Copper dependence of the biotin switch assay: modified assay for measuring cellular and blood nitrosated proteins

Studies have shown that modification of critical cysteine residues in proteins leads to the regulation of protein function. These modifications include disulfide bond formation, glutathionylation, sulfenic and sulfinic acid formation, and S-nitrosation. The biotin switch assay was developed to specifically detect protein S-nitrosation (S. R. Jaffrey et al., Nat. Cell Biol. 3:193-197; 2001). In this assay, proteins are denatured with SDS in the presence of methyl methane thiosulfonate (MMTS) to block free thiols. After acetone precipitation or Sephadex G25 separation to remove excess MMTS, HPDP-biotin and 1 mM ascorbate are added to reduce the S-nitrosothiol bonds and label the reduced thiols with biotin. The proteins are then separated by nonreducing SDS PAGE and detected using either streptavidin-HRP or anti-biotin-HRP conjugate. Our examination of this labeling scheme has revealed that the extent of labeling depends on the buffer composition and, importantly, on the choice of metal-ion chelator (DTPA vs EDTA). Unexpectedly, using purified S-nitrosated albumin, we have found that "contaminating" copper is required for the ascorbate-dependent degradation of S-nitrosothiol; this is consistent with the fact that ascorbate itself does not rapidly reduce S-nitrosothiols. Removal of copper from buffers by DTPA and other copper chelators preserves approximately 90% of the S-nitrosothiol, whereas the inclusion of copper and ascorbate completely eliminates the S-nitrosothiol in the preparation and increases the specific biotin labeling. These biotin switch experiments were confirmed using triiodide-based and copper-based reductive chemiluminescence. Additional modifications of the assay using N-ethylmaleimide for thiol blockade, ferricyanide pretreatment to stabilize S-nitrosated hemoglobin, and cyanine dye labeling instead of biotin are presented for the measurement of cellular and blood S-nitrosothiols. These results indicate that degradation of S-nitrosothiol in the standard biotin switch assay is metal-ion dependent and that experimental variability in S-nitrosothiol yields using this assay occurs secondary to the inclusion of metal-ion chelators in reagents and variable metal-ion contamination of buffers and labware. The addition of copper to ascorbate allows for a simple assay modification that dramatically increases sensitivity while maintaining specificity.     

Ascorbic acid reduction of microtubule protein disulfides and its relevance to protein S-nitrosylation assays

The biotin switch assay was developed to aid in the identification of S-nitrosylated proteins in different cell types. However, our work with microtubule proteins including tubulin and its associated proteins tau and microtubule-associated protein-2 shows that ascorbic acid is not a selective reductant of protein S-nitrosothiols as described in the biotin switch assay. Herein we show that ascorbic acid reduces protein disulfides in tubulin, tau, and microtubule-associated protein-2 that are formed by peroxynitrite anion. Reduction of microtubule-associated protein disulfides by ascorbic acid following peroxynitrite treatment restores microtubule polymerization kinetics to control levels. We also show that ascorbic acid reduces the disulfide dithiobis(2-nitrobenzoic acid), a reagent commonly used to detect protein thiols. Not only do we describe a new reactivity of ascorbic acid with microtubule proteins but we expose an important limitation when using the biotin switch assay to detect protein S-nitrosylation.     

Identification of protein nitrosothiols using phosphine-mediated selective reduction

Regulation of protein function by S-nitrosation of critical cysteines is known to be an important mechanism for nitric oxide signaling. Evidence for this comes from several different experimental approaches including the ascorbate-based biotin switch method. However technical problems with specificity and sensitivity of ascorbate reduction of S-nitrosothiols limit its usefulness and reliability. In the current study we report the use of triphenylphosphine ester derivatives to selectively reduce SNO bonds in proteins. After triphenylphosphine ester reduction, thiols were tagged with biotin or fluorescently labeled maleimide reagents. Importantly we demonstrate that these compounds are specific reductants of SNO in complex biological samples and do not reduce protein disulfides or protein thiols modified by hydrogen peroxide. Reduction proceeds efficiently in cell extracts and in whole fixed cells. Application of this approach allowed us to demonstrate S-nitrosation of specific cellular proteins, label S-nitrosoproteins in whole fixed cells (especially the nuclear compartment) and demonstrate S-nitrosoprotein formation in cells expressing inducible nitric oxide synthase.     

