Affinity maturation of anti-TNF-alpha scFv with somatic hypermutation in non-B cells

Activation-induced cytidine deaminase(AID)is required for the generation of antibody diversity through initiat-ing both somatic hypermutation(SHM)and class switch recombination.A few research groups have success-fully used the feature of AID for generating mutant li-braries in directed evolution of target proteins in B cells in vitro.B cells,cultured in suspension,are not con-venient for transfection and cloning.In this study,we established an AID-based mutant accumulation and sorting system in adherent human cells.Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells,and a stable cell clone(H1299-AID)was selected.Afterwards,anti-hTNF-αscFv(ATscFv)was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells.By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha,two ATscFv mutant gene clones were isolated.Compared with the wild type ATscFv,the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha.The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells,which makes directed evolution in mammalian cells easier and more efficient.

Antibody-Like Phosphorylation Sites in Focus of Statistically Based Bilingual Approach

In accordance with previous reports, the sequences related to phosporylated protein segments occur in conserved variable domains of immunoglobulins including first of all certain N-terminally located segments. Consequently, we look here for the sequences 1) composing human and mouse proteins different from antigen receptors, 2) identical with or highly similar to nucleotide sequence representatives of conserved variable immunoglobulin segments and 3) identical with or closely related to phosphorylation sites. More precisely, we searched for the corresponding actual pairs of DNA and protein sequence segments using five-step bilingual approach employing among others a) different types of BLAST searches, b) two in-principle-different machine-learning methods predicting phosphorylated sites and c) two large databases recording existing phosphorylation sites. The approach identified seven existing phosphorylation sites and thirty-seven related human and mouse segments achieving limits for several predictions or phylogenic parameters. Mostly serines phosporylated with ataxia-telangiectasia-related kinase (involved in regulation of DNA-double-strand-break repair) were indicated or predicted in this study. Hypermutation motifs, located in effective positions of the selected sequence segments, occurred significantly less frequently in transcribed than non-transcribed DNA strands suggesting thus the incidence of mutation events. In addition, marked differences between the numbers and proportions of human and mouse cancer-related sequence items were found in different steps of selection process. The possible role of hypermutation changes within the selected segments and the observed structural relationships are discussed here with respect to DNA damage, carcinogenesis, cancer vaccination, ageing and evolution. Taken together, our data represent additional and sometimes perhaps complementary information to the existing databases of empirically proven phosphorylation sites or pathogenically important spots.

Antibody-Like Phosphorylation Sites. Theme for Studies of Cancer, Aging and Evolution

Sequence similarities were found between protein and DNA sequences encoding certain part of conserved variable immunoglobulin domains (i.e. conserved IgV) and phosphorylation sites. Hypermutation motifs were then indicated in the majority of the corresponding non-IgV nucleotide sequences. According to database confirmations or double prediction of phosphorylation sites, 80% of the selected human and mouse IgV-related phosphorylation sites or their highly probable candidates exhibited substrate relationship to ataxia-telangiectasia-mutated kinase known as ATM. In accordance with literature data, inactivation of ATM by mutations can participate in the mechanisms of carcinogenesis, neurodegeneration and possibly also in aging. In agreement with this relationship, some of the selected IgV-/ATM-related segments formed molecules specifically involved in carcinogenesis. The selected IgV-related sequence segments were also similar to certain segments of higher plants containing immunoglobulin-like repeats and related regions. Bioinformatic analysis of some selected plant sequences then indicated the presence of catalytic domains composing serine/threonine/tyrosine receptor/receptor-like kinases, which are considered important structures for evolution of very early and part of later Ig-domain-related immunity. The analyzed conserved domain similarities also suggested certain interesting structural and phylogenic relationships, which need to be further investigated. This review in fact briefly summarizes the findings on the subject from the last twenty years.

Antiviral Warrior—APOBEC3G

This paper is to further understand how APOBEC3G can defend the retroviruses and to find new approaches to AIDs （acquired immure deficiency syndrome）.The viral infectivity factor （Vif） induces rapid degradation of APOBEC3G by ubiquitination, which is a proteosome-dependent pathway. Precisely speaking, only in the virus-producing cell Vif expression is necessary; in its absence, infection of a subsequent target cell terminates at a postentry step through the action of the human APOBEC3G antiviral mechanism. Hf protein has two domains： one binds to APOBEC3G and the other regulates the degradation of APOBEC3G by a conserved sequence, SLQ （Y/F） LA motif. Recently, the research on Vif has also revealed APOBEC3G is a novel component of innate immune system. In fact, APOBEC3G not only acts in DNA editing to block the replication of retroviruses such as HIV-1, but also is able to defend a wide spectrum of distantly related retroviruses and interferes with HBV through a different mechanism from HIE.

A dual function for the chromatin organizer Special A-T rich Binding Protein 1 in B-lineage cells

SATB1(Special A-T rich Binding protein 1)is a cell type-specific factor that regulates the genetic network in developing T cells and neurons.In T cells,SATB1 is required for lineage commitment,VDJ recombination,development and maturation.Considering that its expression varies during B-cell differentiation,the involvement of SATB1 needs to be clarified in this lineage.Using a KO mouse model in which SATB1 was deleted from the pro-B-cell stage,we examined the consequences of SATB1 deletion in naive and activated B-cell subsets.Our model indicates first,unlike its essential function in T cells,that SATB1 is dispensable for B-cell development and the establishment of a broad IgH repertoire.Second,we show that SATB1 exhibits an ambivalent function in mature B cells,acting sequentially as a positive and negative regulator of Ig gene transcription in naive and activated cells,respectively.Third,our study indicates that the negative regulatory function of SATB1 in B cells extends to the germinal center response,in which this factor limits somatic hypermutation of Ig genes.

