Characterization of the venom of the vermivorous cone snail Conus fulgetrum

Over 200 components with molecular mass ranging mainly from 400 to 4000 Da were characterized from the venom of the vermivorous cone snail Conus fulgetrum that inhabit Egyptian Red Sea. One major component having a molecular mass of 2946 Da was purified by HPLC, and its primary structure was determined by a combination of Edman degradation and MS/MS analysis.     

Optimization of oxidative folding methods for cysteine‐rich peptides: a study of conotoxins containing three disulfide bridges

The oxidative folding of small, cysteine‐rich peptides to selectively achieve the native disulfide bond connectivities is critical for discovery and structure‐function studies of many bioactive peptides. As the propensity to acquire the native conformation greatly depends on the peptide sequence, numerous empirical oxidation methods are employed. The context‐dependent optimization of these methods has thus far precluded a generalized oxidative folding protocol, in particular for peptides containing more than two disulfides. Herein, we compare the efficacy of optimized solution‐phase and polymer‐supported oxidation methods using three disulfide‐bridged conotoxins, namely µ‐SIIIA, µ‐KIIIA and ω‐GVIA. The use of diselenide bridges as proxies for disulfide bridges is also evaluated. We propose the ClearOx‐assisted oxidation of selenopeptides as a fairly generalized oxidative folding protocol. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

The influence of self‐fertilization and grouping on fitness attributes in the freshwater snail Physa acuta: population and individual inbreeding depression

The genetic structure, selfing rate and inbreeding depression of the hermaphroditic freshwater snail Physa acuta were jointly analysed in a population near Montpellier, France. Allozymic markers revealed moderate gene diversity (0.138), and no heterozygote deficiency. The mean outcrossing rate, estimated by using progeny arrays, was 0.9, with substantial variation among families. This also suggests that the number of fathers among outcrossed offspring of a given mother is low. Inbreeding depression was estimated over more than one generation using 83 first‐laboratory‐generation (G₁) families. The main parameters measured were parental (G₁) fecundity, offspring (G₂) survival and fecundity. Size and growth were also monitored. Parental fecundity was analysed under several conditions (isolation, pair and quadruplet outcrossing). The self‐fertilization depression, including parental fecundity, offspring survival and fecundity, was about 0.9 at the population level. The genetic data obtained in the same population indicate a value of about 0.3 using Ritland’s (1990) technique, suggesting that the depression over the whole life‐cyle might be even higher than 0.9. Grouping affected neither fecundity nor self‐fertilization depression. Substantial variation in depression for survival was detected among individuals, from no survival in some selfed families to better survival than that of outbred families in others. The overall result (outbred population structure, high outcrossing rate and high self‐fertilization depression) is consistent with what is expected in large outcrossing populations in which inbreeding depression is maintained by mutation‐selection balance.     

Molecular properties of gp100‐reactive T‐cell receptors drive the cytokine profile and antitumor efficacy of transgenic host T cells

To study the contribution of T‐cell receptors (TCR) to resulting T‐cell responses, we studied three different human αβ TCRs, reactive to the same gp100‐derived peptide presented in the context of HLA‐A*0201. When expressed in primary CD8 T cells, all receptors elicited classic antigen‐induced IFN‐γ responses, which correlated with TCR affinity for peptide–MHC in the order T4H2 > R6C12 > SILv44. However, SILv44 elicited superior IL‐17A release. Importantly, in vivo, SILv44‐transgenic T cells mediated superior antitumor responses to 888‐A2 + human melanoma tumor cells upon adoptive transfer into tumor‐challenged mice while maintaining IL‐17 expression. Modeling of the TCR ternary complexes suggested architectural differences between SILv44 and the other complexes, providing a potential structural basis for the observed differences. Overall, the data reveal a more prominent role for the T‐cell receptor in defining host T‐cell physiology than traditionally assumed, while parameters beyond IFN‐γ secretion and TCR affinity ultimately determine the reactivity of tumor‐reactive T cells.     