Sinapinic acid can replace ascorbate in the biotin switch assay

Background

Protein S-nitrosation is an important post-translational modification altering protein function. Interaction of nitric oxide with thiols is an active area of research, and is one of the mechanisms by which NO exerts its biological effects. Biotin switch assay is the method, which has been developed to identify S-nitrosated proteins. The major concern with biotin switch assay includes reducing disulfide which may lead to false positives. We report a modification of the biotin switch assay where sinapinic acid is utilized instead of ascorbate to eliminate potential artifacts in the detection of S-nitrosated proteins.

Methods

The denitrosation ability of sinapinic acid was assessed by monitoring either the NO or NO₂^- released by chemiluminescent NO detection or by the griess assay, respectively. DTNB assay was used to compare disulfide reduction by ascorbate and sinapinic acid. Sinapinic acid and ascorbate were compared in the biotin switch detection of S-nitrosoproteins in RAW 264.7 cells ± S-nitrosocysteine (CysNO) exposure.

Results

We show that sinapinic acid has the ability to denitrosate S-nitrosothiols at pH 7.0 and denitrate plus denitrosate at pHs 8 and 8.5. Unlike ascorbate, sinapinic acid degrades S-nitrosothiols, but it does not reduce disulfide bridges.

Conclusions

Sinapinic acid denitrosate RSNO and does not reduce disulfides. Thus can readily replace ascorbate in detection of S-nitrosated proteins in biotin switch assay.

General significance

The work described is important in view of protein S-nitrosation. In this study we provide an important modification that eliminates artifacts in widely used technique for detecting the S-nitrosoproteome, the biotin switch assay.     

Persistent S-nitrosation of complex I and other mitochondrial membrane proteins by S-nitrosothiols but not nitric oxide or peroxynitrite: implications for the interaction of nitric oxide with mitochondria

S-nitrosation of mitochondrial proteins has been proposed to contribute to the pathophysiological interactions of nitric oxide (NO) and its derivatives with mitochondria but has not been shown directly. Furthermore, little is known about the mechanism of formation or the fate of these putative S-nitrosothiols. Here we have determined whether mitochondrial membrane protein thiols can be S-nitrosated on exposure to free NO from 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA-NONOate) by interaction with S-nitrosoglutathione or S-nitroso-N-acetylpenicillamine (SNAP) and by the NO derivative peroxynitrite. S-Nitrosation of protein thiols was measured directly by chemiluminescence detection. S-Nitrosoglutathione and S-nitroso-N-acetylpenicillamine led to extensive protein thiol oxidation, with about 30% of the modified protein thiols persistently S-nitrosated. In contrast, there was no protein thiol oxidation or S-nitrosation on exposure to 3,3-bis (aminoethyl)-1-hydroxy-2-oxo-1-triazene. Peroxynitrite extensively oxidized protein thiols but produced negligible amounts of S-nitrosothiols. Therefore, mitochondrial membrane protein thiols are S-nitrosated by preformed S-nitrosothiols but not by NO or by peroxynitrite. These S-nitrosated protein thiols were readily reduced by glutathione, so S-nitrosation will only persist when the mitochondrial glutathione pool is oxidized. Respiratory chain complex I was S-nitrosated by S-nitrosothiols, consistent with it being an important target for S-nitrosation during nitrosative stress. The S-nitrosation of complex I correlated with a significant loss of activity that was reversed by thiol reductants. S-Nitrosation was also associated with increased superoxide production from complex I. These findings point to a significant role for complex I S-nitrosation and consequent dysfunction during nitrosative stress in disorders such as Parkinson disease and sepsis.     

