Cysteine In Position Research Articles

Distinct types of VHHs in Alpaca.

VHHs (VH of heavy-chain-only antibodies) represent a unique alternative to Q7 conventional antibodies because of their smaller size, comparable binding affinity and biophysical properties. In this study, we systematically analyzed VHH NGS sequences from 22 Alpacas and structure data from public database. VHHs in Alpaca can be grouped into five main types with multiple distinct sequence and structure features. Based on the existence of hallmark residues in FR2 region, VHHs can be classified into two groups: nonclassical VHHs (without hallmark residues) and classical VHHs (with hallmark residues). Based on VHH hallmark residues at 42 position (IMGT numbering, FR2 region) and number of cysteines, we found that Alpaca classical VHHs can be further separated into three main types: F_C2 VHHs with F (phenylalanine) at position 42 and having 2 cysteines within sequences, Y_C2 VHHs with Y (tyrosine) at position 42 and having 2 cysteines, and F_C4 with F at position 42 and having 4 cysteines. Non-classical VHHs can be further separated into 2 types based on germlines mapped: N_V3 for VHHs mapped to V3 germlines and N_V4 for V4 germlines. Based on whether FR2 residues are involved in binding, two kinds of paratopes can be identified. Different types of VHHs showed distinct associations with these two paratopes and displayed significant differences in paratope size, residue usage and other structure features. Such results will have significant implications in VHH discovery, engine e ring, and design for innovative therapeutics.

Development of QSARs for cysteine-containing di- and tripeptides with antioxidant activity:influence of the cysteine position.

Antioxidants agents play an essential role in the food industry for improving the oxidative stability of food products. In the last years, the search for new natural antioxidants has increased due to the potential high toxicity of chemical additives. Therefore, the synthesis and evaluation of the antioxidant activity in peptides is a field of current research. In this study, we performed a Quantitative Structure Activity Relationship analysis (QSAR) of cysteine-containing 19 dipeptides and 19 tripeptides. The main objective is to bring information on the relationship between the structure of peptides and their antioxidant activity. For this purpose, 1D and 2D molecular descriptors were calculated using the PaDEL software, which provides information about the structure, shape, size, charge, polarity, solubility and other aspects of the compounds. Different QSAR model for di- and tripeptides were developed. The statistic parameters for di-peptides model (R2train = 0.947 and R2test = 0.804) and for tripeptide models (R2train = 0.923 and R2test = 0.847) indicate that the generated models have high predictive capacity. Then, the influence of the cysteine position was analyzed predicting the antioxidant activity for new di- and tripeptides, and comparing them with glutathione. In dipeptides, excepting SC, TC and VC, the activity increases when cysteine is at the N-terminal position. For tripeptides, we observed a notable increase in activity when cysteine is placed in the N-terminal position.

Evidence of Gas Phase Glucosyl Transfer and Glycation in the CID/HCD-Spectra of S-Glucosylated Peptides.

Protein cysteine S-glycosylation is a relatively rare and less well characterized post-translational modification (PTM). Creating reliable model proteins that carry this modification is challenging. The lack of available models or natural S-glycosylated proteins significantly hampers the development of mass-spectrometry-based (MS-based) methodologies for detecting protein cysteine S-glycosylation in real-world proteomic studies. There is also limited MS-sequencing data describing it as easier to create synthetic S-glycopeptides. Here, we present the results of an in-depth manual analysis of automatically annotated CID/HCD spectra for model S-glucopeptides. The CID spectra show a long series of y/b-fragment ions with retained S-glucosylation, regardless of the dominant m/z signals corresponding to neutral loss of 1,2-anhydroglucose from the precursor ions. In addition, the spectra show signals manifesting glucosyl transfer from the cysteine position onto lysine, arginine (Lys, Arg) side chains, and a peptide N-terminus. Other spectral evidence indicates that the N-glucosylated initial products of transfer are converted into N-fructosylated (i.e., glycated) structures due to Amadori rearrangement. We discuss the peculiar transfer of the glucose oxocarbenium ion (Glc+) to positively charged guanidinium residue (ArgH+) and propose a mechanism for the gas-phase Amadori rearrangement involving a 1,2-hydride ion shift.

