum. Our approach is able to determine human serum albumin domain structures with good accuracy: root-mean-square deviation to crystal structure are 2.8/5.6/2.9 Å (purified samples) and 4.5/5.9/4.8Å (serum samples) for domains A/B/C for the first selected structure; 2.5/4.9/2.9 Å (purified samples) and 3.5/5.2/3.8 Å (serum samples) for the best out of top five selected structures. Our proof-of-concept study on human serum albumin demonstrates initial potential of our approach for determining the structures of more proteins in the complex biological contexts in which they function and which they may require for correct folding. Data are available via ProteomeXchange with identifier PXD001692.

Surface Modification of Natural Rubber Latex Film Using Photocrosslinkable Chitosan

Surface modifications of natural rubber latex films were carried out to improve the hydrophilicity by UV-induced immobilization of water-soluble butyryl derivative of chitosan using the photosensitive hetero-bifunctional crosslinking reagent, 4-azidobenzoic acid. The effects of the concentration of modified chitosan solution and UV irradiation time on the water contact angle values of natural rubber latex films were studied. ATR-FTIR analyses confirmed that the O-butylrylchitosan molecules were covalently bonded onto the surface of natural rubber latex film. The water contact angle value of natural rubber latex film which is about 95o reduces to 55o significantly with the introduction of O-butylrylchitosan. The results indicate that the hydrophilic behaviour of natural rubber latex film has been improved by immobilization of O-butylrylchitosan.

Synthesis of peptide-oligonucleotide conjugates using a heterobifunctional crosslinker.

Peptide-oligonucleotide conjugates (POCs) are molecular chimeras composed of a nucleic acid moiety covalently attached to a polypeptide moiety. POCs have been used in numerous applications from therapeutics to nanotechnology, and most recently as combinatorial agents in the assembly of bivalent protein affinity reagents. This unit describes the synthesis and purification of POC molecules using the heterobifunctional crosslinking reagent succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), which enables amine-modified oligonucleotides to become covalently linked to cysteine-modified polypeptides. This solution-based protocol consists of a two-step synthesis followed by a single purification step.

Chitosan Modification of Adenovirus to Modify Transfection Efficiency in Bovine Corneal Epithelial Cells

BackgroundThe purpose of this study is to modulate the transfection efficiency of adenovirus (Ad) on the cornea by the covalent attachment of chitosan on adenoviral capsids via a thioether linkage between chitosan modified with 2-iminothiolane and Ad cross-linked with N-[γ-maleimidobutyryloxy]succinimide ester (GMBS).Methodology/Principal FindingsModified Ad was obtained by reaction with the heterobifunctional crosslinking reagent, GMBS, producing maleimide-modified Ad (Ad-GMBS). Then, the chitosan-SH was conjugated to Ad-GMBS via a thioether bond at different ratios of Ad to GMBS to chitosan-SH. The sizes and zeta potentials of unmodified Ad and chitosan-modified Ads were measured, and the morphologies of the virus particles were observed under transmission electron microscope. Primary cultures of bovine corneal epithelial cells were transfected with Ads and chitosan-modified Ads in the absence or presence of anti-adenovirus antibodies. Chitosan modification did not significantly change the particle size of Ad, but the surface charge of Ad increased significantly from −24.3 mV to nearly neutral. Furthermore, primary cultures of bovine corneal epithelial cells were transfected with Ad or chitosan-modified Ad in the absence or presence of anti-Ad antibodies. The transfection efficiency was attenuated gradually with increasing amounts of GMBS. However, incorporation of chitosan partly restored transfection activity and rendered the modified antibody resistant to antibody neutralization.Conclusions/SignificanceChitosan can provide a platform for chemical modification of Ad, which offers potential for further in vivo applications.

Evaluation of Synthetic Cell-Penetrating Peptides, Pro-Rich Peptide and Octaargine Derivatives, as Adenovirus Vector Carrier

Two cell-penetrating peptides, a Pro-rich peptide derivative, acetyl-(Val-Arg-Leu-Pro-Pro-Pro)(3)-Gly-Cys amide, and an octaarginine derivative, acetyl-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Cys amide, were prepared by the solid phase method. Each peptide was coupled to the heterobifunctional cross-linking reagent, 6-maleimidohexanoic acid N-hydroxysuccinimide ester, and then conjugated to the Adenovirus vector containing luciferase gene. Peptide-modified Ad, as compared with wild-type Ad, exhibited excellent luciferase activity in B16BL6 cells.