Sequencing and functional validation of the JGI Brachypodium distachyon T‐DNA collection

Due to a large and growing collection of genomic and experimental resources, Brachypodium distachyon has emerged as a powerful experimental model for the grasses. To add to these resources we sequenced 21 165 T‐DNA lines, 15 569 of which were produced in this study. This increased the number of unique insertion sites in the T‐DNA collection by 21 078, bringing the overall total to 26 112. Thirty‐seven per cent (9754) of these insertion sites are within genes (including untranslated regions and introns) and 28% (7217) are within 500 bp of a gene. Approximately 31% of the genes in the v.2.1 annotation have been tagged in this population. To demonstrate the utility of this collection, we phenotypically characterized six T‐DNA lines with insertions in genes previously shown in other systems to be involved in cellulose biosynthesis, hemicellulose biosynthesis, secondary cell wall development, DNA damage repair, wax biosynthesis and chloroplast synthesis. In all cases, the phenotypes observed supported previous studies, demonstrating the utility of this collection for plant functional genomics. The Brachypodium T‐DNA collection can be accessed at http://jgi.doe.gov/our-science/science-programs/plant-genomics/brachypodium/brachypodium-t-dna-collection/ .     

Screening and characterization of a novel high‐efficiency tumor‐homing cell‐penetrating peptide from the buffalo cathelicidin family

Targeted delivery of antitumor drugs is especially important for tumor therapy. Cell‐penetrating peptides (CPPs) have been shown to be very effective drug carriers for tumor therapy. However, most CPPs lack tumor cell specificity. Here, we identified a highly efficient CPP, CAT, from the newly identified buffalo‐derived cathelicidin family, which exhibits a preferential binding capacity for multiple tumor cell lines and delivers carried drug molecules into cells. CAT showed an approximately threefold to sixfold higher translocation efficiency than some reported cell‐penetrating antimicrobial peptides, including the well‐known classical CPP TAT. Moreover, the delivery efficiency of CAT was greater in a variety of tested tumor cells than in normal cells, especially for the human hepatoma cell line SMMC‐7721, for which delivery was 7 times more efficient than the normal human embryonic lung cell line MRC‐5, according to fluorescent labeling experiment results. CAT was conjugated to the Momordica charantia‐derived type‐I ribosome‐inactivating protein MAP 30, and the cytotoxicity of the MAP 30‐CAT fusion protein in the tumor cell line SMMC‐7721 was significantly enhanced compared with that of the unconjugated MAP 30. The IC50 value of MAP 30‐CAT was approximately 83 times lower than the IC50 value of the original MAP 30. Interestingly, the IC50 value of MAP 30 alone for MRC‐5 was approximately twofold higher than the value for SMMC‐7721, showing a small difference. However, when MAP 30 was conjugated to CAT, the difference in IC50 values between the two cell lines was significantly increased by 38‐fold. The results of the flow cytometric detection of apoptosis revealed that the increase in cytotoxicity after CAT conjugation was mainly caused by the increased induction of apoptosis by the fusion protein. These results suggest that CAT, as a novel tumor‐homing CPP, has great potential in drug delivery applications in vivo and will be beneficial to the development of tumor therapeutics.     

A Survey Investigating the Infection of Fusarium langsethiae and Production of HT‐2 and T‐2 Mycotoxins in UK Oat Fields

Fusarium langsethiae is a toxigenic fungal species that has been reported in European small‐grain cereal crops such as oats, wheat and barley. Although its relative contribution to fusarium head blight (FHB) symptoms is not well understood, it is reported to contaminate these cereals with high levels of HT‐2 and T‐2 trichothecenes mycotoxins that are currently under consideration for legislation by the European Commission. Ten commercial oat fields in Shropshire and Staffordshire (two adjacent counties in the Midlands) in the UK were surveyed in the 2006/2007 growing season. Samples were taken from predetermined field locations at Zadoks growth stages 32/33, 69, 77‐85 and 90‐92 for F. langsethiae biomass and HT‐2 and T‐2 toxins quantification. The results from this study showed that oats can be heavily infected with F. langsethiae and have high concentrations of HT‐2 and T‐2 toxins with no apparent FHB symptoms. The regression of HT‐2 + T‐2 toxins on F. langsethiae DNA concentration was highly significant (P < 0.001, r² = 0.55). The results indicated that although F. langsethiae had no direct effect on crop yield, it may result in indirect economic losses where the grain can be rejected or downgraded as a result of intolerable levels of HT‐2 and T‐2 toxins, which are of human food and animal feed safety concern. The influence of cultural field practices on the infection and HT‐2 and T‐2 toxins accumulation in oats was not clear and warrants further studies to identify the sources of F. langsethiae inoculum and conditions favourable for infection and mycotoxin production.     