Dichotomy in TCR V-domain dynamics binding the opposed inclined planes of pMHC-II and pMHC-I α-helices

Ligand recognition by the human α/β T-cell antigen receptor (TCR) heterodimer protein, unlike the surface immunoglobulin (sIg) B-cell receptor, is not governed by relative binding affinity. Its interaction with the peptide (p) plus major histocompatibility complex (MHC) protein (abbrev. pMHC) likely involves some different molecular mechanism linking pMHC binding to T-cell functions. Recent analytical geometry of TCR:pMHC-II solved crystallographic structures (n = 40) revealed that each variable (V)-domain is bound in similar, yet mathematically unique orientations to its target pMHC groove. The relative position of the central cysteine of each V-domain was examined by multivariable calculus in spherical coordinates, where a simple volume element (dV) was found to describe clonotypic geometry with pMHC-II. Here, the study was expanded to include TCR:pMHC-I structures, and to model a physical mechanism, specifically involving the two directionally opposed inclined planes (IP) manifest by the two major α-helices prominent in both MHC-I and MHC-II proteins. Calculations for rotational torque of each V-domain, together with acceleration up and down the slopes of both MHC α-helices were used to estimate the time a given V-domain spends sliding down its cognate MHC IP. This V-domain rotation/sliding mechanism appears to be quantitatively unique for each TCR:pMHC V-domain (n = 40). However, there is an apparent and common dichotomy between the mobility of each V-domain with respect to the two classes of MHC proteins. Evolutionary motifs in the MHC helices support that the V-domains negotiate the opposed inclined planes of pMHC ligands in clonotypic fashion. Thus, this model is useful in understanding how mechanical forces are linked to TCR function.

Functional characterization of rice metallothionein OsMT-I-Id: Insights into metal binding and heavy metal tolerance mechanisms

Metallothioneins (MTs) are cysteine-rich proteins known for their strong metal-binding capabilities, making them effective in detoxifying heavy metals (HMs). This study focuses on characterizing the functional properties of OsMT-I-Id, a type-I Metallothionein found in rice. Using a HM-responsive yeast cup1Δ (DTY4), ycf1∆ (for cadmium), and acr3∆ mutants (for trivalent arsenic), we assessed the impact of OsMT-I-Id on metal accumulation and cellular resilience. Our results demonstrated that yeast cells expressing OsMT-I-Id showed increased tolerance and accumulated higher levels of copper (Cu), arsenic (As), and cadmium (Cd), compared to control cells. This can be attributed to the protein's ability to chelate and bind HMs. Site-directed mutagenesis was employed to investigate the specific contributions of cysteine residues. The study revealed that yeast cells with a mutated C-domain displayed heightened HM sensitivity, while cells with a mutated N-domain exhibited reduced sensitivity. This underscores the critical role of C-cysteine-rich domains in metal binding and tolerance of type-I rice MTs. Furthermore, the study identified the significance of the 12th cysteine position at the N-domain and the 68th and 72nd cysteine positions at the C-domain in influencing OsMT-I-Id metal-binding capacity. This research provides novel insights into the structure-function relationship and metal binding properties of type-I plant MTs.

Vitellogenin receptor transports the 30K protein LP1 without cell-penetrating peptide, into the oocytes of the silkworm, Bombyx mori.

