Biomedical subjects

Annette G Beck-Sickinger

Publications and source records attributed to Annette G Beck-Sickinger.

At least 37 records · Page 2Linked to original sources

PubMed2005

Developing novel hCT derived cell-penetrating peptides with improved metabolic stability.

Many promising therapeutics are currently awaiting their clinical application. Due to their low capability of cell membrane crossing, these compounds do not reach their site of action. One way to overcome this problem might be the fusion of these agents to cell-penetrating peptides (CPP), which are able to shuttle various cargoes across cellular membranes. One disadvantage in using CPP in drug delivery is their low metabolic stability. The aim of our work was to increase the proteolytic resistance of the CPP hCT(9-32), a truncated C-terminal fragment of human calcitonin. Thus, we synthesised six modified N-terminally carboxyfluorescein labelled hCT(9-32) derivatives by replacing positions 12 and/or 16 of hCT(9-32) with either N-methylphenylalanine or d-phenylalanine, respectively. By using confocal laser scanning microscopy we showed that the modifications did neither affect the peptide internalisation efficiency in HeLa nor HEK 293T cells. The metabolic stability of the peptides was investigated in human blood plasma and HEK 293T cell culture supernatant. To analyse the degradation patterns, we used RP-HPLC and MALDI-TOF mass spectrometry. However, we found for all of the new derivatives high metabolic stabilities. In blood plasma, the half-lives for five of the six peptides increased compared to unmodified hCT(9-32). The degradation patterns showed a distinct stabilisation in the N-terminal part of the modified peptides, in the C-terminal part, we found some cleavage to a minor extent. Furthermore, we studied the conformation of the peptides by CD spectroscopy and demonstrated that they possess no cell toxicity. Since our metabolically more stable compounds are still able to pass the cell membrane they provide powerful tools as drug delivery vectors.

Amino Acid Sequence↗

PubMed2005

Membrane surface-associated helices promote lipid interactions and cellular uptake of human calcitonin-derived cell penetrating peptides.

hCT(9-32) is a human calcitonin (hCT)-derived cell-penetrating peptide that has been shown to translocate the plasma membrane of mammalian cells. It has been suggested as a cellular carrier for drugs, green fluorescent protein, and plasmid DNA. Because of its temperature-dependent cellular translocation resulting in punctuated cytoplasmatic distribution, its uptake is likely to follow an endocytic pathway. To gain insight into the molecular orientation of hCT(9-32) when interacting with lipid models, and to learn more about its mode of action, various biophysical techniques from liposome partitioning to high-resolution NMR spectroscopy were utilized. Moreover, to establish the role of individual residues for the topology of its association with the lipid membrane, two mutants of hCT(9-32), i.e., W30-hCT(9-32) and A23-hCT(9-32), were also investigated. Although unstructured in aqueous solution, hCT(9-32) adopted two short helical stretches when bound to dodecylphosphocholine micelles, extending from Thr10 to Asn17 and from Gln24 to Val29. A23-hCT(9-32), in which the helix-breaking Pro23 was replaced by Ala, displayed a continuous alpha-helix extending from residue 12 to 26. Probing with the spin label 5-doxylstearate revealed that association with dodecylphosphocholine micelles was such that the helix engaged in parallel orientation to the micelle surface. Moreover, the Gly to Trp exchange in W30-hCT(9-32) resulted in a more stable anchoring of the C-terminal segment close to the interface, as reflected by a twofold increase in the partition coefficient in liposomes. Interestingly, tighter binding to model membranes was associated with an increase in the in vitro uptake in human cervix epithelial adenocarcinoma cell line cells. Liposome leakage studies excluded pore formation, and the punctuated fluorescence pattern of internalized peptide indicated vesicular localization and, in conclusion, strongly suggested an endocytic pathway of translocation.

Calcitonin↗

PubMed2005

Membrane interaction of neuropeptide Y detected by EPR and NMR spectroscopy.