Small‐molecule reductants inhibit multicatalytic activity of AA‐NADase from Agkistrodon acutus venom by reducing the disulfide‐bonds and Cu(II) of enzyme

AA‐NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA‐NADase contains Cu(II) and has disulfide‐bond linkages between two peptide chains. The effects of the reduction of the disulfide‐bonds and Cu(II) in AA‐NADase by small‐molecule reductants on its NADase and ADPase activities have been investigated by polyacrylamide gel electrophoresis, high performance liquid chromatography, electron paramagnetic resonance spectroscopy and isothermal titration calorimetry. The results show that AA‐NADase has six disulfide‐bonds and fifteen free cysteine residues. L‐ascorbate inhibits AA‐NADase on both NADase and ADPase activities through the reduction of Cu(II) in AA‐NADase to Cu(I), while other reductants, dithiothreitol, glutathione and tris(2‐carboxyethyl)phosphine inhibit both NADase and ADPase activities through the reduction of Cu(II) to Cu(I) and the cleavage of disulfide‐bonds in AA‐NADase. Apo‐AA‐NADase can recover its NADase and ADPase activities in the presence of 1 mM Zn(II). However, apo‐AA‐NADase does not recover any NADase or ADPase activity in the presence of 1 mM Zn(II) and 2 mM TCEP. The multicatalytic activity relies on both disulfide‐bonds and Cu(II), while Cu(I) can not activate the enzyme activities. AA‐NADase is probably only active as a dimer. The inhibition curves for both ADPase and NADase activities by each reductant share a similar trend, suggesting both ADPase and NADase activities probably occur at the same site. In addition, we also find that glutathione and L‐ascorbate are endogenous inhibitors to the multicatalytic activity of AA‐NADase. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 141–149, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Cloning and expression of the gene for an insect haemocyte anti‐aggregation protein (VPr3), from the venom of the endoparasitic wasp,Pimpla hypochondriaca

A venom protein from the endoparasitic wasp, Pimpla hypochondriaca, was recently biochemically isolated. This protein possessed haemocyte anti‐aggregation activity in vitro and shares the same N‐terminal amino acid sequence as that deduced from a gene termed vpr3. The vpr3 gene was identified by sequence analysis of randomly isolated cDNAs from a P. hypochondriaca venom gland library. Presently, the gene for the full‐length sequence of mature VPr3 protein was amplified from the P. hypochondriaca venom gland cDNA library by PCR. The amplicon was directionally cloned into a pET expression vector so that recombinant VPr3 (rVPr3) would have an N‐terminal polyhistidine (His) tag. High levels of target protein expression were obtained following addition of IPTG (1 mM) and growth of the bacteria at 37°C for 5 h, or at 24°C for 20 h. Following lysis of bacteria grown at 37°C, the target protein partitioned into the insoluble fraction. However, at 24°C, a small amount of soluble protein was consistently detected. The amount of soluble rVPr3 was subsequently increased when the transformed bacteria were grown in Overnight Express Instant TB medium at 24°C. Soluble rVPr3 was purified utilizing the MagneHis Protein Purification System. Recombinant VPr3 was determined to have adverse effects on the cytoskeleton of Lacanobia oleracea haemocytes and to inhibit the ability of these cells to form aggregates in vitro. © 2009 Wiley Periodicals, Inc.     

Acquired TET2 mutation in one patient with familial platelet disorder with predisposition to AML led to the development of pre‐leukaemic clone resulting in T2‐ALL and AML‐M0