Phospholemman Recruits Peroxiredoxin 6 to the Cardiac Sodium Pump

Phospholemman (FXYD1, PLM), the principal sarcolemmal substrate for protein kinases A and C in the heart, is a regulator of the cardiac sodium pump. We investigated proteins that interact with PLM in adult rat ventricular myocytes using bifunctional crosslinking reagents and co-immunoprecipitation.Digitonin-permeabilized ventricular myocytes were treated with the heterobifunctional crosslinking reagent sulfo-lc-smpt (distance between reactive groups 2nm, reactive towards amino and sulfhydryl amino acid side chains), and cell lysates immunoblotted for PLM. We found sulfo-lc-smpt quantitatively crosslinked PLM to a 20-25kDa protein (electrophoretic mobility of PLM 15kDa, electrophoretic mobility of crosslinked adduct 37kDa). Co-immunoprecipitation experiments indicated that the crosslinked adduct was PLM linked to the anti-oxidant protein peroxiredoxin 6 (prdx6). PLM phosphorylation at serine 68 (protein kinase A activation with 10μM forskolin) or at serines 63, 68 and threonine 69 (protein kinase C activation with 300nM PMA) had no effect on the ability of sulfo-lc-smpt to crosslink PLM to prdx6. Hydrogen peroxide treatment of ventricular myocytes (1-100μM) was also without effect on the binding of prdx6 to PLM.In conclusion, our data suggest a new role for PLM in the heart. As well as being responsible for kinase-mediated regulation of the cardiac sodium pump, it is responsible for recruitment of prdx6 to this ion transporter. Prdx6 catalyzes the reduction of hydrogen peroxide, fatty acid hydroperoxides and phospholipid hydroperoxides using glutathione. Given the well-established sensitivity of the alpha subunit of the sodium pump to cysteine oxidation, recruitment of prdx6 to the sodium pump by PLM may be important to maintain pump activity during periods of oxidative stress.

Enzyme-independent, orientation-selective conjugation of whole human complement C3 to protein surfaces

Complement C3 is a central component of the humoral immune system. Upon triggering of the complement cascade, proteolytic fragments of C3 mediate important processes such as opsonization and lymphocyte activation. C3 possesses an internal thioester that mediates covalent attachment of proteolytically activated C3 to target surfaces. Treatment of native C3 with methylamine cleaves the thioester bond and exposes a free sulfhydryl group at the target-binding face of the protein. Through the use of sulfhydryl-reactive heterobifunctional cross-linking and biotinylation reagents, we demonstrate the capacity to form stable, multimeric whole human C3-protein conjugates in a fashion reflecting the orientation of physiologically-activated C3. We speculate that this C3 conjugation strategy presents a route for targeting dendritic cells and macrophages. In addition, manipulation of the thioester bond could enhance the study of biological roles of C3 and related proteins such as C4, and also of transmissible agents that exploit complement function such as prions.

Preparation of oligonucleotide microarrays on modified glass using a photoreactive heterobifunctional reagent, 1- N-(maleimidohexanoyl)-6- N-(anthraquinon-2-oyl) hexanediamine (MHAHD)

A novel approach for the construction of oligonucleotide microarrays under the influence of light and microwaves is described. For that purpose, a new heterobifunctional crosslinking reagent, 1- N-(maleimidohexanoyl)-6- N-(anthraquinon-2-oyl) hexanediamine (MHAHD), possessing a photoactive anthraquinone moiety on one terminus and an electrophilic maleimide group on the other one, was developed. The immobilization of oligonucleotides using the reagent, MHAHD, was realized via two routes (A and B). In order to speed up the immobilization procedure, the reaction between 3′- or 5′-mercaptoalkylated oligonucleotides and maleimide moiety of MHAHD was carried out under microwaves in just 15 min. The oligonucleotide arrays produced by both the routes were analyzed by hybridization experiments (hybridization efficiency 30.13%) and subsequently used for the discrimination of base mismatches. The constructed microarrays were found to possess good thermal stability (only ∼4.5% loss of fluorescence intensity observed after ten cycles).

