Gliadin T cell epitope selection by tissue transglutaminase in celiac disease. Role of enzyme specificity and pH influence on the transamidation versus deamidation process

Tissue transglutaminase (TG2) can modify proteins by transamidation or deamidation of specific glutamine residues. TG2 has a major role in the pathogenesis of celiac disease as it is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides that are recognized by CD4(+), DQ2-restricted T cells from the celiac lesions. Capillary electrophoresis with fluorescence-labeled gliadin peptides was used to separate and quantify deamidated and transamidated products. In a competition assay, the affinity of TG2 to a set of overlapping gamma-gliadin peptides was measured and compared with their recognition by celiac lesion T cells. Peptides differed considerably in their competition efficiency. Those peptides recognized by intestinal T cell lines showed marked competition indicating them as excellent substrates for TG2. The enzyme fine specificity of TG2 was characterized by synthetic peptide libraries and mass spectrometry. Residues in positions -1, +1, +2, and +3 relative to the targeted glutamine residue influenced the enzyme activity, and proline in position +2 had a particularly positive effect. The characterized sequence specificity of TG2 explained the variation between peptides as TG2 substrates indicating that the enzyme is involved in the selection of gluten T cell epitopes. The enzyme is mainly localized extracellularly in the small intestine where primary amines as substrates for the competing transamidation reaction are present. The deamidation could possibly take place in this compartment as an excess of primary amines did not completely inhibit deamidation of gluten peptides at pH 7.3. However, lowering of the pH decreased the reaction rate of the TG2-catalyzed transamidation, whereas the rate of the deamidation reaction was considerably increased. This suggests that the deamidation of gluten peptides by TG2 more likely takes place in slightly acidic environments.     

Transglutaminase 2 mediates polymer formation of I-kappaBalpha through C-terminal glutamine cluster

Recently we reported that transglutaminase 2 (TGase 2) activates nuclear factor-kappaB (NF-kappaB) independently of I-kappaB kinase (IKK) activation, by inducing cross-linking and protein polymer formation of inhibitor of nuclear factor-kappaBalpha (I-kappaBalpha). TGase 2 catalyzes covalent isopeptide bond formation between the peptide bound-glutamine and the lysine residues. Using matrix-assisted laser desorption ionization time-of-flight mass spectra analysis of I-kappaBalpha polymers cross-linked by TGase 2, as well as synthetic peptides in an in vitro competition assay, we identified a glutamine cluster at the C terminus of I-kappaBalpha (amino acids 266-268) that appeared to play a key role in the formation of I-kappaBalpha polymers. Although there appeared to be no requirement for specific lysine residues, we found a considerably higher preference for the use of lysine residues at positions 21, 22, and 177 in TGase 2-mediated cross-linking of I-kappaBalpha. We demonstrated that synthetic peptides encompassing the glutamine cluster at amino acid positions 266-268 reversed I-kappaBalpha polymerization in vitro. Furthermore, the depletion of free I-kappaBalpha in EcR/TG cells was completely rescued in vivo by transfection of mutant I-kappaBalphas in glutamine sites (Q266G, Q267G, and Q313G) as well as in a lysine site (K177G). These findings provide additional clues into the mechanism by which TGase 2 contributes to the inflammatory process via activation of NF-kappaB.     

Enhanced B-Cell Receptor Recognition of the Autoantigen Transglutaminase 2 by Efficient Catalytic Self-Multimerization