A fluorescence resonance energy transfer-based binding assay for characterizing kinase inhibitors: Important role for C-terminal biotin tagging of the kinase

Novel biochemical strategies are needed to identify the next generation of protein kinase inhibitors. One promising new assay format is a competition binding approach that employs time-resolved fluorescence resonance energy transfer (TR–FRET). In this assay, a FRET donor is bound to the kinase via a purification tag, whereas a FRET acceptor is bound via a tracer-labeled inhibitor. Displacement of the tracer by an unlabeled inhibitor eliminates FRET between the fluorophores and provides a readout on binding. Although promising, this technique has so far been limited in applicability in part by a lack of signal strength is some cases and also by an inability to predict whether a particular tagging strategy will show robust FRET. In this work, we sought to better understand the factors that give rise to a strong FRET signal in this assay. We determined the magnitude of FRET for several tyrosine kinases using different purification tags (biotin, glutathione S-transferase [GST], and His) placed at either the N terminus or C terminus of the kinase. It was observed that coupling the FRET acceptor to the kinase C terminus using a biotin/streptavidin interaction resulted in the greatest increase in FRET. Specifically, for multiple kinases, the signal/background ratio was at least 3-fold better using C-terminal biotinylation compared with tagging at the N terminus using a His/anti-His antibody or GST/anti-GST antibody interaction. In one case, the FRET signal using C-terminal biotin tagging was more than 150-fold over background. This strong FRET signal facilitated development of improved inhibitor binding assays that required only tens of picomolar enzyme or tracer-labeled inhibitor. Together, these results indicate that C-terminal biotinylation is a promising tagging strategy for developing an optimal FRET-based competition binding assay for tyrosine kinases.     

Mechanism of S-nitrosation of recombinant human brain calbindin D28K

Mass spectrometry and UV-vis absorption results support a mechanism for NO donation by S-nitrosoglutathione (GSNO) to recombinant human brain calbindin D(28K) (rHCaBP) that requires the presence of trace copper, added as either Cu,Zn-superoxide dismutase (CuZnSOD) or CuSO(4). The extent of copper-catalyzed rHCaBP S-nitrosation depends on the ratio of protein to GSNO and on the reaction time, and NO-transfer is prevented when copper chelators are present. CuZnSOD is an efficient catalyst of rHCaBP S-nitrosation, and the mechanism of CuZnSOD-catalyzed S-nitrosation involves reduction of the active-site Cu(II) by a number of the five free thiols in rHCaBP, giving rise to thiyl radicals. The Cu(I)ZnSOD formed catalyzes the reductive cleavage of GSNO present in solution to give GSH and release NO. rHCaBP thiyl radicals react with NO to yield the S-nitrosoprotein. Cu(II)ZnSOD is also reduced by GSH in a concentration-dependent manner up to 5 mM but not at higher GSH concentrations. However, unlike the rHCaBP thiyl radicals, GS(*) radicals dimerize to GSSG faster than their reaction with NO. The data presented here provide a biologically relevant mechanism for protein S-nitrosation by small S-nitrosothiols. S-nitrosation is rapidly gaining recognition as a major form of protein posttranslational modification, and the efficient S-nitrosation of CaBP by CuZnSOD/GSNO is speculated to be of neurochemical importance given that CaBP and CuZnSOD are abundant in neurons.     

