Biomedical subjects

A Buku

Publications and source records attributed to A Buku.

At least 19 recordsLinked to original sources

PubMed2005

[Ala12]MCD peptide: a lead peptide to inhibitors of immunoglobulin E binding to mast cell receptors.

An effort was made to discover mast cell degranulating (MCD) peptide analogs that bind with high affinity to mast cell receptors without triggering secretion of histamine or other mediators of the allergic reaction initiated by immunoglobulin E (IgE) after mast cell activation. Such compounds could serve as inhibitors of IgE binding to mast cell receptors. An alanine scan of MCD peptide reported previously showed that the analog [Ala12]MCD was 120-fold less potent in histamine-releasing activity and fivefold more potent in binding affinity to mast cell receptors than the parent MCD peptide. Because this analog showed marginal intrinsic activity and good binding affinity it was subsequently tested in the present study as an IgE inhibitor. In contrast to MCD peptide, [Ala12]MCD showed a 50% inhibition of IgE binding to the Fc epsilon RI alpha mast cell receptor by using rat basophilic leukemia (RBL-2H3) mast cells and fluorescence polarization. Furthermore, in a beta-hexosaminidase secretory assay, the peptide also showed a 50% inhibition of the secretion of this enzyme caused by IgE. An attempt was made to relate structural changes and biologic differences between the [Ala12]MCD analog and the parent MCD peptide. The present results show that [Ala12]MCD may provide a base for designing agents to prevent IgE/Fc epsilon RI alpha interactions and, consequently, allergic conditions.

Alanine↗

PubMed2003

Histamine-releasing activity and binding to the FcepsilonRI alpha human mast cell receptor subunit of mast cell degranulating peptide analogues with alanine substitutions.

We have investigated the effects on mast cell binding and the histamine-releasing activity of l-alanine substitutions for the five lysine residues and the proline residue in the MCD peptide (1) sequence. All synthesized analogues Ala(2) (2), Ala(6) (3), Ala(11) (4), Ala(12) (5), Ala(17) (6), and Ala(21) (7) showed a loss of histamine release compared to the parent MCD peptide 1. The order of decreased potency was 1 > 6 > 7 > 4 > 2 > 3 > 5. The alanine-substituted analogues showed a 5- to 6-fold decrease in histamine release for analogues 6, 7, and 4 and a 10-fold decrease for analogue 2. A more significant loss was observed in analogue 3 with a 75-fold loss of activity. The greatest loss of activity was observed with alanine substituting for proline in position 12. This analogue 5 showed a 130-fold loss of histamine release compared to the parent peptide 1. The ability of each analogue to interact with the FcepsilonRIalpha subunit of the human mast cell receptor was analyzed by competitive binding of the fluorescent peptide 1 and the alanine analogues using fluorescence polarization. The binding affinities of analogues 4, 6, and 7 for the mast cell receptor were less than the affinity of the native peptide 1. Analogues 2, 3, and 5 showed an increase in binding affinity, with analogue 5 showing the highest increase compared to the native peptide 1. The order of increased affinity was 5 > 3 > 2 > 1 > 4, 6, 7. On the basis of these results, the possibility that analogue 5 inhibits peptide 1-stimulated histamine release was examined. We found that peptide 5 did not inhibit histamine release by peptide 1. The analogues 2, 3, and especially analogue 5 may be useful leads toward study of agents that prevent binding of IgE to mast cell receptors.

Alanine↗

PubMed2001

Further studies on the structural requirements for mast cell degranulating (MCD) peptide-mediated histamine release.

Mast cell degranulating (MCD) peptide was modified in its two disulfide bridges and in the two arginine residues in order to measure the ability of these analogs to induce histamine release from mast cells in vitro. Analogs prepared were [Ala(3,15)]MCD, [Ala(5,19)]MCD, [Orn(16)]MCD, and [Orn(7,16)]MCD. Their histamine-releasing activity was determined spectrofluorometrically with peritoneal mast cells. The monocyclic analogs in which the cysteine residues were replaced pairwise with alanine residues showed three-to ten-fold diminished histamine-releasing activity respectively, compared with the parent MCD peptide. Substantial increases in activity were observed where arginine residues were replaced by ornithines. The ornithine-mono substituted analog showed an almost six-fold increase and the ornithine-doubly substituted analog three-fold increase in histamine-releasing activity compared with the parent MCD peptide. The structural changes associated with these activities were followed by circular dichroism (CD) spectroscopy. Changes in the shape and ellipticity of the CD spectra reflected a role for the disulfide bonds and the two arginine residues in the overall conformation and biological activity of the molecule.