A hallmark of the gluten-driven enteropathy celiac disease is autoantibody production towards the enzyme transglutaminase 2 (TG2) that catalyzes the formation of covalent protein-protein cross-links. Activation of TG2-specific B cells likely involves gluten-specific CD4 T cells as production of the antibodies is dependent on disease-associated HLA-DQ allotypes and dietary intake of gluten. IgA plasma cells producing TG2 antibodies with few mutations are abundant in the celiac gut lesion. These plasma cells and serum antibodies to TG2 drop rapidly after initiation of a gluten-free diet, suggestive of extrafollicular responses or germinal center reactions of short duration. High antigen avidity is known to promote such responses, and is also important for breakage of self-tolerance. We here inquired whether TG2 avidity could be a feature relevant to celiac disease. Using recombinant enzyme we show by dynamic light scattering and gel electrophoresis that TG2 efficiently utilizes itself as a substrate due to conformation-dependent homotypic association, which involves the C-terminal domains of the enzyme. This leads to the formation of covalently linked TG2 multimers. The presence of exogenous substrate such as gluten peptide does not inhibit TG2 self-cross-linking, but rather results in formation of TG2-TG2-gluten complexes. The celiac disease autoantibody epitopes, clustered in the N-terminal part of TG2, are conserved in the TG2-multimers as determined by mass spectrometry and immunoprecipitation analysis. TG2 multimers are superior to TG2 monomer in activating A20 B cells transduced with TG2-specific B-cell receptor, and uptake of TG2-TG2-gluten multimers leads to efficient activation of gluten-specific T cells. Efficient catalytic self-multimerization of TG2 and generation of multivalent TG2 antigen decorated with gluten peptides suggest a mechanism by which self-reactive B cells are activated to give abundant numbers of plasma cells in celiac disease. Importantly, high avidity of the antigen could explain why TG2-specific plasma cells show signs of an extrafollicular generation pathway.     

Gluten T cell epitope targeting by TG3 and TG6; implications for dermatitis herpetiformis and gluten ataxia

Transglutaminase 2 (TG2) is well characterized as the main autoantigen of celiac disease. The ability of TG2 to deamidate and crosslink gluten peptides is essential for the gluten-dependent production of TG2 specific autoantibodies. In patients with primarily extraintestinal manifestation of gluten sensitivity the repertoire of autoantibodies may be different. In dermatitis herpetiformis (DH), TG3 appears to be the target autoantigen whereas in gluten ataxia (GA) autoantibodies reactive with TG6 are present. A functional role for TG3 and TG6 in these diseases has yet to be described. It is also not known whether these enzymes can use gluten peptides implicated in the pathology as substrates. We here report that similar to TG2, TG3 and TG6 can specifically deamidate gluten T cell epitopes. However, the fine specificities of the enzymes were found to differ. TG2 can form covalent complexes with gluten by iso-peptide and thioester bonds. We found that both TG3 and TG6 were able to complex with gluten peptides through thioester linkage although less efficiently than TG2, whereas TG6 but not TG3 was able to form iso-peptide linked complexes. Our findings lend credence to the notion that TG3 and TG6 are involved in the gluten-induced autoimmune responses of DH and GA.     

Oligo(methionyl) proteins. Enzymatic hydrolysis of the model isopeptides N epsilon-oligo(L-methionyl)-L-lysine

A number of model isopeptides containing oligo(methionine) chains varying in length (2-5 residues) covalently linked to the epsilon-amino group of lysine were synthesized by solid-phase procedures. Hydrolysis of these peptides by pepsin, chymotrypsin, cathepsin C (dipeptidyl peptidase IV) and intestinal aminopeptidase N was investigated using high-performance liquid chromatography to identify and quantify the hydrolysis products. Methionine oligomers grafted onto lysine were cleaved to tripeptides by pepsin. Chymotrypsin preferentially hydrolyzed the methionyl-methionine bond preceding the isopeptide bond. Cathepsin C released dimethionyl units from the covalently attached polymers. Intestinal aminopeptidase caused efficient hydrolysis of both peptides and isopeptide bonds although free methionine decreased the cleavage of the latter bond. Hydrophobic characteristics of oligo(methionine) chains promoted enzyme-catalyzed transpeptidations resulting probably from acyl-transfer-type reactions. Complementary hydrolysis of the isopeptides by these digestive enzymes suggests that covalent attachment of oligo(amino acid)s to food proteins may improve their nutritional value.     