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammals. It belongs to the best-conserved peptides in nature, i.e., the amino acid sequences of even evolutionary widely separated species are very similar to each other. Using porcine NPY, which differs from human NPY only at position 17 (a leucine residue exchanged for a methionine), labeled with a TOAC spin probe at the 2nd, 32nd, or 34th positions of the peptide backbone, the membrane binding and penetration of NPY was determined using EPR and NMR spectroscopy. The vesicular membranes were composed of phosphatidylcholine and phosphatidylserine at varying mixing ratios. From the analysis of the EPR line shapes, the spectral contributions of free, dimerized, and membrane bound NPY could be separated. This analysis was further supported by quenching experiments, which selected the contributions of the bound NPY fraction. The results of this study give rise to a model where the alpha-helical part of NPY (amino acids 13-36) penetrates the membrane interface. The unstructured N-terminal part (amino acids 1-12) extends into the aqueous phase with occasional contacts with the lipid headgroup region. Besides the mixing ratio of zwitterionic and negatively charged phospholipid species, the electrostatic peptide membrane interactions are influenced by the pH value, which determines the net charge of the peptide resulting in a modified membrane binding affinity. The results of these variations indicate that NPY binding to phospholipid membranes depends strongly on the electrostatic interactions. An estimation of the transfer energy of the peptide from aqueous solution to the membrane interface DeltaG supports the preferential interaction of NPY with negatively charged membranes.

Amino Acid Sequence↗

PubMed2005

Bilayer interaction and localization of cell penetrating peptides with model membranes: a comparative study of a human calcitonin (hCT)-derived peptide with pVEC and pAntp(43-58).

Cell-penetrating peptides (CPPs) are able to translocate problematic therapeutic cargoes across cellular membranes. The exact mechanisms of translocation are still under investigation. However, evidence for endocytic uptake is increasing. We investigated the interactions of CPPs with phospholipid bilayers as first step of translocation. To this purpose, we employed four independent techniques, comprising (i) liposome buffer equilibrium dialysis, (ii) Trp fluorescence quenching, (iii) fluorescence polarization, and (iv) determination of zeta-potentials. Using unilamellar vesicles (LUVs) of different phospholipid composition, we compared weakly cationic human calcitonin (hCT)-derived peptides with the oligocationic CPPs pVEC and penetratin (pAntp). Apparent partition coefficients of hCT-derived peptides in neutral POPC LUVs were dependent on amino acid composition and secondary structure; partitioning in negatively charged POPC/POPG (80:20) LUVs was increased and mainly governed by electrostatic interactions. For hCT(9-32) and its derivatives, D values raised from about 100-200 in POPC to about 1000 to 1500 when negatively charged lipids were present. Localization profiles of CPPs obtained by Trp fluorescence quenching were dependent on the charge density of LUVs. In POPC/POPG, hCT-derived CPPs were located on the bilayer surface, whereas pVEC and pAntp resided deeper in the membrane. In POPG LUVs, an increase of fluorescence polarization was observed for pVEC and pAntp but not for hCT-derived peptides. Generally, we found strong peptide-phospholipid interactions, especially when negatively charged lipids were present.

Acrylamide↗

PubMed2005

Strongly altered receptor binding properties in PP and NPY chimeras are accompanied by changes in structure and membrane binding.

Neuropeptide Y (NPY) and the pancreatic polypeptide (PP) are members of the neuropeptide Y family of hormones. They bind to the Y receptors with very different affinities: Whereas PP is highly selective for the Y(4) receptor, NPY displays highest affinites for Y(1), Y(2), and Y(5) receptor subtypes. Introducing the NPY segment 19-23 into PP leads to an increase in affinity at the Y(1) and Y(2) receptor subtypes whereas the exchange of this segment from PP into NPY leads to a large decrease in affinity at all receptor subtypes. PP displays a very stable structure in solution, with the N terminus being back-folded onto the C-terminal alpha-helix (the so-called PP-fold). The helix of NPY is less stable and the N terminus is freely diffusing in solution. The exchange of this segment, however, does not alter the PP-fold propensities of the chimeric peptides in solution. The structures of the phospholipid micelle-bound peptides serving to mimic the membrane-bound species display segregation into a more flexible N-terminal region and a well-defined alpha-helical region. The introduction of the [19-23]-pNPY segment into hPP leads to an N-terminal extension of the alpha-helix, now starting at Pro(14) instead of Met(17). In contrast, a truncated helix is observed in [(19)(-)(23)hPP]-pNPY, starting at Leu(17) instead of Ala(14). All peptides display moderate binding affinities to neutral membranes (K(assoc) in the range of 1.7 to 6.8 x 10(4) mol(-)(1) as determined by surface plasmon resonance) with the differences in binding being most probably related to the exchange of Arg-19 (pNPY) by Glu-23 (hPP). Differences in receptor binding properties between the chimeras and their parental peptides are therefore most likely due to changes in the conformation of the micelle-bound peptides.