Vitellogenin receptors (VgRs) transport vitellogenin (Vg) into oocytes, thereby promoting egg growth and embryonic development. VgRs recognize and transport multiple ligands in oviparous animals, but their role in insects is rarely reported. In this study, we investigated whether Bombyx mori VgR (BmVgR) binds and transports lipoprotein-1 (BmLP1) and lipoprotein-7 (BmLP7) of the 30kDa lipoproteins (30K proteins), which are essential for egg formation and embryonic development in B. mori. Protein sequence analysis showed BmLP7, similar to reported lipoprotein-3 (BmLP3), contains the cell-penetrating peptides and Cysteine position, while BmLP1 has not. Assays using Spodoptera frugiperda ovary cells (sf9) indicated the direct entry of BmLP7 into the cells, whereas BmLP1 failed to enter. However, co-immunoprecipitation (Co-IP) assays indicated that BmVgR could bind BmLP1. Western blotting and immunofluorescence assays further revealed that over-expressed BmVgR could transport BmLP1 into sf9 cells. Co-IP assays showed that SE11C (comprising LBD1+EGF1+OTC domains of BmVgR) or SE22C (comprising LBD2+EGF2+OTC domains of BmVgR) could bind BmLP1. Over-expressed SE11C or SE22C could also transport BmLP1 into sf9 cells. Western blotting revealed that the ability of SE11C to transport BmLP1 might be stronger than that of SE22C. In the vit mutant with BmVgR gene mutation (vit/vit), SDS-PAGE and western blotting showed the content of BmLP1 in the ovary, like BmVg, was lower than that in the normal silkworm. When transgenic with hsp70 promoter over-expressed BmVgR in the vit mutant, we found that the phenotype of the vit mutant was partly rescued after heat treatment. And contents of BmLP1 and BmVg in vit mutant over-expressed BmVgR were higher than in the vit mutant. We conclude that BmVgR and its two repeat domains could bind and transport BmLP1 into the oocytes of the silkworm, besides BmVg. These results will provide a reference for studying the molecular mechanism of VgR transporting ligands in insects.

Germline-Encoded Positional Cysteine Polymorphisms Enhance Diversity in Antibody Ultralong CDR H3 Regions.

Ab "ultralong" third H chain complementarity-determining regions (CDR H3) appear unique to bovine Abs and may enable binding to difficult epitopes that shorter CDR H3 regions cannot easily access. Diversity is concentrated in the "knob" domain of the CDR H3, which is encoded by the DH gene segment and sits atop a β-ribbon "stalk" that protrudes far from the Ab surface. Knob region cysteine content is quite diverse in terms of total number of cysteines, sequence position, and disulfide bond pattern formation. We investigated the role of germline cysteines in production of a diverse CDR H3 structural repertoire. The relationship between DH polymorphisms and deletions relative to germline at the nucleotide level, as well as diversity in cysteine and disulfide bond content at the structural level, was ascertained. Structural diversity is formed through (1) DH polymorphisms with altered cysteine positions, (2) DH deletions, and (3) new cysteines that arise through somatic hypermutation that form new, unique disulfide bonds to alter the knob structure. Thus, a combination of mechanisms at both the germline and somatic immunogenetic levels results in diversity in knob region cysteine content, contributing to remarkable complexity in knob region disulfide patterns, loops, and Ag binding surface.

A Review of Methodologies for the Detection, Quantitation, and Localization of Free Cysteine in Recombinant Proteins: A Focus on Therapeutic Monoclonal Antibodies

Free-cysteine residues in recombinant biotherapeutics such as monoclonal antibodies can arise from incorrect cellular processing of disulfide bonds during synthesis or by reduction of disulfide bonds during the harvest and purification stage of manufacture. Free cysteines can affect potency, induce aggregation, and decrease the stability of therapeutic proteins, and the levels and positions of free cysteines in proteins are closely monitored by both manufacturers and regulators to ensure safety and efficacy. This review summarizes the latest methodologies for the detection and quantification of free cysteines.

Effect of Conjugation Site and Technique on the Stability and Pharmacokinetics of Antibody-Drug Conjugates.