Familial platelet disorder with predisposition to acute myeloid leukaemia (FPD/AML) is characterized by germline RUNX1 mutations, thrombocytopaenia, platelet dysfunction and a risk of developing acute myeloid and in rare cases lymphoid T leukaemia. Here, we focus on a case of a man with a familial history of RUNX1^R174Q mutation who developed at the age of 42 years a T2‐ALL and, 2 years after remission, an AML‐M0. Both AML‐M0 and T2‐ALL blast populations demonstrated a loss of 1p36.32‐23 and 17q11.2 regions as well as other small deletions, clonal rearrangements of both TCRγ and TCRδ and a presence of 18 variants at a frequency of more than 40%. Additional variants were identified only in T2‐ALL or in AML‐M0 evoking the existence of a common original clone, which gave rise to subclonal populations. Next generation sequencing (NGS) performed on peripheral blood‐derived CD34⁺ cells 5 years prior to T2‐ALL development revealed only the missense TET2^P1962T mutation at a frequency of 1%, which increases to more than 40% in fully transformed leukaemic T2‐ALL and AML‐M0 clones. This result suggests that TET2^P1962T mutation in association with germline RUNX1^R174Q mutation leads to amplification of a haematopoietic clone susceptible to acquire other transforming alterations.     

Kinetic and functional characterisation of the heparin‐binding peptides from human transglutaminase 2

Transglutaminase 2 (TG2) is an autoantigen in celiac disease (CD) and it has multiple biologic functions including involvement in cell adhesion through interactions with integrins, fibronectin (FN), and heparan sulfate proteoglycans. We aimed to delineate the heparin‐binding regions of human TG2 by studying binding kinetics of the predicted heparin‐binding peptides using surface plasmon resonance method. In addition, we characterized immunogenicity of the TG2 peptides and their effect on cell adhesion. The high‐affinity binding of human TG2 to the immobilized heparin was observed, and two TG2 peptides, P1 (amino acids 202–215) and P2 (261–274), were found to bind heparin. The amino acid sequences corresponding to the heparin‐binding peptides were located close to each other on the surface of the TG2 molecule as part of the α‐helical structures. The heparin‐binding peptides displayed increased immunoreactivity against serum IgA of CD patients compared with other TG2 peptides. The cell adhesion reducing effect of the peptide P2 was revealed in Caco‐2 intestinal epithelial cell attachment to the FN and FN‐TG2 coated surfaces. We propose that TG2 amino acid sequences 202–215 and 261–274 could be involved in binding of TG2 to cell surface heparan sulfates. High immunoreactivity of the corresponding heparin‐binding peptides of TG2 with CD patient's IgA supports the previously described role of anti‐TG2 autoantibodies interfering with this interaction. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Autoimmunity and the microbiome: T‐cell receptor mimicry of “self” and microbial antigens mediates self tolerance in holobionts

I propose a T‐cell receptor (TcR)‐based mechanism by which immunity mediates both “genetic self” and “microbial self” thereby, connecting microbiome disease with autoimmunity. The hypothesis is based on simple principles. First, TcR are selected to avoid strong cross‐reactivity with “self,” resulting in selection for a TcR repertoire mimicking “genetic self.” Second, evolution has selected for a “microbial self” that mimics “genetic self” so as to share tolerance. In consequence, our TcR repertoire also mimics microbiome antigenicity, providing a novel mechanism for modulating tolerance to it. Also, the microbiome mimics the TcR repertoire, acting as a secondary immune system. I call this TcR‐microbiome mimicry “holoimmunity” to denote immune tolerance to the “holobiont self.” Logically, microbiome‐host mimicry means that autoimmunity directed at host antigens will also attack components of the microbiome, and conversely, an immunological attack on the microbiome may cross‐react with host antigens producing “holoautoimmunity.”

     

Isolation and characterization of SsmTx‐I,a Specific Kv2.1 blocker from the venom of the centipede Scolopendra Subspinipes Mutilans L. Koch

Scolopendra subspinipes mutilans, also known as Chinese red‐headed centipede, is a venomous centipede from East Asia and Australasia. Venom from this animal has not been researched as thoroughly as venom from snakes, snails, scorpions, and spiders. In this study, we isolated and characterized SsmTx‐I, a novel neurotoxin from the venom of S. subspinipes mutilans. SsmTx‐I contains 36 residues with four cysteines forming two disulfide bonds. It had low sequence similarity (<10%) with other identified peptide toxins. By whole‐cell recording, SsmTx‐I significantly blocked voltage‐gated K⁺ channels in dorsal root ganglion neurons with an IC₅₀ value of 200 nM, but it had no effect on voltage‐gated Na⁺ channels. Among the nine K⁺ channel subtypes expressed in human embryonic kidney 293 cells, SsmTx‐I selectively blocked the Kv2.1 current with an IC₅₀ value of 41.7 nM, but it had little effect on currents mediated by other K⁺ channel subtypes. Blockage of Kv2.1 by SsmTx‐I was not associated with significant alteration of steady‐state activation, suggesting that SsmTx‐I might act as a simple inhibitor or channel blocker rather than a gating modifier. Our study reported a specific Kv2.1‐blocker from centipede venom and provided a basis for future investigations of SsmTx‐I, for example on structure–function relationships, mechanism of action, and pharmacological potential. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Identification and characterization of two novel antimicrobial peptides,temporin‐Ra and temporin‐Rb,from skin secretions of the marsh frog (Rana ridibunda)