The Heparin/Heparan Sulfate Sequence That Interacts with Cyclophilin B Contains a 3-O-Sulfated N-Unsubstituted Glucosamine Residue

Many of the biological functions of heparan sulfate (HS) proteoglycans can be attributed to specialized structures within HS moieties, which are thought to modulate binding and function of various effector proteins. Cyclophilin B (CyPB), which was initially identified as a cyclosporin A-binding protein, triggers migration and integrin-mediated adhesion of peripheral blood T lymphocytes by a mechanism dependent on interaction with cell surface HS. Here we determined the structural features of HS that are responsible for the specific binding of CyPB. In addition to the involvement of 2-O,6-O, and N-sulfate groups, we also demonstrated that binding of CyPB was dependent on the presence of N-unsubstituted glucosamine residues (GlcNH2), which have been reported to be precursors for sulfation by 3-O-sulfotransferases-3 (3-OST-3). Interestingly, 3-OST-3B isoform was found to be the main 3-OST isoenzyme expressed in peripheral blood T lymphocytes and Jurkat T cells. Moreover, down-regulation of the expression of 3-OST-3 by RNA interference potently reduced CyPB binding and consequent activation of p44/42 mitogen-activated protein kinases. Altogether, our results strongly support the hypothesis that 3-O-sulfation of GlcNH2 residues could be a key modification that provides specialized HS structures for CyPB binding to responsive cells. Given that 3-O-sulfation of GlcNH2-containing HS by 3-OST-3 also provides binding sites for glycoprotein gD of herpes simplex virus type I, these findings suggest an intriguing structural linkage between the HS sequences involved in CyPB binding and viral infection.

Studies on Heterobifunctional Cross-Linking Reagents, 6-Maleimidohexanoic Acid Active Esters

6-maleimidohexanoic acid N-hydroxysuccinimide ester has been used widely for preparation of enzyme immunoconjugates as a unique heterobifunctional cross-linking reagent. Its heterobifunctional reactivity is good, but its ester portion hydrolyzes easily in the presence of water. Several 6-maleimidohexanoic acid active esters (6-maleimidohexanoic acid 4-nitrophenyl ester, 6-maleimidohexanoic acid N-hydroxy-5-norbornene-endo-2,3-dicarboximide ester, and 6-maleimidohexanoic acid pentafluorophenyl ester) were prepared and their reactivity and stability in an aqueous media were tested. Of the synthetic esters, the pentafluorophenyl ester exhibited the highest reactivity and stability in aqueous media.

Triple helix stabilization by covalently linked DNA–bisbenzimidazole conjugate synthesized by maleimide–thiol coupling chemistry

Tethering of BBZPNH 2, an analogue of the Hoechst 33258, with a 14 nucleotide long DNA sequence with the help of succinimidyl-4-( N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), a heterobifunctional crosslinking reagent, using DMF/ water as solvent yields a conjugate which effectively stabilizes the triple helix. The above conjugate was hybridized with 26 bp long double stranded (ds) DNA having 14 bp long polypurine–polypyrimidine stretch to form a pyrimidine motif triple helix. The above conjugate increases the thermal stability of both the transitions, that is, triple helix to double helix by 12 °C and double helix to single strand transition by 16 °C for the triple helix formed with conjugated TFO over the triple helix made from non-conjugated TFO. Fluorescence and circular dichroism spectra recorded at different temperatures confirm the presence of minor groove binding bisbenzimidazole in the AT-rich minor groove of dsDNA even after the major groove bound TFO separates out.

Dantrolene Stabilizes Domain Interactions within the Ryanodine Receptor

Interdomain interactions between N-terminal and central domains serving as a "domain switch" are believed to be essential to the functional regulation of the skeletal muscle ryanodine receptor-1 Ca(2+) channel. Mutational destabilization of the domain switch in malignant hyperthermia (MH), a genetic sensitivity to volatile anesthetics, causes functional instability of the channel. Dantrolene, a drug used to treat MH, binds to a region within this proposed domain switch. To explore its mechanism of action, the effect of dantrolene on MH-like channel activation by the synthetic domain peptide DP4 or anti-DP4 antibody was examined. A fluorescence probe, methylcoumarin acetate, was covalently attached to the domain switch using DP4 as a delivery vehicle. The magnitude of domain unzipping was determined from the accessibility of methylcoumarin acetate to a macromolecular fluorescence quencher. The Stern-Volmer quenching constant (K(Q)) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22 degrees C and was unaffected by calmodulin. [(3)H]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca(2+) release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37 degrees C, yet required the presence of calmodulin at 22 degrees C. These results suggest that the mechanism of action of dantrolene involves stabilization of domain-domain interactions within the domain switch, preventing domain unzipping-induced channel dysfunction. We suggest that temperature and calmodulin primarily affect the coupling between the domain switch and the downstream mechanism of regulation of Ca(2+) channel opening rather than the domain switch itself.

