Edman Degradation Research Articles

Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization.

Caspase-2 is the most specific protease of all caspases and therefore highly suitable as tag removal enzyme creating an authentic N-terminus of overexpressed tagged proteins of interest. The wild type human caspase-2 is a dimer of heterodimers generated by autocatalytic processing which is required for its enzymatic activity. We designed a circularly permuted caspase-2 (cpCasp2) to overcome the drawback of complex recombinant expression, purification and activation, cpCasp2 was constitutively active and expressed as a single chain protein. A 22 amino acid solubility tag and an optimized fermentation strategy realized with a model-based control algorithm further improved expression in Escherichia coli and 5.3 g/L of cpCasp2 in soluble form were obtained. The generated protease cleaved peptide and protein substrates, regardless of N-terminal amino acid with high activity and specificity. Edman degradation confirmed the correct N-terminal amino acid after tag removal, using Ubiquitin-conjugating enzyme E2 L3 as model substrate. Moreover, the generated enzyme is highly stable at −20 °C for one year and can undergo 25 freeze/thaw cycles without loss of enzyme activity. The generated cpCasp2 possesses all biophysical and biochemical properties required for efficient and economic tag removal and is ready for a platform fusion protein process.

Isolation, heterologous expression, and purification of a novel antifungal protein from Bacillus subtilis strain Z-14

BackgroundWheat sheath blight, a soil borne fungal disease caused by Rhizoctonia cerealis, is considered as one of the most serious threats to wheat worldwide. Bacillus subtilis Z-14 was isolated from soil sampled from a wheat rhizosphere and was confirmed to have strong antifungal activity against R. cerealis.ResultsAn antifungal protein, termed F2, was isolated from the culture supernatant of Z-14 strain using precipitation with ammonium sulfate, anion exchange chromatography, and reverse phase chromatography. Purified F2 had a molecular mass of approximately 8 kDa, as assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Edman degradation was used to determine the amino acid sequence of the N-terminus, which was NH2ASGGTVGIYGANMRS. This sequence is identical to a hypothetical protein RBAM_004680 (YP_001420098.1) synthesized by B. amyloliquefaciens FZB42. The recombinant F2 protein (rF2) was heterologously expressed in the yeast host Pichia pastoris, purified using a Niaffinity column, and demonstrated significant antifungal activity against R. cerealis. The purified rF2 demonstrated broad spectrum antifungal activity against different varieties of fungi such as Fusarium oxysporum, Verticillium dahliae, Bipolaris papendorfii, and Fusarium proliferatum. rF2 was thermostable, retaining 91.5% of its activity when incubated for 30 min at 100 °C. Meanwhile, rF2 maintained its activity under treatment by proteinase K and trypsin and over a wide pH range from 5 to 10.ConclusionsA novel antifungal protein, F2, was purified from biocontrol Bacillus subtilis Z-14 strain fermentation supernatant and heterologously expressed in Pichia pastoris to verify its antifungal activity against R. cerealis and the validity of the gene encoding F2. Considering its significant antifungal activity and stable characteristics, protein F2 presents an alternative compound to resist fungal infections caused by R. cerealis.

Mannose-binding C-type lectins as defense molecules on the body surface of the sea urchin Pseudocentrotus depressus

We found that the extract of the body wall of the sea urchin, Pseudocentrotus depressus, agglutinate Escherichia coli and is inhibited by mannose. A mannose-binding protein of 22 kDa was purified via affinity chromatography using mannose-agarose. Amino acid sequences obtained by Edman degradation and liquid chromatography quadrupole time-of-flight mass spectrometry followed by de novo sequencing suggested that the protein is a C-type lectin. Products of PCR with a degenerate primer pair and of RACE PCR for the cDNA of the 22 kDa protein were sequenced and produced two full-length cDNA sequences encoding C-type lectins. These two lectins, named P. depressus mannose-binding C-type lectin (PdMBCL) 1 and 2 are composed of 187 and 189 amino acid residues, including signal peptides, respectively, and share 86% identity in their mature form. PdMBCLs agglutinated Lactococcus garvieae, a Gram-positive fish pathogen. Reverse transcription PCR showed that both the genes for the PdMBCLs were expressed in the body wall and in other tissues. Furthermore, the lectins were detected from a rinse of the body surface. Taken together, the present study showed that PdMBCLs function as anti-microbial agents on the body surface of P. depressus.