In this study, two novel antimicrobial peptides from the skin secretions of the marsh frog, Rana ridibunda, named temporin‐Ra and temporin‐Rb, were identified and purified using RP‐HPLC. Temporin‐Ra and temporin‐Rb are composed of 14 and 12 amino acids, respectively. Our results show that these peptides have inhibitory effects on both gram‐negative and gram‐positive bacteria, especially antibiotic resistant strains prevalent in hospitals, such as Staphylococcus aureus and Streptococcus agalactiae. The sequences and molecular weights of these peptides were determined using tandem MS. The molecular masses were found to be 1242.5 Da for temporin‐Rb and 1585.1 Da for temporin‐Ra. Human red blood cells tolerated well exposure to temporin‐Ra and temporin‐Rb, which, at a concentration of 60 µg/ml, induced 1.3% and 1.1% hemolysis, respectively. MIC values of these peptides are suitable for potent antimicrobial peptides. The low hemolytic effect and wide‐spectrum antimicrobial activity suggest a possible therapeutic application of these novel peptides. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Novel Ca2+‐independent carbohydrate recognition of the C‐type lectins,SPL‐1 and SPL‐2, from the bivalve Saxidomus purpuratus

Novel Ca²⁺‐independent C‐type lectins, SPL‐1 and SPL‐2, were purified from the bivalve Saxidomus purpuratus. They are composed of dimers with either identical (SPL‐2 composed of two B‐chains) or distinct (SPL‐1 composed of A‐ and B‐chains) polypeptide chains, and show affinity for N‐acetylglucosamine (GlcNAc)‐ and N‐acetylgalactosamine (GalNAc)‐containing carbohydrates, but not for glucose or galactose. A database search for sequence similarity suggested that they belong to the C‐type lectin family. X‐ray crystallographic analysis revealed definite structural similarities between their subunits and the carbohydrate‐recognition domain (CRD) of the C‐type lectin family. Nevertheless, these lectins (especially SPL‐2) showed Ca²⁺‐independent binding affinity for GlcNAc and GalNAc. The crystal structure of SPL‐2/GalNAc complex revealed that bound GalNAc was mainly recognized via its acetamido group through stacking interactions with Tyr and His residues and hydrogen bonds with Asp and Asn residues, while widely known carbohydrate‐recognition motifs among the C‐type CRD (the QPD [Gln‐Pro‐Asp] and EPN [Glu‐Pro‐Asn] sequences) are not involved in the binding of the carbohydrate. Carbohydrate‐binding specificities of individual A‐ and B‐chains were examined by glycan array analysis using recombinant lectins produced from Escherichia coli cells, where both subunits preferably bound oligosaccharides having terminal GlcNAc or GalNAc with α‐glycosidic linkages with slightly different specificities.     

Distribution and characterization of more than 1000 T‐DNA tags in the genome of Brachypodium distachyon community standard line Bd21

A collection of 4117 fertile T‐DNA lines has been generated by Agrobacterium‐mediated transformation of the diploid community standard line Bd21 of Brachypodium distachyon. The regions flanking the T‐DNA left and right borders of the first 741 transformed plants were isolated by adapter‐ligation PCR and sequenced. A total of 1005 genomic sequences (representing 44.1% of all flanking sequences retrieved) characterized 660 independent T‐DNA loci assigned to a unique location in the Brachypodium genome sequence. Seventy‐six percent of the fertile plant lines contained at least one anchored T‐DNA locus (1.17 loci per tagged line on average). Analysis of the regions flanking both borders of the T‐DNA increased the number of T‐DNA loci tagged and the number of tagged lines by approximately 50% when compared to a single border analysis. T‐DNA integration (2.4 insertions per Mb on average) was proportional to chromosome size, however, varied greatly along each chromosome with often low insertion level around centromeres. The frequency of insertion within transposable elements (5.3%) was fivefold lower than expected if random insertion would have occurred. More than half of the T‐DNAs inserted in genic regions. On average, one gene could be tagged for every second fertile plant line produced and more than one plant line out of three contained a T‐DNA insertion directly within or 500 bp around the coding sequence. Approximately, 60% of the genes tagged corresponded to expressed genes. The T‐DNA lines generated by the BrachyTAG programme are available as a community resource and have been distributed internationally since 2008 via the BrachyTAG.org web site.     