The propensity for deamidation and transamidation of peptides by transglutaminase 2 is dependent on substrate affinity and reaction conditions

Transglutaminase 2 (TG2) catalyzes cross-linking or deamidation of glutamine residues in peptides and proteins. The in vivo deamidation of gliadin peptides plays an important role in the immunopathogenesis of celiac disease (CD). Although deamidation is considered to be a side-reaction occurring in the absence of suitable amines or at a low pH, a recent paper reported the selective deamidation of the small heat shock protein 20 (Hsp20), suggesting that deamidation could be a substrate dependent event. Here we have measured peptide deamidation and transamidation in the same reaction to reveal factors that affect the relative propensity for the two possible products. We report that the propensity for deamidation by TG2 is both substrate dependent and influenced by the reaction conditions. Direct deamidation is favored for poor substrates and at low concentrations of active TG2, while indirect deamidation (i.e. hydrolysis of transamidated product) can significantly contribute to the deamidation of good peptide substrates at higher enzyme concentrations. Further, we report for the first time that TG2 can hydrolyze iso-peptide bonds between two peptide substrates. This was observed also for gliadin peptides introducing a novel route for the generation of deamidated T cell epitopes in celiac disease.     

Protein cross-links: universal isolation and characterization by isotopic derivatization and electrospray ionization mass spectrometry.

A general method of unequivocally identifying and obtaining sequence information on cross-linked peptides derived by proteolytic digestion of cross-linked proteins has been developed. The method is based on isotopic labeling of alpha-amino groups with 2, 4-dinitrofluorobenzene (DNFB) coupled with electrospray ionization mass spectrometry. Proteins containing covalent cross-link(s) are reductively methylated to convert lysine residues to dimethyl lysine. The methylated protein is partially hydrolyzed and the liberated alpha-amino termini are derivatized with an equimolar mixture of DNFB and [(2)H(3)]DNFB. Dinitrophenyl (DNP)-labeled peptides may be fractionated into mono- and bis-DNP pools by chromatography on phenyl media. The bis-DNP peptides are further separated by reverse-phase HPLC and analyzed by electrospray ionization mass spectrometry. The molecular ions of cross-linked peptides are unambiguously identified as 1:2:1 triplets in the mass spectrum resulting from the binomial distribution of isotopic label in the bis-DNP derivative. Sequence information can be elucidated from the unique product ion patterns which are generated from in-source fragmentation at an elevated cone voltage. Analysis of the disulfide cross-linked peptide (VTCG)(2) was undertaken as a proof of concept and the generality of the method was demonstrated by isolating and sequencing the isopeptide bond of polyubiquitin.     

Cyclic and dimeric gluten peptide analogues inhibiting DQ2-mediated antigen presentation in celiac disease

Celiac disease is an immune mediated enteropathy elicited by gluten ingestion. The disorder has a strong association with HLA-DQ2. This HLA molecule is involved in the disease pathogenesis by presenting gluten peptides to T cells. Blocking the peptide-binding site of DQ2 may be a way to treat celiac disease. In this study, two types of peptide analogues, modeled after natural gluten antigens, were studied as DQ2 blockers. (a) Cyclic peptides. Cyclic peptides containing the DQ2-alphaI gliadin epitope LQPFPQPELPY were synthesized with flanking cysteine residues introduced and subsequently crosslinked via a disulfide bond. Alternatively, cyclic peptides were prepared with stable polyethylene glycol bridges across internal lysine residues of modified antigenic peptides such as KQPFPEKELPY and LQLQPFPQPEKPYPQPEKPY. The effect of cyclization as well as the length of the spacer in the cyclic peptides on DQ2 binding and T cell recognition was analyzed. Inhibition of peptide-DQ2 recognition by the T cell receptor was observed in T cell proliferation assays. (b) Dimeric peptides. Previously we developed a new type of peptide blocker with much enhanced affinity for DQ2 by dimerizing LQLQPFPQPEKPYPQPELPY through the lysine side chains. Herein, the effect of linker length on both DQ2 binding and T cell inhibition was investigated. One dimeric peptide analogue with an intermediate linker length was found to be especially effective at inhibiting DQ2 mediated antigen presentation. The implications of these findings for the treatment of celiac disease are discussed.     