Construction of an immunotoxin with the pore forming protein StI and ior C5, a monoclonal antibody against a colon cancer cell line

Sticholysin I (StI), a potent cytolysin isolated from the sea anemone Stichodactyla helianthus, was linked to the monoclonal antibody (mAb) ior C5. StI acts by forming hydrophilic pores in the membrane of the attacked cells leading to osmotic lysis. ior C5 is a murine IgG 1, which recognizes the tumor associated antigen (TAA) ior C2. The cytolysin and the mAb were coupled by using the heterobifunctional cross-linking reagent sulfosuccinimidyl 4-( N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). Two hybrid molecules composed by one ior C5 and one or two StI molecules were obtained (named conjugated I and II, respectively). The purified conjugates were evaluated by a binding affinity assay against an ior C2-positive colon cancer cell line (SW948). Both molecules were able to recognize the antigen (Ag) in the same way that unconjugated ior C5 does. The activity of both conjugates against human erythrocytes and SW948 cells was assessed. They lost most of their hemolytic activity but their residual activity was very similar. Nevertheless, when their cytotoxicity was studied on the SW948 cell line, only conjugate II killed efficiently the cells, indicating a specific mAb-Ag interaction. In this chimeric molecule the ratio between the cytotoxic and the hemolytic activity was larger than that of the free cytolysin. This fact indicates an increase of the specificity of the toxic effect toward the SW948 cell line and consequently an increase of the difference between its hemolytic and cytotoxic doses. The results herein support the feasibility of directing StI to the surface of cancer cells expressing ior C2 Ag via the mAb ior C5.

Biologically Active Decorin Is a Monomer in Solution

It has been reported that decorin and its protein core can have molecular masses nearly double the size of those previously published, suggesting a dimeric structure. In this study we tested whether biologically active decorin and its glycoprotein core would form dimers in solution. We used homo- and hetero-bifunctional chemical cross-linking reagents, BS3 and sulfo-SMPB, respectively, as well as glutaraldehyde and found no preferential dimer formation, whether chemical cross-linking was performed in the presence or absence of live cells. Under the same experimental conditions, we easily detected dimers of epidermal growth factor receptor and basic fibroblast growth factor, two glycoproteins known to dimerize. Only at very high cross-linker to decorin molar ratios (2000:1) were trimers and multimers observed, but performing the chemical cross-linking in the presence of a reducing agent abolished these. The elution of decorin protein core in Superose 6 gel chromatography gave masses compatible with monomeric proteins, both before and after denaturation with 2.5 M guanidine HCl. Matrix-assisted laser desorption ionization gave a mass of 44,077 Da for decorin protein core, without any evidence of dimers or oligomers. Extensive oligomerization of the decorin protein core was observed only after dialysis against water and freeze-drying. These oligomers were considered artifacts because they were independent of chemical cross-linking and were resistant to heat denaturation and disulfide-bond reduction. Oligomeric preparations showed markedly reduced biological activity in both phosphorylation and collagen fibrillogenesis assays. Thus, biologically active decorin is a monomer in solution and, as such, is a monovalent ligand for various extracellular matrix proteins, growth factors, and cell surface receptors.

Preparation of immunoconjugates using antibody oligosaccharide moieties.

Heterobifunctional crosslinking reagents are small molecular weight chemicals containing two different reactive groups that have become important tools in generating conjugates of two different biomolecules, such as two proteins. The resulting bioconjugates are hybrid molecules or proteins, a new category of biomolecules that exhibit the combined functions of the two parent biomolecules. An important category of hybrid proteins are conjugates of antibodies with other effector molecules, such as drugs or toxins. These antibody conjugates or immunoconjugates have a variety of the applications in medicine, with particular emphasis on the treatment of cancer. The most commonly used heterobifunctional crosslinking reagents for the synthesis of antibody conjugates contain an N-hydroxysuccinimide ester moiety, which allows derivatization of amino groups in proteins. The chemical modification of a functionally important amino group in the antigen-binding region of an antibody causes impairment or loss of the antigen binding function, resulting in a defective antibody conjugate that lacks one of its component functions. Furthermore, even if the chemical derivatization does not affect the antigen binding function, the subsequent coupling of an effector protein at or near the antigen-binding region can also cause the loss of the antigen binding function for steric reasons. In this chapter, heterobifunctional crosslinking reagents are described that allow the generation of antibody conjugates where the effector proteins are coupled to the antibody carbohydrate moieties. Because antibody carbohydrate moieties are distal from the antigen-binding region, the use of carbohydrate-directed heterobifunctional crosslinking reagents, such as S-(2-thiopyridyl)-L-cysteine hydrazide (TPCH), prevents inactivation of the antigen-binding function. The synthesis of two carbohydrate-directed heterobifunctional crosslinking reagents is described. Coupling protocols for the preparation of antibody conjugates with effector proteins of different sizes using carbohydrate-directed heterobifunctional crosslinking reagents are also provided.