Appropriate selection of conjugation sites and conjugation technologies is now widely accepted as crucial for the success of antibody-drug conjugates (ADCs). Herein, we present ADCs conjugated by different conjugation methods to different conjugation positions being systematically characterized by multiple in vitro assays as well as in vivo pharmacokinetic (PK) analyses in transgenic Tg276 mice. Conjugation to cysteines, genetically introduced at positions N325, L328, S239, D265, and S442, was compared to enzymatic conjugation via microbial transglutaminase (mTG) either to C-terminal light (LC) or heavy chain (HC) recognition motifs or to endogenous position Q295 of a native antibody. All conjugations yielded homogeneous DAR 2 ADCs with similar hydrophobicity, thermal stability, human neonatal Fc receptor (huFcRn) binding, and serum stability properties, but with pronounced differences in their PK profiles. mTG-conjugated ADC variants conjugated either to Q295 or to LC recognition motifs showed superior PK behavior. Within the panel of engineered cysteine variants L328 showed a similar PK profile compared to previously described S239 but superior PK compared to S442, D265, and N325. While all positions were first tested with trastuzumab, L328 and mTG LC were further evaluated with additional antibody scaffolds derived from clinically evaluated monoclonal antibodies (mAb). Based on PK analyses, this study confirms the newly described position L328 as favorable site for cysteine conjugation, comparable to the well-established engineered cysteine position S239, and emphasizes the favorable position Q295 of native antibodies and the tagged LC antibody variant for enzymatic conjugations via mTG. In addition, hemizygous Tg276 mice are evaluated as an adequate model for ADC pharmacokinetics, facilitating the selection of suitable ADC candidates early in the drug discovery process.

Crystallographic Structures of Titin Immunoglobulin-Like I21 Domains Involved in Dilated Cardiomyopathy

The protein titin is the main responsible for the passive elasticity of the sarcomere, determining the stiffness and contractile function of cardiomyocytes. Titin is also a key factor in the etiology of heart disease, given that truncating mutations in the titin gene are the most common cause of dilated cardiomyopathy (DCM). However, missense mutations have commonly been classified as variants of uncertain significance because of their high frequency in the general population and the absence of functional annotation. We have recently identified a missense mutation targeting a conserved cysteine in the I21 domain of titin as a cause of DCM in humans. Here, we present the structural characterization of the wild type domain, that follows a fold characteristic for the intermediate set (type I) of the immunoglobulin superfamily: 2 antiparallel 4-strand-β-sheets packed in a β-sandwich shape. Interestingly, we observed that the I21 titin domain contains a second cysteine located 5Å away from the cysteine position mutated in dilated cardiomyopathy patients. We also report the structural and biophysical characterization of the mutated domain. The mutated domain presents unaltered overall structure, although its thermal stability is severely compromised, showing a decrease of 17ºC in its melting temperature when compared to the wild type domain. Our results suggest that titin domain destabilization by missense variants can lead to DCM, even if the structure of the native state is preserved. Future research will investigate the origin of the thermal destabilization in the mutated domain, and the potential influence of intramolecular disulfide bonds.

Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtii

Protein S-nitrosylation plays a fundamental role in cell signaling and nitrosoglutathione (GSNO) is considered as the main nitrosylating signaling molecule. Enzymatic systems controlling GSNO homeostasis are thus crucial to indirectly control the formation of protein S-nitrosothiols. GSNO reductase (GSNOR) is the key enzyme controlling GSNO levels by catalyzing its degradation in the presence of NADH. Here, we found that protein extracts from the microalga Chlamydomonas reinhardtii catabolize GSNO via two enzymatic systems having specific reliance on NADPH or NADH and different biochemical features. Scoring the Chlamydomonas genome for orthologs of known plant GSNORs, we found two genes encoding for putative and almost identical GSNOR isoenzymes. One of the two, here named CrGSNOR1, was heterologously expressed and purified. Its kinetic properties were determined and the three-dimensional structures of the apo-, NAD+- and NAD+/GSNO-forms were solved. These analyses revealed that CrGSNOR1 has a strict specificity towards GSNO and NADH, and a conserved folding with respect to other plant GSNORs. The catalytic zinc ion, however, showed an unexpected variability of the coordination environment. Furthermore, we evaluated the catalytic response of CrGSNOR1 to thermal denaturation, thiol-modifying agents and oxidative modifications as well as the reactivity and position of accessible cysteines. Despite being a cysteine-rich protein, CrGSNOR1 contains only two solvent-exposed/reactive cysteines. Oxidizing and nitrosylating treatments have null or limited effects on CrGSNOR1 activity and folding, highlighting a certain resistance of the algal enzyme to redox modifications. The molecular mechanisms and structural features underlying the response to thiol-based modifications are discussed.