APOBEC deaminases-mutases with defensive roles for immunity

In recent years, tremendous progress has been made in the elucidation of the biological roles and molecular mechanisms of the apolioprotein B mRNA-editing enzyme catalytic polypeptide (APOBEC) family of enzymes. The APOBEC family of cytidine deaminases has important functional roles within the adaptive and innate immune system. Activation induced cytidine deaminase (AID) plays a central role in the biochemical steps of somatic hypermutation and class switch recombination during antibody maturation, and the APOBEC 3 enzymes are able to inhibit the mobility of retroelements and the replication of retroviruses and DNA viruses, such as the human immunodeficiency virus type-1 and hepatitis B virus. Recent advances in structural and functional studies of the APOBEC enzymes provide new biochemical insights for how these enzymes carry out their biological roles. In this review, we provide an overview of these recent advances in the APOBEC field with a special emphasis on AID and APOBEC3G.

Generation and repair of AID-initiated DNA lesions in B lymphocytes

Activation-induced deaminase （AID） initiates the secondary antibody diversification process in B lymphocytes. In mammalian B cells, this process includes somatic hypermutation （SHM） and class switch recombination （CSR）, both of which require AID. AID induces U：G mismatch lesions in DNA that are subsequently converted into point mutations or DNA double stranded breaks during SHM/CSR. In a physiological context, AID targets immunogiobulin （Ig） loci to mediate SHM/CSR. However, recent studies reveal genome-wide access of AID to numerous non-Ig loci. Thus, AID poses a threat to the genome of B cells if AID-initiated DNA lesions cannot be properly repaired. In this review, we focus on the molecular mechanisms that regulate the specificity of AID targeting and the repair pathways responsible for processing AID-initiated DNA lesions.

Rigorous Running Time Analysis of a Simple Immune-Based Multi-Objective Optimizer for Bi-Objective Pseudo-Boolean Functions

A simple immune-based multi-objective optimizer(IBMO) is proposed, and a rigorous running time analysis of IBMO on three proposed bi-objective pseudo-Boolean functions(Bi-Trap, Bi-Plateau and Bi-Jump) is presented. The running time of a global simple evolutionary multi-objective optimizer(GSEMO) using standard bit mutation operator with IBMO using somatic contiguous hypermutation(CHM) operator is compared with these three functions. The results show that the immune-based hypermutation can significantly beat standard bit mutation on some well-known multi-objective pseudo-Boolean functions. The proofs allow us to understand the relationship between the characteristics of the problems and the features of the algorithms more deeply. These analysis results also give us a good inspiration to analyze and design a bio-inspired search heuristics.

Developing controllable hypermutable Clostridium cells through manipulating its methyl-directed mismatch repair system

Development of controllable hypermutable cells can greatly benefit understanding and harnessing microbial evolution.However,there have not been any similar sys-tems developed for Clostridium,an important bacterial genus.Here we report a novel two-step strategy for de-veloping controllable hypermutable cells of Clostridium acetobutylicum,an important and representative indus-trial strain.Firstly,the mutS/L operon essential for methyl-directed mismatch repair(MMR)activity was inactivated from the genome of C.acetobutylicum to generate hy-permutable cells with over 250-fold increased mutation rates.Secondly,a proofreading control system carrying an inducibly expressed mutS/L operon was constructed.The hypermutable cells and the proofreading control system were integrated to form a controllable hypermut-able system SMBMutC,of which the mutation rates can be regulated by the concentration of anhydrotetracycline(aTc).Duplication of the miniPthl-tetR module of the proof-reading control system further signifi cantly expanded the regulatory space of the mutation rates,demonstrating hypermutable Clostridium cells with controllable mutation rates are generated.The developed C.acetobutylicum strain SMBMutC2 showed higher survival capacities than the control strain facing butanol-stress,indicating greatly increased evolvability and adaptability of the controllable hypermutable cells under environmental challenges.

Detecting genetic hypermutability of gastrointestinal tumor by using a forensic STR kit

Growing evidence suggests that somatic hypermutational status and programmed cell death-1 overexpression are potential predictive biomarkers indicating treatment benefits from immunotherapy using immune checkpoint inhibitors.However,biomarker-matched trials are still limited,and many of the genomic alterations remain difficult to target.To isolate the potential somatic hypermutational tumor from microsatellite instability low/microsatellite stability(MSI-L/MSS)cases,we employed two commercial kits to determine MSI and forensic short tandem repeat(STR)alternations in 250 gastrointestinal(GI)tumors.Three types of forensic STR alternations,namely,allelic loss,Aadd,and Anew,were identified.62.4%(156/250)of the patients with GI exhibited STR alternation,including 100%(15/15)and 60%(141/235)of the microsatellite high instability and MSI-L/MSS cases,respectively.30%(75/250)of the patients exhibited STR instability with more than 26.32%(26.32%–84.21%)STR alternation.The cutoff with 26.32%of the STR alternations covered all 15 MSI cases and suggested that it might be a potential threshold.Given the similar mechanism of the mutations of MSI and forensic STR,the widely used forensic identifier STR kit might provide potential usage for identifying hypermutational status in GI cancers.