Introduction of O-butyrylchitosan with a photosensitive hetero-bifunctional crosslinking reagent to silicone rubber film by radiation grafting and its blood compatibility

Based on an in vitro test for an improvement of the blood compatibility of silicone rubber (SR) films by grafting O-butyrylchitosan (OBCS), OBCS was covalently immobilized onto SR film surface using the photosensitive hetero-bifunctional crosslinking reagent, 4-azidobenzoic acid, which was previously bonded to OBCS by reaction between an acid group of the crosslinking reagent and a free amino group of OBCS. Surface properties of SR film were investigated by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), electron spectroscopy for chemical analysis (ESCA) and the water contact angle measurements. The blood compatibility of SR film was evaluated by platelet rich plasma (PRP) contacting experiments and the results were observed by scanning electron microscopy (SEM). The state of platelet adhesion was described. The suitable modifications could be carried out to tailor SR films biomaterial to meet the specific needs of different biomedical applications. These results suggest that the blood compatible of SR films/OBCS films show their suitability as potential biomaterials.

Covalent immobilization of O-butyrylchitosan with a photosensitive hetero-bifunctional crosslinking reagent on biopolymer substrate surface and bloodcompatibility characterization

O-Butyrylchitosan (OCS) was covalently immobilized onto a substrate (suture and PTFE) surface using the photosensitive hetero-bifunctional crosslinking reagent 4-azidobenzoic acid, which was previously bonded to OCS by the reaction between an acid group of the crosslinking reagent and a free amino group of OCS. The immobilization was accomplished by irradiating the modified OCS coated on the substrate surface with ultraviolet light to photolyze azide groups, thus the crosslinking OCS was immobilized on the substrate surface. The result indicated that OCS molecules immobilized on the substrate, and significantly reduced the fibrinogen adsorption and the deposition and spreading of platelets, demonstrating superior bloodcompatibility. Therefore, OCS could be developed into bloodcompatible biomaterial that used to modify the biomedical devices, such as PTFE and Dacron vascular prostheses surface.

One-End Immobilization of Individual DNA Molecules on a Functional Hydrophobic Glass Surface

We demonstrate an effective method for DNA immobilization on a hydrophobic glass surface. The new DNA immobilizing technique is extremely simple compared with conventional techniques that require heterobifunctional crosslinking reagent between DNA and substrate surface that are both modified chemically. In the first process, a coverslip was treated with dichlorodimethylsilane resulting in hydrophobic surface. γ DNA molecules were ligated with 3′-terminus disulfide-modified 14 mer oligonucleotides at one cohesive end. After reduction of the disulfide to sulfhydryl (thiol) groups the resulting thiol-modified γ DNA molecules were reacted on silanized coverslip. Fluorescent observation showed that the thiol-modified γ DNA molecules were anchored specifically to the hydrophobic surface at one terminus, although non-specific binding of the DNA molecules was suppressed. It was observed that the one-end-attached DNA molecule was bound firmly to the surface and stretched reversibly in one direction when a d.c. electric field was applied.

Covalent immobilization of chitosan/heparin complex with a photosensitive hetero-bifunctional crosslinking reagent on PLA surface

Chitosan (CS) was covalently immobilized onto polylactic acid (PLA) film surface using the photosensitive hetero-bifunctional crosslinking reagent, 4-azidobenzoic acid, which was previously bonded to chitosan by reaction between an acid group of the crosslinking reagent and a free amino group of chitosan. The immobilization was accomplished by irradiating with ultraviolet light, the modified chitosan being coated on the film surface to photolyze azide groups, thus crosslinking chitosan and PLA together. The hydroxyl and amino groups of chitosan may provide the opportunity for them being further derived by chemically immobilizing a variety of functional groups. These modifications could be carried out to tailor PLA biomaterial to meet the specific needs of different biomedical applications. For example, chitosan molecules immobilized on the PLA could be modified by heparin (Hp) solution to form a polyelectrolyte complex on the PLA surface. Platelet adhesion assay showed that PLA surface modified by chitosan/heparin complex could inhibit platelet adhesion and activation. Cell culture assay indicated that PLA surface with CS/Hp complex showed enhanced cell adhesion.