A conserved catalytic residue in the ubiquitin-conjugating enzyme family

Ubiquitin (Ub) regulates diverse functions in eukaryotes through its attachment to other proteins. The defining step in this protein modification pathway is the attack of a substrate lysine residue on Ub bound through its C-terminus to the active site cysteine residue of a Ub-conjugating enzyme (E2) or certain Ub ligases (E3s). So far, these E2 and E3 cysteine residues are the only enzyme groups known to participate in the catalysis of conjugation. Here we show that a strictly conserved E2 asparagine residue is critical for catalysis of E2- and E2/RING E3-dependent isopeptide bond formation, but dispensable for upstream and downstream reactions of Ub thiol ester formation. In contrast, the strictly conserved histidine and proline residues immediately upstream of the asparagine are dispensable for catalysis of isopeptide bond formation. We propose that the conserved asparagine side chain stabilizes the oxyanion intermediate formed during lysine attack. The E2 asparagine is the first non-covalent catalytic group to be proposed in any Ub conjugation factor.     

Identification of transglutaminase-mediated deamidation sites in a recombinant alpha-gliadin by advanced mass-spectrometric methodologies

Celiac disease is a permanent immune-mediated food intolerance triggered by ingestion of wheat gliadins in genetically susceptible individuals. It has been reported that tissue transglutaminase plays an important role in the onset of celiac disease by converting specific glutamine residues within gliadin fragments into glutamic acid residues. This process increases binding affinity of gliadin peptides to HLA-DQ2/DQ8 molecules, thus enhancing the immune response. The aim of the present study was to achieve a detailed structural characterization of modifications induced by transglutaminase on gliadin peptides. Therefore, structural analyses were carried out on a recombinant alpha-gliadin and on a panel of 26 synthetic peptides, overlapping the complete protein sequence. Modified glutamine residues were identified by means of advanced mass-spectrometric methodologies on the basis of MALDI-TOF-MS and tandem mass spectrometry. Results led to the identification of 19 of 94 glutamine residues present in the recombinant alpha-gliadin, which were converted into glutamic acid residues by a transglutaminase-mediated reaction. This allowed us to achieve a global view of the modifications induced by the enzyme on this protein. Furthermore, results gathered could likely be utilized as relevant information for a better understanding of processes leading to T-cell recognition of gliadin peptides involved in celiac disease.     

Susceptibility to transglutaminase of gliadin peptides predicted by a mass spectrometry-based assay

A peptidomics approach was developed to identify transglutaminase-susceptible Q residues within a pepsin-trypsin gliadin digest. Based on tagging with a monodansylcadaverine fluorescent probe, six alpha/beta-, gamma-gliadin, and low molecular weight glutenin peptides were identified by nanospray tandem mass spectrometry. In functioning as an acyl acceptor, tissue transglutaminase was able to form complexes with the glutamine-rich gliadin peptides, whereas by lowering pH, the peptides were deamidated by transglutaminase at the same Q residues, which were previously transamidated. The main common feature shared by the peptides was the consensus sequence Q-X-P. Our findings offer relevant information for the understanding of how dietary peptides interact with the host organism in celiac disease.     

Screening for transglutaminase-catalyzed modifications by peptide mass finger printing using multipoint recalibration on recognized peaks for high mass accuracy.

Detection of posttranslational modifications is expected to be one of the major future experimental challenges for proteomics. We describe herein a mass spectrometric procedure to screen for protein modifications by peptide mass fingerprinting that is based on post-data acquisition improvement of the mass accuracy by exporting the peptide mass values into analytical software for multipoint recalibration on recognized peaks. Subsequently, the calibrated peak mass data set is used in searching for modified peptides, i.e., peptides possessing specific mass deviations. In order to identify the location of Lys- and Gln-residues available for transglutaminase-catalyzed isopeptide bond formation, mammalian small heat shock proteins (sHsps) were screened for labeling with the two hexapeptide probes GQDPVR and GNDPVK in presence of transglutaminase. Peptide modification due to cross-linking of the GQDPVR hexa-peptide probe was detected for C-terminal Lys residues. Novel transglutaminase-susceptible Gln sites were identified in two sHsps (Q31/Q27 in Hsp20 and HspB2, respectively), by cross-linking of the GNDPVK hexapeptide probe. Deamidation of specific Gln residues was also detected, as well an isopeptide derived from intramolecular Gln-Lys isopeptide bond formation. We conclude that peptide mass fingerprinting can be an efficient way of screening for various posttranslational modifications. Basically any instrumentation for MALDI mass spectrometry can be used, provided that post-data acquisition recalibration is applied.     