Animals↗

PubMed2005

Comparative analysis of the signaling capabilities of the insulin receptor-related receptor.

Although insulin receptor (InsR) and type I insulin-like growth factor receptor (IGF-IR) elicit different physiological effects in their target tissues, their signaling capabilities are similar to a large extent. In the present work, we investigated the potential of the third member of the family, insulin receptor-related receptor (IRR), to associate with known interaction partners of the InsR and the IGF-I receptor in a yeast two-hybrid assay. Using the intracellular part of the IRR we found no association with any of the tested signaling molecules. Phosphotyrosine detection revealed a lack in the constitutive activation of the IRR described for analogous constructs of the two other members of the family. Replacement of the kinase domain of the IGF-IR or its C-terminal lobe alone into the IRR caused a complete restoration of the tyrosine phosphorylation of the IRR. The reestablishment of autophosphorylation was paralleled by restoration of interaction with a specific range of signaling molecules.

Humans↗

PubMed2005

Decoding the entry of two novel cell-penetrating peptides in HeLa cells: lipid raft-mediated endocytosis and endosomal escape.

Cellular entry of peptide, protein, and nucleic acid biopharmaceuticals is severely impeded by the cell membrane. Linkage or assembly of such agents and cell-penetrating peptides (CPP) with the ability to cross cellular membranes has opened a new horizon in biomedical research. Nevertheless, the uptake mechanisms of most CPP have been controversially discussed and are poorly understood. We present data on two recently developed oligocationic CPP, the sweet arrow peptide SAP, a gamma-zein-related sequence, and a branched human calcitonin derived peptide, hCT(9-32)-br, carrying a simian virus derived nuclear localization sequence in the side chain. Uptake in HeLa cells and intracellular trafficking of N-terminally carboxyfluorescein labeled peptides was studied by confocal laser scanning microscopy and flow cytometry using biochemical markers in combination with quenching and colocalization approaches. Both peptides were readily internalized by HeLa cells through interaction with the extracellular matrix followed by lipid raft-mediated endocytosis as confirmed by reduced uptake at lower temperature, in the presence of endocytosis inhibitors and through cholesterol depletion by methyl-beta-cyclodextrin, supported by colocalization with markers for clathrin-independent pathways. In contrast to the oligocationic SAP and hCT(9-32)-br, interaction with the extracellular matrix, however, was no prerequisite for the observed lipid raft-mediated uptake of the weakly cationic, unbranched hCT(9-32). Transient involvement of endosomes in intracellular trafficking of SAP and hCT(9-32)-br prior to endosomal escape of both peptides was revealed by colocalization and pulse-chase studies of the peptides with the early endosome antigen 1. The results bear potential for CPP as tools for intracellular drug delivery.

Biological Transport↗

PubMed2005

The anti-epileptic actions of neuropeptide Y in the hippocampus are mediated by Y and not Y receptors.

Neuropeptide Y (NPY) potently inhibits glutamate release and seizure activity in rodent hippocampus in vitro and in vivo, but the nature of the receptor(s) mediating this action is controversial. In hippocampal slices from rats and several wild-type mice, a Y2-preferring agonist mimicked, and the Y2-specific antagonist BIIE0246 blocked, the NPY-mediated inhibition both of glutamatergic transmission and of epileptiform discharges in two different slice models of temporal lobe epilepsy, stimulus train-induced bursting (STIB) and 0-Mg2+ bursting. Whereas Y5 receptor-preferring agonists had small but significant effects in vitro, they were blocked by BIIE0246, and a Y5 receptor-specific antagonist did not affect responses to any agonist tested in any preparation. In slices from mice, NPY was without effect on evoked potentials or in either of the two slice seizure models. In vivo, intrahippocampal injections of Y2- or Y5-preferring agonists inhibited seizures caused by intrahippocampal kainate, but again the Y5 agonist effects were insensitive to a Y5 antagonist. Neither Y2- nor Y5-preferring agonists affected kainate seizures in mice. A Y5-specific antagonist did not displace the binding of two different NPY ligands in WT or mice, whereas all NPY binding was eliminated in the mouse. Thus, we show that Y2 receptors alone mediate all the anti-excitatory actions of NPY seen in the hippocampus, whereas our findings do not support a role for Y5 receptors either in vitro or in vivo. The results suggest that agonists targeting the Y2 receptor may be useful anticonvulsants.