New Sea Anemone Toxin RTX-VI Selectively Modulates Voltage-Gated Sodium Channels.

A new neurotoxin RTX-VI that modulates the voltage-gated sodium channels (NaV) was isolated from the ethanolic extract of the sea anemone Heteractis crispa. Its amino acid sequence was determined using the combination of Edman degradation and tandem mass spectrometry. RTX-VI turned out to be an unusual natural analogue of the previously described sea anemone toxin RTX-III. The RTX-VI molecule consists of two disulfide-linked peptide chains and is devoid of Arg13, which is important for the selectivity and affinity of such peptides for the NaV channels. Electrophysiological screening of RTV-VI on NaV channel subtypes showed its selective interaction with the central nervous system (NaV1.2, NaV1.6) and insect (BgNaV1, VdNaV1) sodium channels.

Ovarian Cancer: Tumor-Specific Urinary Micro-Peptides Profiling as Potential Biomarkers for Early Diagnosis.

Ovarian cancer is the second major lethal gynecologic malignancy in developing countries. This study aimed to characterize urinary micro-peptides as potential diagnostic biomarkers for ovarian cancer. In a prospective, longitudinal and case-controlled study and following informed consent, urine and plasma samples were collected from 112 women with histologically-proven ovarian cancer and 200 apparently healthy age-matched volunteers. Urinary micro-peptides were detected and sequenced using SDS-PAGE and Edman degradation technique. Serum CA125 was detected in less than a quarter (23.2%, 26/112) of patients. One or more urinary micro-peptides were detected in about two thirds of the patients (62.5%, 70/112). A total of 40 patients had three bands (57.1%, 40/70), while two bands (15 and 35 kDa) were detected in 28.6% (20/70) of the patients. Isolated 45 kDa band was seen in 14.3% (10/70). No urinary micro-peptide was detected in the volunteers. The 15 and 35 kDa bands disappeared after 6 months of regular chemotherapy, while the 45 kDa band persisted in 2.9% (2/70) of the patients after treatment. The micro-peptides were identified as: Catalase (45 kDa), α-1 Acid Glycoprotein (35 kDa) and Peroxiredoxin-2 (15 kDa). Urinary catalase, α-1 Acid Glycoprotein and Peroxiredoxin-2 can be useful biomarkers for early detection and treatment response of ovarian cancer.

Production of functional human nerve growth factor from the submandibular glands of mice using a CRISPR/Cas9 genome editing system.

Nerve growth factor (NGF) is an essential trophic factor for the growth and survival of neurons in the central and peripheral nervous systems. For many years, mouse NGF (mNGF) has been used to treat various neuronal and non-neuronal disorders. However, the biological activity of human NGF (hNGF) is significantly higher than that of mNGF in human cells. Using the CRISPR/Cas9 system, we constructed the transgenic mice expressing hNGF specifically in their submandibular glands. As demonstrated by fluorescence immunohistochemical staining, these mice produced hNGF successfully, with 0.8mg produced per gram of submandibular glands. hNGF with 99% purity was successfully extracted by two-step ion-exchange chromatography and one-step size-exclusion chromatography from the submandibular glands of these transgenic mice. Further, the purified hNGF was verified by LC-MS/MS. We analyzed the NH2-terminus of hNGF using both Edman degradation and LC-MS/MS-based methods. Both results showed that the obtained hNGF lost the NH2-terminal octapeptide (SSSHPIFH). Moreover, the produced hNGF demonstrated a strong promotion in the proliferation of TF1 cells.

Adaptive nanopores: A bioinspired label-free approach for protein sequencing and identification