Insights into the catalytic mechanism of type VI sulfide:quinone oxidoreductases

Sulfide oxidation is catalyzed by ancient membrane-bound sulfide:quinone oxidoreductases (SQR) which are classified into six different types. For catalysis of sulfide oxidation, all SQRs require FAD cofactor and a redox-active centre in the active site, usually formed between conserved essential cysteines. SQRs of different types have variation in the number and position of cysteines, highlighting the potential for diverse catalytic mechanisms. The photosynthetic purple sulfur bacterium, Thiocapsa roseopersicina contains a type VI SQR enzyme (TrSqrF) having unusual catalytic parameters and four cysteines likely involved in the catalysis. Site-directed mutagenesis was applied to identify the role of cysteines in the catalytic process of TrSqrF. Based on biochemical and kinetic characterization of these TrSqrF variants, Cys121 is identified as crucial for enzyme activity. The cofactor is covalently bound via a heterodisulfide bridge between Cys121 and the C8M group of FAD. Mutation of another cysteine present in all SQRs (Cys332) causes remarkably decreased enzyme activity (14.6% of wild type enzyme) proving important, but non-essential role of this residue in enzyme catalysis. The sulfhydril-blocking agent, iodoacetamide can irreversibly inactivate TrSqrF but only if substrates are present and the enzyme is actively catalyzing its reaction. When the enzyme is inhibited by iodoacetamide, the FAD cofactor is released. The inhibition studies support a mechanism that entails opening and reforming of the heterodisulfide bridge during the catalytic cycle of TrSqrF. Our study thus reports the first detailed structure-function analysis of a type VI SQR enzyme which enables the proposal of a distinct mechanism of sulfide oxidation for this class.

Entropy based analysis of vertebrate sperm protamines sequences: evidence of potential dityrosine and cysteine-tyrosine cross-linking in sperm protamines

BackgroundSpermatogenesis is the process by which germ cells develop into spermatozoa in the testis. Sperm protamines are small, arginine-rich nuclear proteins which replace somatic histones during spermatogenesis, allowing a hypercondensed DNA state that leads to a smaller nucleus and facilitating sperm head formation. In eutherian mammals, the protamine-DNA complex is achieved through a combination of intra- and intermolecular cysteine cross-linking and possibly histidine-cysteine zinc ion binding. Most metatherian sperm protamines lack cysteine but perform the same function. This lack of dicysteine cross-linking has made the mechanism behind metatherian protamines folding unclear.ResultsProtamine sequences from UniProt’s databases were pulled down and sorted into homologous groups. Multiple sequence alignments were then generated and a gap weighted relative entropy score calculated for each position. For the eutherian alignments, the cysteine containing positions were the most highly conserved. For the metatherian alignment, the tyrosine containing positions were the most highly conserved and corresponded to the cysteine positions in the eutherian alignment.ConclusionsHigh conservation indicates likely functionally/structurally important residues at these positions in the metatherian protamines and the correspondence with cysteine positions within the eutherian alignment implies a similarity in function. One possible explanation is that the metatherian protamine structure relies upon dityrosine cross-linking between these highly conserved tyrosines. Also, the human protamine P1 sequence has a tyrosine substitution in a position expecting eutherian dicysteine cross-linking. Similarly, some members of the metatherian Planigales genus contain cysteine substitutions in positions expecting plausible metatherian dityrosine cross-linking. Rare cysteine-tyrosine cross-linking could explain both observations.