Identification of the peptide-binding site in the heat shock chaperone/tumor rejection antigen gp96 (Grp94)

Heat shock protein (HSP)-peptide complexes from tumor cells elicit specific protective immunity when injected into inbred mice bearing the same specific type of tumor. The HSP-mediated specific immunogenicity also occurs with virus-infected cells. The immune response is solely due to endogenous peptides noncovalently bound to HSP. A vesicular stomatitis virus capsid-derived peptide ligand bearing a photoreactive azido group was specifically bound by and cross-linked to murine HSP glycoprotein (gp) 96. The peptide-binding site was mapped by specific proteolysis of the cross-links followed by analysis of the cross-linked peptides using a judicious combination of SDS-gel electrophoresis, mass spectrometry, and amino acid sequencing. The minimal peptide-binding site was mapped to amino acid residues 624-630 in a highly conserved region of gp96. A model of the peptide binding pocket of gp96 was constructed based on the known crystallographic structure of major histocompatibility complex class I molecule bound to a similar peptide. The gp96-peptide model predicts that the peptide ligand is held in a groove formed by alpha-helices and lies on a surface consisting of antiparallel beta-sheets. Interestingly, in this model, the peptide binding pocket abuts the dimerization domain of gp96, which may have implications for the extraordinary stability of peptide-gp96 complexes, and for the faithful relay of peptides to major histocompatibility complex class I molecule for antigen presentation.     

Synthetic peptide substrates for the erythrocyte protein carboxyl methyltransferase. Detection of a new site of methylation at isomerized L-aspartyl residues

Four hexapeptides of sequence L-Val-L-Tyr-L-Pro-(Asp)-Gly-L-Ala containing D- or L-aspartyl residues in normal or isopeptide linkages have been synthesized by the Merrifield solid-phase method as potential substrates of the erythrocyte protein carboxyl methyltransferase. This enzyme has been shown to catalyze the methylation of D-aspartyl residues in proteins in red blood cell membranes and cytosol. Using a new vapor-phase methanol diffusion assay, we have found that the normal hexapeptides containing either D- or L-aspartyl residues were not substrates for the human erythrocyte methyltransferase. On the other hand, the L-aspartyl isopeptide, in which the glycyl residue was linked in a peptide bond to the beta-carboxyl group of the aspartyl residue, was a substrate for the enzyme with a Km of 6.3 microM and was methylated with a maximal velocity equal to that observed when ovalbumin was used as a methyl acceptor. The enzyme catalyzed the transfer of up to 0.8 mol of methyl groups/mol of this peptide. Of the four synthetic peptides, only the L-isohexapeptide competitively inhibits the methylation of ovalbumin by the erythrocyte enzyme. This peptide also acts as a substrate for both of the purified protein carboxyl methyltransferases I and II which have been previously isolated from bovine brain (Aswad, D. W., and Deight, E. A. (1983) J. Neurochem. 40, 1718-1726). The L-isoaspartyl hexapeptide represents the first defined synthetic substrate for a eucaryotic protein carboxyl methyltransferase. These results demonstrate that these enzymes can not only catalyze the formation of methyl esters at the beta-carboxyl groups of D-aspartyl residues but can also form esters at the alpha-carboxyl groups of isomerized L-aspartyl residues. The implications of these findings for the metabolism of modified proteins are discussed.     