Characterization and application of the biotin-switch assay for the identification of S-nitrosated proteins

S-Nitrosation of protein cysteinyl residues has been suggested to be an important nitric oxide-dependent posttranslational modification. The so-called biotin-switch method has been developed to identify S-nitrosated proteins. This method relies on the selective reduction of S-nitrosothiols by ascorbate. In this study we have assessed the ability of ascorbate to reduce S-nitrosothiols and show that ascorbate is a very inefficient reducing agent. We show that higher concentrations of ascorbate and longer incubation times can significantly improve immunological detection of S-nitrosothiols. We have compared immunological detection of S-nitrosothiols with the level of intracellular S-nitrosothiols measured by tri-iodide chemiluminescence and show that the biotin-switch method is capable of detecting only high (nmol/mg protein) levels of intracellular S-nitrosothiols obtained after exposing cells to S-nitrosocysteine, but not the low levels observed during physiological nitric oxide formation. Preliminary proteomic analysis of protein S-nitrosothiols has identified elongation factor 2, heat shock protein 90 beta, and a 65-kDa macrophage protein homologous to human L-plastin as major nitrosation targets at high intracellular nitrosation levels in the murine macrophage-derived RAW 264.7 cell line. While the biotin-switch method may be a useful tool to aid in the positive identification of protein S-nitrosothiols, it cannot match the sensitivity of chemiluminescence-based methods and its use in proteomic studies likely suffers from selective detection of more easily reducible S-nitrosothiols.     

Nitrosative stress leads to protein glutathiolation, increased s-nitrosation, and up-regulation of peroxiredoxins in the heart

Nitric oxide (NO) is produced by different isoforms of nitric oxide synthases (NOSs) and operates as a mediator of important cell signaling pathways, such as the cGMP signaling cascade. Another mechanism by which NO exerts biological effects is mediated through S-nitrosation of target proteins. To explore thiol-based protein modifications in a situation of defined nitrosative stress, we used a transgenic mouse model with cardiac specific overexpression of inducible nitric oxide synthase (iNOS) and concomitant myoglobin deficiency (iNOS(+)/myo(-/-)). In comparison with the wild type hearts, protein glutathiolation detected by immunoblotting was significantly enhanced in iNOS(+)/myo(-/-) hearts, whereas protein S-nitrosation as measured by the biotin switch assay and two-dimensional PAGE revealed that nearly all of the detected proteins ( approximately 60) remained unchanged with the exception of three proteins. Tandem mass spectrometry revealed these proteins to be peroxiredoxins (Prxs), which are known to possess peroxidase activity, whereby hydrogen peroxide, peroxynitrite, and a wide range of organic hydroperoxides are reduced and detoxified. Immunoblotting with specific antibodies revealed up-regulation of Prx VI in the iNOS(+)/myo(-/-) hearts, whereas expression of Prx II and Prx III remained unchanged. Furthermore, the analysis of the cardiac S-nitrososubproteome identified several new proteins possibly being involved in NO-signaling pathways. Our data indicate that S-nitrosation and glutathiolation of cardiac proteins may contribute to the phenotype of NO-induced heart failure. The up-regulation of antioxidant proteins like Prx VI appears to be an additional mechanism to antagonize an excess of reactive oxygen/nitrogen species. Furthermore, S-nitrosation of Prxs may serve a new function in the signaling cascade of nitrosative stress.     

Effect of ionic strength on the binding of ascorbate to albumin

A fluorophore-nitroxide free radical dual-functional probe (FN) was utilized to study the kinetics of ascorbate (AH(-)) binding to Bovine Serum Albumin (BSA). Since the free radical fragment in the FN probe intramolecularly quenches fluorescence, ascorbate reduction of the nitroxide function is accompanied by a concomitant fluorescence intensity increase from the fluorophore. Thus, both fluorescence and the EPR techniques could be utilized to measure the reaction rate. In the presence of BSA protein, the observed rate of the overall process is the sum of that from at least two reactions: the reaction between free ascorbate and free probe, and the reaction between bound ascorbate and bound probe. Our findings show that the observed rate is strongly dependent on the ionic strength of the medium. A corollary of this observation is the indication of a purely electrostatic interaction between ascorbate and the BSA protein. This conclusion was further corroborated by 1H NMR measurement of the transverse relaxation time, T(2), of ascorbate protons in BSA solutions. Ascorbate ion was released from the ascorbate/BSA ensemble in the presence of increasing concentrations of NaCl. Binding constants of AH(-) to BSA were calculated at different ionic strengths at pH 7.4. Furthermore, an increase in ionic strength did not affect the ability of albumin to protect ascorbate against autoxidation. This suggests that the protein's protective antioxidant effect may be attributed to BSA binding of trace quantities of transition-metal cations (rather than ascorbate binding to BSA). This conclusion is supported by ascorbate UV-absorption measurements in the presence of albumin and Cu(2+) ions as a function of ionic strength.     