CMV seropositivity and T‐cell senescence predict increased cardiovascular mortality in octogenarians: results from the Newcastle 85+ study

Although chronic infection with cytomegalovirus (CMV) is known to drive T lymphocytes toward a senescent phenotype, it remains controversial whether and how CMV can cause coronary heart disease (CHD). To explore whether CMV seropositivity or T‐cell populations associated with immunosenescence were informative for adverse cardiovascular outcome in the very old, we prospectively analyzed peripheral blood samples from 751 octogenarians (38% males) from the Newcastle 85+ study for their power to predict survival during a 65‐month follow‐up (47.3% survival rate). CMV‐seropositive participants showed a higher prevalence of CHD (37.7% vs. 26.7%, P = 0.030) compared to CMV‐seronegative participants together with lower CD4/CD8 ratio (1.7 vs. 4.1, P < 0.0001) and higher frequencies of senescence‐like CD4 memory cells (41.1% vs. 4.5%, P < 0.001) and senescence‐like CD8 memory cells (TEMRA, 28.1% vs. 6.7%, P < 0.001). CMV seropositivity was also associated with increased six‐year cardiovascular mortality (HR 1.75 [1.09–2.82], P = 0.021) or death from myocardial infarction and stroke (HR 1.89 [107–3.36], P = 0.029). Gender‐adjusted multivariate Cox regression analysis revealed that low percentages of senescence‐like CD4 T cells (HR 0.48 [0.32–0.72], P < 0.001) and near‐senescent (CD27 negative) CD8 T cells (HR 0.60 [0.41–0.88], P = 0.029) reduced the risk of cardiovascular death. For senescence‐like CD4, but not near‐senescent CD8 T cells, these associations remained robust after additional adjustment for CMV status, comorbidities, and inflammation markers. We conclude that CMV seropositivity is linked to a higher incidence of CHD in octogenarians and that senescence in both the CD4 and CD8 T‐cell compartments is a predictor of overall cardiovascular mortality as well as death from myocardial infarction and stroke.     

Specific anti‐integrase abzymes from HIV‐infected patients: a comparison of the cleavage sites of intact globular HIV integrase and two 20‐mer oligopeptides corresponding to its antigenic determinants

HIV‐infected patients possess anti‐integrase (IN) IgGs and IgMs that, after isolation by chromatography on IN‐Sepharose, unlike canonical proteases, specifically hydrolyze only IN but not many other tested proteins. Hydrolysis of intact globular IN first leads to formation of many long fragments of protein, while its long incubation with anti‐IN antibodies, especially in the case of abzymes (Abzs) with a high proteolytic activity, results in the formation of short and very short oligopeptides (OPs). To identify all sites of IgG‐mediated proteolysis corresponding to known AGDs of integrase, we have used a combination of reverse‐phase chromatography, matrix‐assisted laser desorption/ionization spectrometry, and thin‐layer chromatography to analyze the cleavage products of two 20‐mer OPs corresponding to these AGDs. Both OPs contained 9–10 mainly clustered major, medium, and minor sites of cleavage. The main superficial cleavage sites of the AGDs in the intact IN and sites of partial or deep hydrolysis of the peptides analyzed do not coincide. The active sites of anti‐IN Abzs are localized on their light chains, whereas the heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of Abzs provide high specificity of IN hydrolysis. The affinity of anti‐IN Abzs for intact integrase was ~1000‐fold higher than for the OPs. The data suggest that both OPs interact mainly with the light chains of different monoclonal Abzs of the total pool of IgGs, which possesses lower affinity for substrates; and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific and remarkably different in comparison with the cleavage of intact globular IN. Copyright © 2013 John Wiley & Sons, Ltd.