Animals↗

PubMed2005

Neuropeptide Y stimulates neuronal precursor proliferation in the post-natal and adult dentate gyrus.

Adult dentate neurogenesis is important for certain types of hippocampal-dependent learning and also appears to be important for the maintenance of normal mood and the behavioural effects of antidepressants. Neuropeptide Y (NPY), a peptide neurotransmitter released by interneurons in the dentate gyrus, has important effects on mood, anxiety-related behaviour and learning and memory. We report that adult NPY receptor knock-out mice have significantly reduced cell proliferation and significantly fewer immature doublecortin-positive neurons in the dentate gyrus. We also show that the neuroproliferative effect of NPY is dentate specific, is Y1-receptor mediated and involves extracellular signal-regulated kinase (ERK)1/2 activation. NPY did not exhibit any effect on cell survival in vitro but constitutive loss of the Y1 receptor in vivo resulted in greater survival of newly generated neurons and an unchanged total number of dentate granule cells. These results show that NPY stimulates neuronal precursor proliferation in the dentate gyrus and suggest that NPY-releasing interneurons may modulate dentate neurogenesis.

Animals↗

PubMed2005

Expressed protein ligation for protein semisynthesis and engineering.

Over the past decade, a significant methodological development in peptide ligation strategies has been elaborated that now permits the assembly of peptides and proteins. Native chemical ligation (NCL) has been introduced to join synthetic unprotected peptides by using the chemoselective reaction between a C-terminal thioester and an N-terminal cysteine residue to result in a native peptide bond. Although this method has been applied to obtain peptides, small proteins, or protein domains (up to approx 150 residues), larger proteins could not been easily received because of the limited size of the ligated fragments. Intein technologies benefit from the opportunity to participate in the production of polypeptides with the reactive groups necessary for NCL aside from the rapid isolation of highly pure recombinant proteins. Expressed protein ligation extends the scope of NCL by overcoming the size limitation of target proteins accessible to synthesis. The intein splicing and EPL have been already proven to be useful for protein semisynthesis and for various investigations, including the studies of protein-protein interactions, segmental isotopic labeling for protein structure determination, synthesis of cytotoxic proteins, protein cyclization, and site-specific incorporation of noncanonical amino acids and biophysical probes into a protein sequence.

Inteins↗

PubMed2005

Expressed protein ligation to obtain selectively modified aldo/keto reductases.

Specific enzyme immobilization has moved into the focus for many applications in biochemical research fields. Expressed Protein Ligation (EPL) has been proven to be ideal to selectively label proteins at single positions. Applying this technique to enzymes of the aldo/keto reductase superfamily provides a new approach to generate native or modified redox enzymes for direct and indirect immobilization.

Alcohol Oxidoreductases↗

PubMed2004

Structural investigations of a human calcitonin-derived carrier peptide in a membrane environment by solid-state NMR.

Previous studies have shown that human calcitonin (hCT) and its C-terminal fragment hCT(9-32) translocate in nasal epithelium. Moreover, hCT(9-32) was used as a carrier to internalize efficiently the green fluorescent protein, drugs, and plasmid DNA. To understand the mechanism of the membrane crossing process, we determined structural parameters of the carrier peptide hCT(9-32) in a membrane environment using solid-state NMR. For that purpose, we synthesized a multiply labeled hCT(9-32) peptide comprising four positions with fully (15)N- and (13)C-labeled amino acids. Multilamellar vesicle samples containing varying mixing ratios of hCT(9-32) and phospholipids found in the plasma membrane of nasal epithelium were prepared. The typical axially symmetric powder patterns of (31)P NMR spectra confirmed the presence of lamellar bilayers in our samples. The chemical shift anisotropy of the (31)P NMR spectra of the samples in the presence of hCT(9-32) is slightly reduced, revealing weak interaction of the peptide with the lipid headgroups. The peptide does not penetrate the lipid membrane as indicated by very similar (2)H NMR order parameters of the phospholipid fatty acid chains in the absence and presence of the carrier peptide. This membrane topology was confirmed by measurements of paramagnetic enhancement of relaxation rates. The conformation of hCT(9-32) was investigated by cross polarization magic angle spinning NMR methods. All peptide signals were resolved and fully assigned in two-dimensional proton-driven (13)C spin diffusion experiments. The isotropic chemical shifts of (13)CO, (13)Calpha, and (13)Cbeta provide information about the secondary structure of the carrier peptide. The conformation of hCT(9-32) was further corroborated by quantitative phi torsion angle measurements. Two monomeric structural models are consistent with the data: (i) a linear backbone conformation of hCT(9-32) and (ii) an antiparallel beta-sheet structure. These structures are maintained over a wide range of peptide:lipid mixing ratios. No direct indications for fibril formation of hCT(9-32) were found. Dipolar coupling measurements indicate rather high amplitudes of motion of the peptide.