The role of a formaldehyde dehydrogenase-glutathione pathway in protein S-nitrosation in mammalian cells.

Intracellular sulfhydryls, both protein and non-protein, are potential targets of nitric oxide-related species. S-Nitrosation of proteins can occur in vivo and can affect their activity. Metabolic pathways that regulate protein S-nitrosation are therefore likely to be biologically important. We now report that formaldehyde dehydrogenase, an enzyme that decomposes S-nitrosoglutathione, can indirectly regulate the level of cellular protein S-nitrosation. Nitrogen oxide donors induced high levels of protein S-nitrosation in HeLa cells and lower levels in Mutatect fibrosarcoma cells, as determined by Saville-Griess assay and Western-dot-blot analysis. Depletion of glutathione by treatment with buthionine sulfoximine markedly increased protein S-nitrosation in both cell lines. Glutathione depletion also increased cytokine-induced S-nitrosation in brain endothelial cells. Formaldehyde dehydrogenase activity was 2-fold higher in Mutatect than in HeLa cells. We downregulated formaldehyde dehydrogenase activity in Mutatect cells by stably expressing antisense RNA and short-interfering RNA. In these cells, both protein S-nitrosation and S-nitrosoglutathione levels were significantly enhanced after exposure to nitrogen oxide donors as compared to parental cells. Overall, a strong inverse correlation between total S-nitrosothiols and formaldehyde dehydrogenase activity was seen. Inhibition of glutathione reductase, the enzyme that converts oxidized to reduced glutathione, by dehydroepiandrosterone similarly increased protein S-nitrosation and S-nitrosoglutathione levels in both cell lines. Our results provide the first evidence that formaldehyde dehydrogenase-dependent decomposition of S-nitrosoglutathione plays a role in protecting against nitrogen oxide-mediated protein S-nitrosation. We propose that formaldehyde dehydrogenase and glutathione reductase participate in a glutathione-dependent metabolic cycle that decreases protein S-nitrosation following exposure of cells to nitric oxide.     

Characterization of low affinity Fcγ receptor biotinylation under controlled reaction conditions by mass spectrometry and ligand binding analysis

Chemical protein biotinylation and streptavidin or anti‐biotin‐based capture is regularly used for proteins as a more controlled alternative to direct coupling of the protein on a biosensor surface. On biotinylation an interaction site of interest may be blocked by the biotin groups, diminishing apparent activity of the protein. Minimal biotinylation can circumvent the loss of apparent activity, but still a binding site of interest can be blocked when labeling an amino acid involved in the binding. Here, we describe reaction condition optimization studies for minimal labeling. We have chosen low affinity Fcγ receptors as model compounds as these proteins contain many lysines in their active binding site and as such provide an interesting system for a minimal labeling approach. We were able to identify the most critical parameters (protein:biotin ratio and incubation pH) for a minimal labeling approach in which the proteins of choice remain most active toward analyte binding. Localization of biotinylation by mass spectrometric peptide mapping on minimally labeled material was correlated to protein activity in binding assays. We show that only aiming at minimal labeling is not sufficient to maintain an active protein. Careful fine‐tuning of critical parameters is important to reduce biotinylation in a protein binding site.     