Substrate preference of transglutaminase 2 revealed by logistic regression analysis and intrinsic disorder examination

Tissue transglutaminase (TG2) catalyzes the Ca²⁺-dependent posttranslational modification of proteins via formation of isopeptide bonds between their glutamine and lysine residues. Although substrate specificity of TG2 has been studied repeatedly at the sequence level, no clear consensus sequences have been determined so far. With the use of the extensive structural information on TG2 substrate proteins listed in TRANSDAB Wiki database†, a slight preference of TG2 for glutamine and lysine residues situated in turns could be observed. When the spatial environment of the favored glutamine and lysine residues was analyzed with logistic regression, the presence of specific amino acid patterns was identified. By using the occurrence of the predictor amino acids as selection criteria, several polypeptides were predicted and later identified as novel in vitro substrates for TG2. By studying the sequence of TG2 substrate proteins lacking available crystal structure, the strong favorable influence on substrate selection of the presence of substrate glutamine and lysine residues in intrinsically disordered regions could also be revealed. The collected structural data have provided novel understanding of how this versatile enzyme selects its substrates in various cell compartments and tissues.     

Localization of the human complement component C3 binding site on the IgG heavy chain

The location of the covalent binding site of the third component of complement (C3) on the IgG heavy chain was determined by sequence analysis of peptides generated by cyanogen bromide digestion of C3-IgG adducts. Activation of the alternative pathway by incubation of heat-aggregated human IgG1 with fresh normal human plasma formed covalent adducts of C3b-IgG. CNBr peptides of these adducts were transferred to a polyvinylidene difluoride membrane, and amino-terminal sequences were determined. A 40-kDa dipeptide containing the covalent bond was identified by labeling the free thiol group (generated during activation of the internal thioester of C3b) with iodo[1-14C]acetamide and analyzed by amino acid sequencing. The resulting double sequence suggested an adduct with NH2 termini at residue 938 (pro-C3 numbering) of C3 (75 residues NH2-terminal to the thioester) and residue 84 in the variable region of the IgG heavy chain. These results combined with results from hydroxylamine treatment (splits ester linkage between C3b and IgG) imply that this adduct peptide consists of a 22-kDa C3 fragment and an 18-kDa IgG fragment. Therefore, C3 binds covalently within the region extending from the last 20 residues of the variable region through the first 20 residues of CH2.     

Identification of a critical lysine residue in apolipoprotein B-100 that mediates noncovalent interaction with apolipoprotein(a)

We have previously shown that lipoprotein(a) (Lp(a)) assembly involves an initial noncovalent interaction between sequences within apolipoprotein(a) (apo(a)) kringle IV types 5-8 and the amino terminus of apolipoprotein B-100 (sequences between amino acids 680 and 781 in apoB-100), followed by formation of a disulfide bond. In the present study, citraconylation of lysine residues in apoB-100 abolished the ability of the modified low density lipoprotein to associate with apo(a), thereby demonstrating a direct role for lysine residues in apoB in the first step of Lp(a) assembly. To identify specific lysine residues in the amino terminus of apoB that are required for the noncovalent interaction, we initially used an affinity chromatography method in which recombinant forms of apo(a) (r-apo(a)) were immobilized on Sepharose beads. Assessment of the ability of carboxyl-terminal truncations of apoB-18 to bind to r-apo(a)-Sepharose revealed that a 25-amino acid sequence in apoB (amino acids 680-704) bound specifically to apo(a) in a lysine-dependent manner; citraconylation of the lysine residues in the apoB derivative encoding this sequence abolished the binding interaction. Using fluorescence spectrometry, we found that a synthetic peptide corresponding to this sequence bound directly to apo(a); the peptide also reduced covalent Lp(a) formation. Lysine residues present in this sequence (Lys(680) and Lys(690)) were mutated to alanine in the context of apoB-18. We found that the apoB-18 species containing the Lys(680) mutation was incapable of binding to r-apo(a)-Sepharose columns, whereas the apoB-18 species containing the Lys(690) mutation exhibited slightly reduced binding to these columns. Taken together, our data indicate that Lys(680) is critical for the noncovalent interaction of apo(a) and apoB-100 that precedes covalent Lp(a) formation.     