Single molecule protein sequencing would tremendously impact in proteomics and human biology and it would promote the development of novel diagnostic and therapeutic approaches. However, its technological realization can only be envisioned, and huge challenges need to be overcome. Major difficulties are inherent to the structure of proteins, which are composed by several different amino-acids. Despite long standing efforts, only few complex techniques, such as Edman degradation, liquid chromatography and mass spectroscopy, make protein sequencing possible. Unfortunately, these techniques present significant limitations in terms of amount of sample required and dynamic range of measurement. It is known that proteins can distinguish closely similar molecules. Moreover, several proteins can work as biological nanopores in order to perform single molecule detection and sequencing. Unfortunately, while DNA sequencing by means of nanopores is demonstrated, very few examples of nanopores able to perform reliable protein-sequencing have been reported so far. Here, we investigate, by means of molecular dynamics simulations, how a re-engineered protein, acting as biological nanopore, can be used to recognize the sequence of a translocating peptide by sensing the “shape” of individual amino-acids. In our simulations we demonstrate that it is possible to discriminate with high fidelity, 9 different amino-acids in a short peptide translocating through the engineered construct. The method, here shown for fluorescence-based sequencing, does not require any labelling of the peptidic analyte. These results can pave the way for a new and highly sensitive method of sequencing.

Antibiofilm Activity of Acidic Phospholipase Isoform Isolated from Bothrops erythromelas Snake Venom.

Introduction: Bacterial resistance is a worldwide public health problem, requiring new therapeutic options. An alternative approach to this problem is the use of animal toxins isolated from snake venom, such as phospholipases A2 (PLA2), which have important antimicrobial activities. Bothrops erythromelas is one of the snake species in the northeast of Brazil that attracts great medical-scientific interest. Here, we aimed to purify and characterize a PLA2 from B. erythromelas, searching for heterologous activities against bacterial biofilms. Methods: Venom extraction and quantification were followed by reverse-phase high-performance liquid chromatography (RP-HPLC) in C18 column, matrix-assisted ionization time-of-flight (MALDI-ToF) mass spectrometry, and sequencing by Edman degradation. All experiments were monitored by specific activity using a 4-nitro-3-(octanoyloxy) benzoic acid (4N3OBA) substrate. In addition, hemolytic tests and antibacterial tests including action against Escherichia coli, Staphylococcus aureus, and Acinetobacter baumannii were carried out. Moreover, tests of antibiofilm action against A. baumannii were also performed. Results: PLA2, after one purification step, presented 31 N-terminal amino acid residues and a molecular weight of 13.6564 Da, with enzymatic activity confirmed in 0.06 µM concentration. Antibacterial activity against S. aureus (IC50 = 30.2 µM) and antibiofilm activity against A. baumannii (IC50 = 1.1 µM) were observed. Conclusions: This is the first time that PLA2 purified from B. erythromelas venom has appeared as an alternative candidate in studies of new antibacterial medicines.

Kunkecin A, a New Nisin Variant Bacteriocin Produced by the Fructophilic Lactic Acid Bacterium, Apilactobacillus kunkeei FF30-6 Isolated From Honey Bees.

Apilactobacillus kunkeei FF30-6 isolated from healthy honey bees synthesizes the bacteriocin, which exhibits antimicrobial activity against Melissococcus plutonius. The bacteriocin, kunkecin A, was purified through three-step chromatography, and mass spectrometry revealed that its relative molecular mass was 4218.3. Edman degradation of purified kunkecin A showed only the N-terminal residue, isoleucine. Hence, alkaline alkylation made the subsequent amino acid residues accessible to Edman degradation, and 30 cycles were sequenced with 11 unidentified residues. Whole genome sequencing of A. kunkeei FF30-6, followed by Sanger sequencing, revealed that the genes encoding the proteins involved in lantibiotic biosynthesis were within the plasmid, pKUNFF30-6. Most of the identified proteins exhibited significant sequence similarities to the biosynthetic proteins of nisin A and its variants, such as subtilin. However, the kunkecin A gene cluster lacked the genes corresponding to nisI, nisR, and nisK of the nisin A biosynthetic gene cluster. A comparison of the gene products of kukA and nisA (kunkecin A and nisin A structural genes, respectively) suggested that they had similar post-translational modifications. Furthermore, the structure of kunkecin A was proposed based on a comparison of the observed and calculated relative molecular masses of kunkecin A. The structural analysis revealed that kunkecin A and nisin A had a similar mono-sulfide linkage pattern. Purified kunkecin A exhibited a narrow antibacterial spectrum, but high antibacterial activity against M. plutonius. Kunkecin A is the first bacteriocin to be characterized in fructophilic lactic acid bacteria and is the first nisin-type lantibiotic found in the family Lactobacillaceae.