SNO spectral counting (SNOSC), a label-free proteomic method for quantification of changes in levels of protein S-nitrosation

Abstract

S-Nitrosation plays an important role in regulation of protein function and signal transduction. Discovering S-nitrosated targets is a prerequisite for further functional study. However, current proteomic methods used to quantify S-nitrosation are limited in their applicability to certain types of samples, or by the need for special reagents and complex procedures to obtain the results. Here we devised a label-free proteomic method for quantification of changes in the level of protein S-nitrosation on the basis of a spectral counting strategy, called S-nitrosothiol (SNO) spectral counting (SNOSC). With this method, samples can be from any source (cells, tissues); there is no need for labelling reagents or procedures, and the results yield quantitative information. Moreover, as it is based on the irreversible biotinylation procedure (IBP) for S-nitrosation protein enrichment, false positive targets caused by the interference of intermolecular disulphide bonds are ruled out. Using SNOSC we studied S-nitrosation in the cell line RAW264.7 induced exogenously with S-nitrosoglutathione (GSNO), or induced endogenously by lipopolysaccharides/interferon-gamma (LPS/IFN-γ). We detected a significant increase in S-nitrosation of 50 proteins after exogenous induction and 17 proteins after endogenous induction. We thus demonstrate that SNOSC is a widely applicable proteomic method for fast screening of SNO proteins.     

A “fluorescence switch” technique increases the sensitivity of proteomic detection and identification of S‐nitrosylated proteins

Protein S‐nitrosylation is a reversible post‐translational modification of protein cysteines that is increasingly being considered as a signal transduction mechanism. The “biotin switch” technique marked the beginning of the study of the S‐nitrosoproteome, based on the specific replacement of the labile S‐nitrosylation by a more stable biotinylation that allowed further detection and purification. However, its application for proteomic studies is limited by its relatively low sensitivity. Thus, typical proteomic experiments require high quantities of protein extracts, which precludes the use of this method in a number of biological settings. We have developed a “fluorescence switch” technique that, when coupled to 2‐DE proteomic methodologies, allows the detection and identification of S‐nitrosylated proteins by using limited amounts of starting material, thus significantly improving the sensitivity. We have applied this methodology to detect proteins that become S‐nitrosylated in endothelial cells when exposed to S‐nitroso‐L ‐cysteine, a physiological S‐nitrosothiol, identifying already known S‐nitrosylation targets, as well as proteins that are novel targets. This “fluorescence switch” approach also allowed us to identify several proteins that are denitrosylated by thioredoxin in cytokine‐activated RAW264.7 (murine macrophage) cells. We believe that this method represents an improvement in order to approach the identification of S‐nitrosylated proteins in physiological conditions.     

Chemical methods to detect S-nitrosation

Nitric oxide (NO) is a cell-signaling molecule involved in a number of physiological and pathophysiological processes. Modification of cysteine residues by NO (or NO metabolites), that is S-nitrosation, changes the function of a broad spectrum of proteins. This reaction represents an important post-translational modification that transduces NO-dependent signals. However, the detection and quantification of S-nitrosation in biological samples remain a challenge mainly because of the lability of S-nitrosation products: S-nitrosothiols (SNO). In this review we summarize recent developments of the methods to detect S-nitrosation. Our focus is on the methods which can be used to directly conjugate the site(s) of S-nitrosation.     

Targeted and proximity-dependent promiscuous protein biotinylation by a mutant Escherichia coli biotin protein ligase

A method for general protein biotinylation by enzymatic means has been developed. A mutant form (R118G) of the biotin protein ligase (BirA) of Escherichia coli is used and biotinylation is thought to proceed by chemical acylation of protein lysine side chains by biotinoyl-5'-AMP released from the mutant protein. Bovine serum albumin, chloramphenicol acetyltransferase, immunoglobulin chains and RNAse A as well as a large number of E. coli proteins have been biotinylated. The biotinylation reaction is proximity dependent in that the extent of biotinylation is much greater when the ligase is coupled to the acceptor protein than when the acceptor is free in solution. This is presumably due to rapid hydrolysis of the acylation agent, biotinoyl-5'-AMP. Therefore, when the mutant ligase is attached to one partner involved in a protein-protein interaction, it can be used to specifically tag the other partner with biotin, thereby permitting facile detection and recovery of the proteins by existing avidin/streptavidin technology.     