The highly divergent Jekyll genes, required for sexual reproduction, are lineage specific for the related grass tribes Triticeae and Bromeae.

SummaryPhylogenetically related groups of species contain lineage‐specific genes that exhibit no sequence similarity to any genes outside the lineage. We describe here that the Jekyll gene, required for sexual reproduction, exists in two much diverged allelic variants, Jek1 and Jek3. Despite low similarity, the Jek1 and Jek3 proteins share identical signal peptides, conserved cysteine positions and direct repeats. The Jek1/Jek3 sequences are located at the same chromosomal locus and inherited in a monogenic Mendelian fashion. Jek3 has a similar expression as Jek1 and complements the Jek1 function in Jek1‐deficient plants. Jek1 and Jek3 allelic variants were almost equally distributed in a collection of 485 wild and domesticated barley accessions. All domesticated barleys harboring the Jek1 allele belong to single haplotype J1‐H1 indicating a genetic bottleneck during domestication. Domesticated barleys harboring the Jek3 allele consisted of three haplotypes. Jekyll‐like sequences were found only in species of the closely related tribes Bromeae and Triticeae but not in other Poaceae. Non‐invasive magnetic resonance imaging revealed intrinsic grain structure in Triticeae and Bromeae, associated with the Jekyll function. The emergence of Jekyll suggests its role in the separation of the Bromeae and Triticeae lineages within the Poaceae and identifies the Jekyll genes as lineage‐specific.

Unravelling enhancement of antibody fragment stability – Role of format structure and cysteine modification

Antibody-based diagnostics and therapeutics have huge commercial value. However, applications of antibodies are often limited by instability, particularly for recombinant antibody formats. This paper describes the conversion of a single-chain variable fragment (scFv) antibody to a single-chain antibody fragment (scAb) with notably improved stability characteristics. This scAb retains antigen-binding activity (i) at high temperature (up to 60 °C), (ii) in guanidine hydrochloride (GdnHCl, up to 1 M), and (iii) when stored at 37 °C for 6 months. However, limited improvement was observed when the original scFv was converted to a larger fragment antigen-binding (Fab) format. Certain Cys-to-Ala mutations in the third complementarity determining region of the antibody heavy chain (CDRH3) also led to stability improvements. Our findings indicate that the stability of an antibody derivative depends on its format and on the positions of cysteines in the CDRs.

Influence of Length and Amino Acid Composition on Dimer Formation of Immunoglobulin based Chimera.

The dimeric immunoglobulin (Ig) chimeras used for drug targeting and delivery are preferred biologics over their monomeric forms. Designing these Ig chimeras involves critical selection of a suitable Ig base that ensures dimer formation. In the present study, we systematically analyzed several factors that influence the formation of dimeric chimera. We designed and predicted 608 cytokine-Ig chimeras where we tested the contributions of (1) different domains of Ig constant heavy chain, (2) length of partner proteins, (3) amino acid (AA) composition and (4) position of cysteine in the formation of homodimer. The sequences of various Ig and cytokines were procured from Uniprot database, fused and submitted to COTH (CO-THreader) server for the prediction of dimer formation. Contributions of different domains of Ig constant heavy chain, length of chimeric proteins, AA composition and position of cysteine to the homodimer formation of 608 cytokine-Ig chimeras were tested. Various in silico approaches were adopted for validating the in silico findings. Experimentally we also validated our approach by expressing the chimeric design of shorter cytokine with Ig domain in CHO cells and analyzing the protein by SDS-PAGE. Our results advocate that while the CH1 region and the Hinge region of Ig heavy chain are critical, the length of partner proteins also crucially influences homodimer formation of the Ig-based chimera. We also report that the CH1 domain of Ig is not required for dimer formation of Ig based chimera in the presence of larger partner proteins. For shorter partner proteins fused to CH2-CH3, careful selection of partner sequence is critical, particularly the hydrophobic AA composition, cysteine content & their positions, disulphide bond formation property, and the linker sequences. We validated our in silico observation by various bioinformatics tools and checked the ability of chimeras to bind with the receptors of native protein by docking studies. As a proof of concept, we have expressed the chimeric proteins in CHO cells and found that our design favors the synthesis of dimeric proteins. Our structural prediction study suggests that extra amino acids in the range of 15-20 added to the CH2 domain of Ig is a critical requirement to make homodimer. This information from our study will have implication in designing efficacious homodimeric chimera.