Expression and Characterization of Rice-produced Recombinant Porcine Lactoferrin and its Antioxidant Activities

Background: Lactoferrin (LF) exhibits multiple beneficial biological activities and thus has been used as a health food and additive. To broaden its application in the food industry, the porcine LF (pLF) gene has been engineered into rice to produce recombinant LF (rpLF) for use as a food additive. The iron-binding and antimicrobial activities of rpLF and its positive effects on early weaned piglets have been previously evaluated, yet several features, such as the signal peptide removal, glycosylation sites and antioxidant activity of rpLF, have not been fully characterized. Objective: In this work, the rice-produced rpLF was purified and its biochemical structure and antioxidant activities characterized. Methods: HPLC, Western blot, PAS/VVL/PNA staining, Edman degradation assay, MALDI-TOF, LC-MS/MS and antioxidant activity assays were performed. Results: The results showed that this purified rpLF is a mature form of LF; its signal peptide was correctly removed, and two N-glycosylation sites located at N365 and N472 were identified. The molecular mass heterogeneity of rpLF could be eliminated by treatment with PNGase glycosidase, suggesting that different degrees of N-glycosylation occur in rpLF. A series of assays including the iron chelating activity, reducing power assay, lipid peroxidase activity and radical-scavenging activity showed that the antioxidant activity of rice-produced rpLF was equivalent to that of bovine LF. Conclusion: Rice-produced rpLF was correctly processed post-translationally and displayed antioxidant activity equivalent to that of bovine LF; thus, rice-produced rpLF can be recognized as a plant-based antioxidant to be used as a functional additive in animal feed and for the food industry.

Preparation and purification of an immunoregulatory peptide from Stolephorus chinensis of the East Sea of China

Immunomodulatory peptides derived from marine organisms are potential sources of new immunomodulating drugs. This study aimed to investigate the optimal enzymatic hydrolysis conditions of immunoregulatory peptides from Stolephorus chinensis. The relative proliferation rate (RPR) of RAW264.7 macrophages was set as the screening index. The immunoregulatory peptides from S. chinensis was prepared via process optimization, ultrafiltration, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography (RP-HLPC). The amino acid sequence of the immunoregulatory peptide from S. chinensis (IPSC) was identified to be Tyr-Val-Met-Arg-Phe (YVMRF; MW 715.4 Da) using Edman degradation and mass spectrometry. In addition, the macrophages became larger with more pseudopods after IPSC treatment, indictating that IPSC stimulated RAW 264.7 differentiation. IPSC also increased productions of nitric oxide (NO), interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α). Our results provide a theoretical basis for preparing immunomodulatory functional food from S. chinensis in future.

GiTx1(β/κ-theraphotoxin-Gi1a), a novel toxin from the venom of Brazilian tarantula Grammostola iheringi (Mygalomorphae, Theraphosidae): Isolation, structural assessments and activity on voltage-gated ion channels

Spider venoms, despite their toxicity, represent rich sources of pharmacologically active compounds with biotechnological potential. However, in view of the large diversity of the spider species, the full potential of their venom molecules is still far from being known. In this work, we report the purification and structural and functional characterization of GiTx1 (β/κ-TRTX-Gi1a), the first toxin purified from the venom of the Brazilian tarantula spider Grammostola iheringi. GiTx1 was purified by chromatography, completely sequenced through automated Edman degradation and tandem mass spectrometry and its structure was predicted by molecular modeling. GiTx1 has a MW of 3.585 Da, with the following amino acid sequence: SCQKWMWTCDQKRPCCEDMVCKLWCKIIK. Pharmacological activity of GiTx1 was characterized by electrophysiology using whole-cell patch clamp on dorsal root ganglia neurons (DRG) and two-electrode voltage-clamp on voltage-gated sodium and potassium channels subtypes expressed in Xenopus laevis oocytes. GiTx1, at 2 μM, caused a partial block of inward (∼40%) and outward (∼20%) currents in DRG cells, blocked rNav1.2, rNav1.4 and mNav1.6 and had a significant effect on VdNav, an arachnid sodium channel isoform. IC50 values of 156.39 ± 14.90 nM for Nav1.6 and 124.05 ± 12.99 nM for VdNav, were obtained. In addition, this toxin was active on rKv4.3 and hERG potassium channels, but not Shaker IR or rKv2.1 potassium channels. In summary, GiTx1 is a promiscuous toxin with multiple effects on different types of ion channels.