SNO spectral counting (SNOSC), a label-free proteomic method for quantification of changes in levels of protein S-nitrosation

S-Nitrosation plays an important role in regulation of protein function and signal transduction. Discovering S-nitrosated targets is a prerequisite for further functional study. However, current proteomic methods used to quantify S-nitrosation are limited in their applicability to certain types of samples, or by the need for special reagents and complex procedures to obtain the results. Here we devised a label-free proteomic method for quantification of changes in the level of protein S-nitrosation on the basis of a spectral counting strategy, called S-nitrosothiol (SNO) spectral counting (SNOSC). With this method, samples can be from any source (cells, tissues); there is no need for labelling reagents or procedures, and the results yield quantitative information. Moreover, as it is based on the irreversible biotinylation procedure (IBP) for S-nitrosation protein enrichment, false positive targets caused by the interference of intermolecular disulphide bonds are ruled out. Using SNOSC we studied S-nitrosation in the cell line RAW264.7 induced exogenously with S-nitrosoglutathione (GSNO), or induced endogenously by lipopolysaccharides/interferon-gamma (LPS/IFN-γ). We detected a significant increase in S-nitrosation of 50 proteins after exogenous induction and 17 proteins after endogenous induction. We thus demonstrate that SNOSC is a widely applicable proteomic method for fast screening of SNO proteins.     

Metabolic biotinylation of recombinant antibody by biotin ligase retained in the endoplasmic reticulum

Due to its strength and specificity, the interaction between avidin and biotin has been used in a variety of scientific and medical applications ranging from immunohistochemistry to drug targeting. The present study describes two methods for biotinylation of proteins secreted from eukaryotic cells using the Escherichia coli biotin protein ligase. In one system the biotin ligase was co-secreted from the cells along with substrate protein enabling extracellular biotinylation of the tagged protein. In the other system, biotin ligase was engineered to be retained in the endoplasmic reticulum (ER) and metabolically biotinylates the secretory protein as it passes through the ER. An engineered antibody fragment, a diabody with specificity for carcinoembryonic antigen (CEA) was fused to the biotin acceptor domain (123 amino acid) of Propionibacterium shermanii. Coexpression of the fusion protein with ER retained biotin ligase showed higher biotinylation efficiency than biotinylation by co-secreted ligase. Biotinylation of the anti-CEA diabody tagged with a short (15 amino acid, Biotin Avitag) biotin acceptor peptide was also successful. Utilization of ER retained biotin ligase for biotinylation of protein is an attractive alternative for efficiently producing uniformly biotinylated recombinant proteins for a variety of avidin-biotin technologies.     

Nonenzymatic biotinylation of a biotin carboxyl carrier protein: unusual reactivity of the physiological target lysine

Enzyme-catalyzed addition of biotin to proteins is highly specific. In any single organism one or a small number of proteins are biotinylated and only a single lysine on each of these proteins is modified. A detailed understanding of the structural basis for the selective biotinylation process has not yet been elucidated. Recently certain mutants of the Escherichia coli biotin protein ligase have been shown to mediate "promiscuous" biotinylation of proteins. It was suggested that the reaction involved diffusion of a reactive activated biotin intermediate, biotinoyl-5'-AMP, with nonspecific proteins. In this work the reactivity of this chemically synthesized intermediate toward the natural target of enzymatic biotinylation, the biotin carboxyl carrier protein, was investigated. The results indicate that the intermediate does, indeed, react with target protein, albeit at a significantly slower rate than the enzyme-catalyzed process. Surprisingly, analysis of the products of nonenzymatic biotinylation indicates that of five lysine residues in the protein only the physiological target side chain is modified. These results indicate that either the environment of this lysine residue or its intrinsic properties render it highly reactive to nonenzymatic biotinylation mediated by biotinoyl-5'-AMP. This reactivity may be important for its selective biotinylation in vivo.