Amino Acid Sequence↗

PubMed2004

Metabolic cleavage of cell-penetrating peptides in contact with epithelial models: human calcitonin (hCT)-derived peptides, Tat(47-57) and penetratin(43-58).

We assessed the metabolic degradation kinetics and cleavage patterns of some selected CPP (cell-penetrating peptides) after incubation with confluent epithelial models. Synthesis of N-terminal CF [5(6)-carboxyfluorescein]-labelled CPP, namely hCT (human calcitonin)-derived sequences, Tat(47-57) and penetratin(43-58), was through Fmoc (fluoren-9-ylmethoxycarbonyl) chemistry. Metabolic degradation kinetics of the tested CPP in contact with three cell-cultured epithelial models, MDCK (Madin-Darby canine kidney), Calu-3 and TR146, was evaluated by reversed-phase HPLC. Identification of the resulting metabolites of CF-hCT(9-32) was through reversed-phase HPLC fractionation and peak allocation by MALDI-TOF-MS (matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry) or direct MALDI-TOF-MS of incubates. Levels of proteolytic activity varied highly between the investigated epithelial models and the CPP. The Calu-3 model exhibited the highest proteolytic activity. The patterns of metabolic cleavage of hCT(9-32) were similar in all three models. Initial cleavage of this peptide occurred at the N-terminal domain, possibly by endopeptidase activity yielding both the N- and the C-terminal counterparts. Further metabolic degradation was by aminopeptidase, endopeptidase and/or carboxypeptidase activities. In conclusion, when in contact with epithelial models, the studied CPP were subject to efficient metabolism, a prerequisite of cargo release on the one hand, but with potential for premature cleavage and loss of the cargo as well on the other. The results, particularly on hCT(9-32), may be used as a template to suggest structural modifications towards improved CPP performance.

Amino Acid Sequence↗

PubMed2004

Central NPY receptor-mediated alteration of heart rate dynamics in mice during expression of fear conditioned to an auditory cue.

Neuropeptide Y (NPY) is involved in the regulation of emotionality including fear and anxiety, which modulate autonomic control of cardiovascular function. We therefore investigated the central effects of porcine NPY, selective Y1, Y2 and Y5 receptor agonists and a Y1 receptor antagonist on heart rate (HR) and HR variability in freely moving mice using auditory fear conditioning. Intracerebroventricular (i.c.v.) injections were applied 15 min before the tone-dependent memory test. NPY dose-dependently induced bradycardia associated with decreased HR variability, and blunted the stress-induced tachycardic response. The selective Y1 receptor antagonist BIBO 3304 blocked the NPY- and Y1-receptor agonist-induced suppression of conditioned tachycardia without affecting basal HR. The tachycardia elicited by both conditioned and unconditioned stressor was effectively attenuated by the Y1 receptor agonist. These results suggest a specific contribution of Y1, but not Y2 and Y5 receptors, to modulation of emotional responses most likely unrelated to impairment or modulation of memory. The NPY-induced bradycardia is attributed to not yet characterized NPY receptor subtypes other than Y1, Y2 and Y5, or a complex receptor interaction. In conclusion, NPY mediates central inhibition of sympathetic outflow, potentially coupled with attenuation of parasympathetic tone, i.e., mechanisms that may be associated with the reported anxiolytic action.