16α-Hydroxyestrone: Mass Spectrometry-Based Methodologies for the Identification of Covalent Adducts Formed with Blood Proteins.

Elevated levels of the estrone metabolite, 16α-hydroxyestrone (16αOHE1), have been linked with multiple diseases. As an electrophilic reactive metabolite, covalent binding to proteins is thought to constitute one of the possible mechanisms in the onset of deleterious health outcomes associated with 16αOHE1. Whereas mass spectrometry (MS)-based methodologies are currently considered the best suited to monitor the formation of protein covalent adducts, the application of these approaches for the identification of covalent adducts of 16αOHE1 is yet to be provided. In the present study, with the ultimate goal of determining the most adequate methodology for searching for 16αOHE1-derived covalent adducts, we explored multiple liquid chromatography-electrospray ionization tandem high-resolution mass spectrometry (LC-ESI-HRMS/MS)-based approaches to investigate the nature and specific locations of the covalent adducts produced in human hemoglobin (Hb) and human serum albumin (HSA) modified in vitro with 16αOHE1. The application of a "bottom up" proteomics approach, involving the nanoLC-ESI-HRMS/MS analysis of tryptic peptides, allowed the identification of multiple sites of 16αOHE1 adduction in Hb and HSA. As expected, the majority of the adducted peptides occurred in lysine residues following stabilization of the Schiff base formed with 16αOHE1 by reduction or via Heyns rearrangement, yielding the stable α-hydroxyamine and ketoamine adducts, respectively. Noteworthy is the fact that a serine residue was also identified to be covalently modified with 16αOHE1, which to our knowledge constitutes a first-hand report of a keto electrophile as target of hydroxyl-based nucleophilic amino acids. The N-alkyl Edman degradation resulted to be unsuitable for the identification of 16αOHE1adducts formed with the N-terminal valine of Hb, most probably due to stereochemical restraints of the tested derivatizing agents (fluorescein isothiocyanate and phenyl isothiocyanate) on assessing these bulky covalent adducts. Nonetheless, the digestion of adducted proteins to amino acids resulted in the detection of 16αOHE1-derived keto and α-hydroxyamine Lys adducts. The simplicity of this methodology might be beneficial for clinical studies, with the possibility of offering quantitative information with the preparation of synthetic standards of these adducts. The results obtained are crucial not only for the identification and quantification of biomarkers of exposure to 16αOHE1 but also for clarifying the role of protein binding in the onset of diseases associated with elevated levels of this reactive metabolite.

Determination of Amino Acid Composition of Ganirelix Acetate in an Injectable Formulation by Pre-column Derivatization with 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate.

Ganirelix is a synthetic decapeptide linked with nine different amino acids. To understand the peptide amino acid sequence or primary structure, the first step is to determine the amino acid composition of the peptide which can be a determining factor for the peptide immunogenicity. Edman degradation is not a suitable analytical technique to identify amino acid sequence present in Ganirelix due to the absence of uncharged N-terminal amino group. To address this challenge, a pre-column derivatization method was developed with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent. In the present work, the Ganirelix active pharmaceutical ingredient present in the injectable formulation was isolated by fraction collection and further purified by flash chromatography. The amino acid composition of Ganirelix is assayed by carrying out acid hydrolysis with 6molL-1 hydrochloric acid solution containing 1% phenol at 100°C for 24h and derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent solution, followed by determination of individual amino acids by reverse-phase chromatography using a C18 column. High resolution was achieved for the nine amino acid mixture. The amino acid composition results of temperature-stressed Ganirelix generic product and reference listed drug are in good agreement with the theoretical molar ratio of label information.

A turripeptide from Polystira nobilis venom inhibits human α3β2 and α7 nicotinic acetylcholine receptors