A novel pentameric phospholipase A2 myotoxin (PophPLA2) from the venom of the pit viper Porthidium ophryomegas

The first toxin isolated from the venomous pit viper Porthidium ophryomegas is a basic pentameric phospholipase A2 (PophPLA2). Elucidation of its amino acid sequence showed that it belongs to the group IIA of secreted PLA2s, with the presence of all 14 conserved cysteine positions. The toxin displayed catalytic activity, in agreement with the presence of Asp49 in its sequence of 121 residues. SDS-PAGE analysis revealed that this toxin is pentameric in non-reducing conditions, a structural organization that has not been described for any viperid PLA2. PophPLA2 displayed moderate myotoxic (in vivo) and cytotoxic (in vitro) activities, as well as anticoagulant activity on human plasma (in vitro). PophPLA2 was not lethal, and did not induce signs of toxicity or distress in mice, when administered intravenously at a dose of up to 100 μg (5.9 μg/g). The toxin showed highest sequence identity with other PLA2s from the venoms of ancestral Asian pit viper species.

PvdO is required for the oxidation of dihydropyoverdine as the last step of fluorophore formation in Pseudomonas fluorescens

Pyoverdines are important siderophores that guarantee iron supply to important pathogenic and non-pathogenic pseudomonads in host habitats. A key characteristic of all pyoverdines is the fluorescent dihydroxyquinoline group that contributes two ligands to the iron complexes. Pyoverdines are derived from the non-ribosomally synthesized peptide ferribactin, and their fluorophore is generated by periplasmic oxidation and cyclization reactions of d-tyrosine and l-diaminobutyric acid. The formation of the fluorophore is known to be driven by the periplasmic tyrosinase PvdP. Here we report that the putative periplasmic oxidoreductase PvdO of Pseudomonas fluorescens A506 is required for the final oxidation of dihydropyoverdine to pyoverdine, which completes the fluorophore. The pvdO deletion mutant accumulates dihydropyoverdine, and this phenotype is fully complemented by recombinant PvdO. The autoxidation of dihydropyoverdine at alkaline pH and the presence of high copper concentrations can mask this phenotype. Mutagenesis of conserved residues with potential catalytic function identified Glu-260 as an essential residue whose mutation abolished function without affecting stability or transport. Glu-260 of PvdO is at the exact position of the active-site cysteine in the structurally related formylglycine-generating enzyme. Evolution thus used the same protein fold for two distinct functionalities. As purified PvdO was inactive, additional factors are required for catalysis.

Immunogenetic factors driving formation of ultralong VH CDR3 in Bos taurus antibodies.