Animals↗

PubMed2004

Structure-activity studies of orexin a and orexin B at the human orexin 1 and orexin 2 receptors led to orexin 2 receptor selective and orexin 1 receptor preferring ligands.

The neuropeptides orexin A and B (also known as hypocretins) play an important role in many physiological and behavioral activities. Orexins are ligands of two closely related G-protein-coupled receptors, that are the named orexin 1 and orexin 2 receptors. To clearly identify the minimal ligand sequences required for receptor activation, we synthesized and analyzed different centrally, C- and N-terminally truncated analogues of orexins A and B. Furthermore, we used the shortest active analogue to screen for important amino acid residues by l-alanine and l-proline replacement scans. For orexin A, only full-length peptides were able to show the same activity as orexin A, but interestingly, reduced orexin A and natural orexin A, which contains the two disulfide bonds, had the same activity. The shortest highly active orexin B analogue was orexin B 6-28. In addition, we identified orexin A 2-33 as the first analogue with orexin 1 receptor preference and orexin B 10-28, [A27]orexin B 6-28, and [P11]orexin B 6-28 as being highly potent orexin 2 receptor selective (>1000-fold) peptides.

Calcium↗

PubMed2004

In vitro gene delivery by a novel human calcitonin (hCT)-derived carrier peptide.

Gene therapy still awaits a broader application, since safe and efficient gene delivery is a major problem. Also for the investigation of signal transduction and intracellular trafficking, delivery systems for hydrophilic macromolecules that are easy to use are needed. Several peptide-based delivery systems have been developed during the last years. We present here a novel carrier peptide derived from human calcitonin that is capable of transfecting human neuroblastoma cells by complex formation with a plasmid. Because of the peptide's physiological origin, cytotoxic effects are not expected.

Amino Acid Sequence↗

PubMed2004

Novel modified and radiolabelled neuropeptide Y analogues to study Y-receptor subtypes.

To better characterize neuropeptide Y (NPY) receptor subtypes, several novel selective analogues of the NPY sequence have been developed in the last few years. These analogues show high selectivity towards single NPY receptors and facilitate insights into the localisation and physiological role of each receptor subtype in health and disease. Besides the important role of NPY in the regulation of food intake and other physiological functions, recently NPY receptors have been identified to play an important role in several types of cancer. To further enlighten all these numerous functions the radioactive labelling of peptides is still an indispensable and versatile tool. By using the appropriate radioisotope and labelling strategy several different radioisotopes can be incorporated into the sequence of subtype selective NPY analogues. Depending on the radioisotope the resulting radiolabelled NPY analogues can be used for diagnostic or therapeutic applications and enable us to better investigate single receptor subtypes in vitro and in vivo.

Amino Acid Sequence↗

PubMed2004

Cellular internalization of human calcitonin derived peptides in MDCK monolayers: a comparative study with Tat(47-57) and penetratin(43-58).

PURPOSE: The objective of this study was to evaluate key motif requirements of human calcitonin (hCT)-derived peptides for the permeation through the plasma membrane of MDCK monolayers, as epithelial model. METHODS: Truncated and sequence-modified fluorescent-labeled hCT-derived peptides were synthesized through Fmoc chemistry. Peptide uptake by confluent MDCK was observed by confocal laser scanning microscopy. The cytotoxic effect of the peptides on cellular integrity was followed by LDH release. For direct comparison we covered the cellular uptake of established cell penetrating peptides, Tat(47-57) and penetratin(43-58). RESULTS: Truncated sequences of hCT, from hCT(9-32) to hCT(18-32), penetrated the plasma membrane and demonstrated a sectoral, punctuated cytoplasmic distribution. The uptake process appeared to be temperature-, time- and concentration-dependent. Amino acid modifications of hCT(18-32) indicated that both the proline in position 23 and the positive charge of lysine in position 18 are crucial for peptide uptake. The reverse sequence hCT(32-18) did not penetrate the membrane, indicating the importance of sequence orientation. Tat(47-57) and penetratin(43-58) showed a similar punctuated cytoplasmic distribution in MDCK and HeLa cell lines. No relevant toxicity was observed. CONCLUSIONS: Selected hCT-derived peptides have cell penetrating properties. The uptake mechanism seems to involve an endocytic pathway.

Amino Acid Sequence↗