Almost all marine snails within superfamily Conoidea produce venoms containing numerous neuroactive peptides. Most toxins characterized from members of this superfamily are produced by species belonging to family Conidae. These toxins (conotoxins) affect diverse membrane proteins, such as voltage- and ligand-gated ion channels, including nicotinic acetylcholine receptors (nAChRs). Family Turridae has been considerably less studied than their Conidae counterpart and, therefore, turrid toxins (turritoxins) have just been barely described. Consequently, in this work the most prominent chromatographic (RP-HPLC) fractions from the East Pacific species Polystira nobilis venom duct extract were isolated. The biological activity of six selected fractions was assayed on human (h) α7 AChRs expressed in Xenopus laevis oocytes. One of these fractions, F21, inhibited the acetylcholine-elicited response by 62 ± 12%. Therefore, this fraction was further purified and the F21-2 peptide was obtained. This peptide (at 5.6 μM) strongly and irreversibly inhibited the acetylcholine-induced response on hα7 and hα3β2 nAChRs, by 55 ± 4 and 91 ± 1%, respectively. Electrospray mass spectrometry indicates that the average molecular mass of this toxin is 12 358.80 Da. The affinity for hα3β2 nAChRs is high (IC50 of 566.2 nM). A partial sequence without cysteines was obtained by automated Edman degradation: WFRSFKSYYGHHGSVYRPNEPNFRSFAS…; blastp search revealed that this sequence has low similarity to some non-Cys-containing turripeptides. This is the first report of a turritoxin from a species of the American Pacific and the second description of a turripeptide inhibiting nAChRs.

Levels of the hemoglobin adduct N-(2,3-Dihydroxypropyl)-valine in cord and maternal blood: Prenatal transfer of glycidol in the ENVIRONAGE birth cohort

BackgroundGlycidol, a probable human carcinogen, is a reactive chemical released in the gastrointestinal tract from glycidyl fatty acid esters, which are heat-induced dietary contaminants. ObjectivesTo investigate the prenatal transfer of glycidol, a specific hemoglobin adduct was measured as a biomarker for internal glycidol exposure in paired cord and maternal blood samples. MethodsIn 100 mother-newborn pairs from the Belgian ENVIRONAGE (ENVIRonmental influence ON AGEing in early life) birth cohort, we studied the correlation between levels of the glycidol-derived hemoglobin adduct N-(2,3-dihydroxypropyl)-valine (2,3-diHOPr-Val) in paired cord and maternal blood samples. The adduct levels were determined after cleavage with a modified Edman degradation by using ultra-high performance liquid chromatography-tandem mass spectrometry and an isotope-labeled reference standard. Results2,3-DiHOPr-Val was detectable in all 100 maternal blood samples and in 96 cord blood samples (LOD =0.5 pmol 2,3-diHOPr-Val/g hemoglobin), with medians of 5.4 (range: 2.3–29.2) and 1.6 (range: LOD – 8.9) pmol/g hemoglobin), respectively. In blood samples of mothers who smoked during pregnancy and in the cord blood samples of their newborns (n = 6), the median 2,3-diHOPr-Val levels were 16.7 (range: 6.4–29.2) and 6.2 (range: LOD – 8.6) pmol/g hemoglobin, respectively. The median ratio of 2,3-diHOPr-Val levels of cord to maternal blood was 0.35 (range: 0.19–1.14) (n = 49). The Spearman correlation coefficient between 2,3-diHOPr-Val levels in cord and maternal blood samples was 0.63 (p < 0.001) among all mother-newborn pairs and 0.59 (p < 0.001) among mother-newborn pairs of non-smoking mothers. DiscussionMaternal data confirm widespread exposure to glycidol, also in non-smokers. Neonatal levels indicate prenatal exposure to glycidol, due to an obviously relatively unhindered passive transfer through the placental barrier. Possible health effects of fetal (and postnatal) glycidol exposure in children may be addressed in epidemiological studies.

Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)-A CleavEx Based Analysis.

Kallikrein-related peptidases (KLKs) and matrix metalloproteinases (MMPs) are secretory proteinases known to proteolytically process components of the extracellular matrix, modulating the pericellular environment in physiology and in pathologies. The interconnection between these families remains elusive. To assess the cross-activation of these families, we developed a peptide, fusion protein-based exposition system (Cleavage of exposed amino acid sequences, CleavEx) aiming at investigating the potential of KLK14 to recognize and hydrolyze proMMP sequences. Initial assessment identified ten MMP activation domain sequences which were validated by Edman degradation. The analysis revealed that membrane-type MMPs (MT-MMPs) are targeted by KLK14 for activation. Correspondingly, proMMP14-17 were investigated in vitro and found to be effectively processed by KLK14. Again, the expected neo-N-termini of the activated MT-MMPs was confirmed by Edman degradation. The effectiveness of proMMP activation was analyzed by gelatin zymography, confirming the release of fully active, mature MT-MMPs upon KLK14 treatment. Lastly, MMP14 was shown to be processed on the cell surface by KLK14 using murine fibroblasts overexpressing human MMP14. Herein, we propose KLK14-mediated selective activation of cell-membrane located MT-MMPs as an additional layer of their regulation. As both, KLKs and MT-MMPs, are implicated in cancer, their cross-activation may constitute an important factor in tumor progression and metastasis.