Intramolecular isopeptide but not internal thioester bonds confer proteolytic and significant thermal stability to the S. pyogenes pilus adhesin Spy0125

Streptococcus pyogenes and other Gram‐positive bacterial pathogens present long macromolecular filaments known as pili on their surface that mediate adhesion and colonization. These pili are covalent polymers, assembled by sortases. Typically, they comprise a putative adhesin at their tip, a backbone subunit present in multiple copies and a basal subunit that is covalently anchored to the peptidoglycan layer of the cell surface. The crystal structures of pilin subunits revealed the presence of unusual covalent linkages in these proteins, including intramolecular isopeptide and internal thioester bonds. The intramolecular isopeptide bonds in backbone pilins are important for protein stability. Here, using both the wild‐type protein and a set of mutants, we assessed the proteolytic and thermal stability of the S. pyogenes pilus tip adhesin Spy0125, in the presence and absence of its intramolecular isopeptide and internal thioester bonds. We also determined a crystal structure of the internal thioester bond variant Spy0125^Cys426Ala. We find that mutations in the intramolecular isopeptide bonds compromise the stability of Spy0125. Using limited proteolysis and thermal denaturation assays, we could separate the contribution of each intramolecular isopeptide bond to Spy0125 stability. In contrast, mutation in the internal thioester bond had a lesser effect on protein stability and the crystal structure is essentially identical to wild type. This work suggests that the internal thioester in Spy0125, although having a minor contributory role, is not required for protein stability and must have a different primary function, most likely mediating a covalent interaction with host cell ligands. Proteins 2014; 82:517–527. © 2013 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Chemical and enzymatic analysis of covalent bonds between peptides and chromosomal DNA.

DNA from Ehrlich ascites tumor (EAT) cells and from human placenta was examined for covalent bonds between hydroxy amino acid residues in peptides and nucleotide phosphate groups. The residual proteinaceous material in highly purified DNA was radiolabelled with 125Iodine and the linking-groups between peptides and nucleotides released by combined protease and nuclease treatment were investigated with respect to their chemical and enzymatic stabilities. The residual nucleotide(s)-peptide(s) fraction from DNA isolated after prolonged alkaline cell lysis and phenol extraction contains mainly alkali and acid-stable but phosphodiesterase-sensitive peptide-nucleotide complexes which indicates phosphodiesters between tyrosyl residues in peptides and nucleotide phosphates. In contrast, the linking-group fraction from DNA isolated under native conditions contains additional peptide components. (a) Phospho-peptides that co-purify with DNA but that are not covalently bound to nucleotides. (b) A fraction of peptides that is released from nucleotides by alkali in a time and concentration-dependent reaction. Evidence is presented indicating that the latter fraction involves phospho-triesters between hydroxy amino acid residues in peptides and internucleotide phosphates. The phosphodiesters between hydroxy amino acids and nucleotide phosphates representing the predominant class of peptide-nucleotide complexes in alkali-denatured DNA are most likely side products of peptide-nucleotide phospho-triester hydrolysis.     

Localization of the transglutaminase cross-linking sites in the Bacillus subtilis spore coat protein GerQ

The Bacillus subtilis spore coat protein GerQ is necessary for the proper localization of CwlJ, an enzyme important in the hydrolysis of the peptidoglycan cortex during spore germination. GerQ is cross-linked into high-molecular-mass complexes in the spore coat late in sporulation, and this cross-linking is largely due to a transglutaminase. This enzyme forms an epsilon-(gamma-glutamyl) lysine isopeptide bond between a lysine donor from one protein and a glutamine acceptor from another protein. In the current work, we have identified the residues in GerQ that are essential for transglutaminase-mediated cross-linking. We show that GerQ is a lysine donor and that any one of three lysine residues near the amino terminus of the protein (K2, K4, or K5) is necessary to form cross-links with binding partners in the spore coat. This leads to the conclusion that all Tgl-dependent GerQ cross-linking takes place via these three lysine residues. However, while the presence of any of these three lysine residues is essential for GerQ cross-linking, they are not essential for the function of GerQ in CwlJ localization.