The antibody repertoire of Bos taurus is characterized by a subset of variable heavy (VH) chain regions with ultralong third complementarity determining regions (CDR3) which, compared to other species, can provide a potent response to challenging antigens like HIV env. These unusual CDR3 can range to over seventy highly diverse amino acids in length and form unique β-ribbon 'stalk' and disulfide bonded 'knob' structures, far from the typical antigen binding site. The genetic components and processes for forming these unusual cattle antibody VH CDR3 are not well understood. Here we analyze sequences of Bos taurus antibody VH domains and find that the subset with ultralong CDR3 exclusively uses a single variable gene, IGHV1-7 (VHBUL) rearranged to the longest diversity gene, IGHD8-2. An eight nucleotide duplication at the 3' end of IGHV1-7 encodes a longer V-region producing an extended F β-strand that contributes to the stalk in a rearranged CDR3. A low amino acid variability was observed in CDR1 and CDR2, suggesting that antigen binding for this subset most likely only depends on the CDR3. Importantly a novel, potentially AID mediated, deletional diversification mechanism of the B. taurus VH ultralong CDR3 knob was discovered, in which interior codons of the IGHD8-2 region are removed while maintaining integral structural components of the knob and descending strand of the stalk in place. These deletions serve to further diversify cysteine positions, and thus disulfide bonded loops. Hence, both germline and somatic genetic factors and processes appear to be involved in diversification of this structurally unusual cattle VH ultralong CDR3 repertoire.

Thiol‐Oxidation Impairs Alpha2‐Adrenergic Receptor Mediated Inhibition of N‐type Ca2+ channels and Results in Receptor Mislocalization

IntroductionOxidative stress and sympathetic hyperactivity contribute to hypertension. In sympathetic neurons, activation of alpha2‐adrenergic receptors (α2AR) decreases nerve terminal release of norepinephrine (NE) by activating Gαi/o proteins to inhibit N‐type voltage‐gated Ca2+channels. We have shown in DOCA‐salt hypertensive rats that α2AR‐mediated inhibition of Ca2+channels is impaired in sympathetic neurons, resulting in increased NE release. Furthermore, blood pressure and α2AR function are preserved following treatment with clodronate (macrophage depletion agent) and apocynin (NADPH oxidase inhibitor). Oxidative stress is associated with adrenergic dysfunction and Gαi/o are targets of oxidative stress. Inhibition of α2AR‐Gαi/o coupling following pertussis toxin (PTX) treatment occurs due to ADP‐ribosylation on the C‐terminal cysteine (position 351) of Gαi/o. It is not known if thiol‐oxidation of this residue results in similar uncoupling. Therefore, we tested the hypothesis that oxidizing agents impair α2‐AR function by thiol‐oxidation of Cys351 on Gαi/o.MethodsThese studies used cultured sympathetic neurons, G1A1 cells (HEK cells stably expressing N‐type Ca2+ channels), and HEK 293T cells. Cell lines were co‐transfected with α2AR and either WT‐Gαo or PTX‐insensitive Gαo (PTXi‐Gαo, Cys351Gly). Oxidative stress was induced with H2O2, t‐BOOH, or diamide (thiol‐specific oxidant). Whole cell patch clamp was used to detect inhibition of Ca2+ current using NE to activate α2AR and GppNHp to inhibit Ca2+ current independent of α2AR activation. Immunohistochemistry was used to localize α2AR and Gαo following oxidant treatment before and after activation of α2AR with NE.ResultsIn sympathetic neurons, α2AR‐mediated inhibition of Ca2+ current was reduced following H2O2, t‐BOOH or diamide treatment vs. untreated neurons. GppNHp inhibited Ca2+ current equally in all neurons independent of oxidative stress. Oxidative stress impaired α2AR‐mediated inhibition of Ca2+ current similarly in G1A1 cells co‐transfected with WT‐Gαo or PTXi‐Gαo. Oxidative stress had no effect on cell capacitance or Ca2+ channel activity in neurons and G1A1 cells. In HEK 293T cells, oxidative stress did not affect co‐localization of α2AR and Gαo, however, oxidative stress resulted in greater cytoplasmic localization of α2AR.ConclusionsThiol‐oxidation results in impaired α2AR‐mediated inhibition of Ca2+ current. Thiol‐oxidation does not affect Gαo coupling to α2AR. Dysfunction may be attributed to thiol‐oxidation of cysteine residues on α2AR, which do not affect Gαo coupling, but may alter structural motifs that could affect receptor localization to the cell membrane.Support or Funding InformationSupported by NIH 2P01HL070687 and American Heart Association (15PRE25090218)