Purification and cDNA Cloning of Antimicrobial Peptides from the Skin Secretion of the Chinese Frog Rana chensinensis

The skin secretions of amphibians are a rich source of bioactive peptides. We isolated chensirin-1 and chensirin-2 from the skin secretion of the Chinese frog Rana chensinensis. Sephadex-G-50 and RP-HPLC were employed to purify these peptides. The amino acid sequences of these peptides were VLPLVGNLLNDLLGE and IIPLPLGYFAKKT, respectively, as determined by Edman degradation. The molecular weights were 1578.7 and 1460.8 Da, respectively, as analyzed by HPLC-ESI-MS. The chensirin cDNA was cloned by 5′ and 3′ amplification of cDNA ends, synthesized and purified. The antibacterial activities of the chensirins were tested using minimum inhibitory concentration, the results indicated that chensirins inhibit the growth of gram-negative and gram-positive bacteria. Among them, chensirin-1 is a novel peptide with a higher antibacterial activity compared to other similar antimicrobial peptides. These low molecular weight peptides with good antimicrobial efficacy are considered potential sources for developing new antimicrobial agents to improve traditional drug resistance.

Sensor Array Based Determination of Edman Degradated Amino Acids Using Poly(p‐phenyleneethynylene)s

A cross‐reactive optical sensor array based on poly(p‐phenyleneethynylene)s (PPEs) determines Edman degraded amino acids. We report a sensor array composed of three anionic PPEs P1–P3, and their electrostatic complexes with metal ions (Fe2+, Cu2+, Co2+). We recorded distinct fluorescence intensity response patterns as “fingerprints” of this chemical tongue toward standard phenylthiohydantoin (PTH) amino acids—degradation products of the Edman process. These “fingerprints” were converted into canonical scores by linear discrimination analysis (LDA), which differentiates all of the PTH‐amino acids. This array discriminates PTH‐amino acid residues degraded from an oligopeptide through Edman sequencing. This approach is complementary to chromatography approaches which rely on mass spectrometry; our array offers the advantage of simplicity.

BmK NSP, a new sodium channel activator from Buthus martensii Karsch, promotes neurite outgrowth in primary cultured spinal cord neurons

Scorpion venom is a rich source of bioactive compounds that affect neuronal excitability by modulating the activities of various channels/receptors. In the current study, guided by a Ca2+ mobilization assay, we purified a new neuroactive peptide designated as BmK NSP (Buthus martensii Karsch neurite-stimulating peptide, MW: 7064.30 Da). The primary structure of BmK NSP was determined by Edman degradation. BmK NSP concentration-dependently elevated intracellular Ca2+ concentration ([Ca2+]i) with an EC50 value of 4.18 μM in primary cultured spinal cord neurons (SCNs). Depletion of extracellular Ca2+ abolished BmK NSP-triggered Ca2+ response. Moreover, we demonstrated that BmK NSP-induced Ca2+ response was partially suppressed by the inhibitors of L-type Ca2+ channels, Na+-Ca2+ exchangers and NMDA receptors and was abolished by voltage-gated sodium channel (VGSC) blocker, tetrodotoxin. Whole-cell patch clamp recording demonstrated that BmK NSP delayed VGSC inactivation (EC50 = 1.10 μM) in SCNs. BmK NSP enhanced neurite outgrowth in a non-monotonic manner that peaked at ~30 nM in SCNs. BmK NSP-promoted neurite outgrowth was suppressed by the inhibitors of L-type Ca2+ channels, NMDA receptors, and VGSCs. Considered together, these data demonstrate that BmK NSP is a new α-scorpion toxin that enhances neurite outgrowth through main routes of Ca2+ influx. Modulation of VGSC activity by α-scorpion toxin might represent a novel strategy to regulate the neurogenesis in SCNs.