Animals↗

PubMed2001

Mast cell degranulating peptide binds to RBL-2H3 mast cell receptors and inhibits IgE binding.

Fluorescent and biotinylated analogs of mast cell degranulating (MCD) peptide were synthesized and the labels fluoresceinisothiocyanate and N-hydroxysuccinimidobiotin were conjugated at position 1 in the MCD peptide sequence. The analogs with these moieties retained histamine-releasing activity as high as that of the parent MCD peptide in rat peritoneal mast cell assays. These labeled analogs were used in rat basophilic leukemia cells (RBL-2H3) to demonstrate by confocal microscopy and flow cytometry the specific binding of MCD peptide to mast cell receptors. Consequently MCD peptide was found to compete with and inhibit the binding of fluorescent IgE on RBL cells as monitored by flow cytometry. Thus MCD peptide may prove to be useful in the study of IgE receptor-bearing cells.

Amino Acid Sequence↗

PubMed1999

Mast cell degranulating (MCD) peptide: a prototypic peptide in allergy and inflammation.

The solid phase synthesis of mast degranulating peptide (MCD peptide) raised the possibility of preparing analogs and examining the pharmacology and the proposed role of this peptide as a potential agent in allergy and inflammation. MCD peptide, a cationic 22-amino acid residue peptide with two disulfide bridges, causes mast cell degranulation and histamine release at low concentrations and has anti-inflammatory activity at higher concentrations. Because of these unique immunologic properties, MCD peptide may serve as a useful tool for studying secretory mechanisms of inflammatory cells such as mast cells, basophils, and leukocytes, leading to the design of compounds with therapeutic potential.

Animals↗

PubMed1999

Distinct secretases, a cysteine protease and a serine protease, generate the C termini of amyloid beta-proteins Abeta1-40 and Abeta1-42, respectively.

The carboxy-terminal ends of the 40- and 42-amino acids amyloid beta-protein (Abeta) may be generated by the action of at least two different proteases termed gamma(40)- and gamma(42)-secretase, respectively. To examine the cleavage specificity of the two proteases, we treated amyloid precursor protein (APP)-transfected cell cultures with several dipeptidyl aldehydes including N-benzyloxycarbonyl-Leu-leucinal (Z-LL-CHO) and the newly synthesized N-benzyloxycarbonyl-Val-leucinal (Z-VL-CHO). All dipeptidyl aldehydes tested inhibited production of both Abeta1-40 and Abeta1-42. Changes in the P1 and P2 residues of these aldehydes, however, indicated that the amino acids occupying these positions are important for the efficient inhibition of gamma-secretases. Peptidyl aldehydes inhibit both cysteine and serine proteases, suggesting that the two gamma-secretases belong to one of these mechanistic classes. To differentiate between the two classes of proteases, we treated our cultures with the specific cysteine protease inhibitor E-64d. This agent inhibited production of secreted Abeta1-40, with a concomitant accumulation of its cellular precursor indicating that gamma(40)-secretase is a cysteine protease. In contrast, this treatment increased production of secreted Abeta1-42. No inhibition of Abeta production was observed with the potent calpain inhibitor I (acetyl-Leu-Leu-norleucinal), suggesting that calpain is not involved. Together, these results indicate that gamma(40)-secretase is a cysteine protease distinct from calpain, whereas gamma(42)-secretase may be a serine protease. In addition, the two secretases may compete for the same substrate. Dipeptidyl aldehyde treatment of cultures transfected with APP carrying the Swedish mutation resulted in the accumulation of the beta-secretase C-terminal APP fragment and a decrease of the alpha-secretase C-terminal APP fragment, indicating that this mutation shifts APP cleavage from the alpha-secretase site to the beta-secretase site.

Aldehydes↗

PubMed1998

Connexin50, a gap junction protein of macrogliaP6n the mammalian retina and visual pathway.

Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunocytochemistry were used to study the expression of gap junction proteins (connexins; Cx) in the rat and rabbit retina. RT-PCR of rabbit total retinal RNA using primers selected for the human Cx50 (alpha 8 Cx) DNA template yielded cDNA fragments of the predicted base pair size. Western blots of rat and rabbit retinal membrane preparations probed with a monoclonal antibody which recognizes Cx50 in the lens of several mammalian species revealed a single band (MW 50 kD), identical to that recognized in lens membrane extracts. In frozen retinal sections of both species, the same monoclonal antibody as well as two polyclonal antisera raised against a synthetic peptide from the C-terminal region of the human Cx50 polypeptide labeled Müller cells and astrocytes. In Müller cells, labeling was strongest in the endfeet and in the filamentous processes ensheathing the photoreceptors. Extending from the neural retina, Cx50-like immuno-reactivity was detected in astrocytes of the optic nerve and along retinal projections within the CNS. Our data indicate that Müller cells and astrocytes of mammalian retinas and throughout the visual pathway are coupled through gap junctions composed of connexin50.

Animals↗

PubMed1998

Bioactivities and secondary structure of mast cell degranulating (MCD) peptide analogs.

Three analogs of Mast Cell Degranulating (MCD) peptide with C-terminal and one analog with N- and C-terminal deletions were synthesized and assayed for histamine-releasing activity in mast cells. Des(20-22)-MCD and des(21)-MCD markedly decreased this activity. In des(16-17,21)-MCD this activity was completely abolished. By contrast, when the C- and N-termini were truncated in des(1-2,20-22)-MCD, the full activity of MCD peptide was restored. The possibility that these analogs trigger or inhibit oxygen radicals from neutrophils was examined in a cell-free system with a chemiluminescence assay. None of the above analogs exhibited inflammatory or anti-inflammatory activity. Changes in biological activities were correlated with structural changes, as seen by circular dichroism (CD) spectroscopy.

Amino Acid Sequence↗

PubMed1998

Differentiation-dependent expression of alpha-2,3-sialyltransferase in rabbit corneal epithelium.

PURPOSE: Lectin studies have shown that in the rabbit corneal epithelium, alpha-2,3 sialylation of O-linked glycans differentiates limbal and corneal epithelial cell phenotypes. Because sialic acid can be regulated at the level of the expression of sialyltransferases (STs), the purpose of the present study was to analyze the expression of alpha-2,3STs in this tissue. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used to generate ST cDNA from total rabbit corneal epithelium RNA using primers selected from the sequences of three previously cloned STs capable of catalyzing the transfer of sialic acid to O-linked oligosaccharides, human placental Galbeta-1,3GalNAc-Galbeta-1,4GluNAcalpha-2,3ST (STZ), and mouse brain Galbeta-1,3GalNAcalpha-2,3ST types I and II (ST3Gal I and ST3Gal II). Tissue distribution of mRNA was assayed by fluorescent in situ hybridization. A synthetic peptide whose sequence was deduced from a cloned cDNA fragment was synthesized and used to prepare an anti-ST goat antiserum. The molecular weights of immunodetectable polypeptides and their distribution in cryostat sections of the limbocorneal area were investigated by western blot analysis and indirect immunofluorescence, respectively. RESULTS: RT-PCR yielded cDNA of expected basepair length for STZ and ST3(Gal II. The rabbit STZ cDNA was 86% identical with its human equivalent. Its mRNA was confined to the cornea, mainly in basal epithelial cells, and was not expressed in the limbus. Western blot analysis identified a band at 37 kDa whose binding was abolished by preincubation of the antiserum with the immunization peptide. Immunohistologic analysis revealed the presence of immunoreactive epitopes in all basal cells of the cornea but not in the limbus. CONCLUSIONS: STZ mRNA and the enzyme itself are expressed in the basal layer of the corneal epithelium but are absent in the limbus. This enzyme's de novo expression seems thus responsible for the differential expression of alpha-2,3 sialylation along the limbocorneal differentiation axes. At least one more alpha-2,3ST is also present in the epithelium.

Animals↗

PubMed1997

Immunohistochemical localization of the neuron-specific glutamate transporter EAAC1 (EAAT3) in rat brain and spinal cord revealed by a novel monoclonal antibody.

Neuronal regulation of glutamate homeostasis is mediated by high-affinity sodium-dependent and highly hydrophobic plasma membrane glycoproteins which maintain low levels of glutamate at central synapses. To further elucidate the molecular mechanisms that regulate glutamate metabolism and glutamate flux at central synapses, a monoclonal antibody was produced to a synthetic peptide corresponding to amino acid residues 161-177 of the deduced sequence of the human neuron-specific glutamate transporter III (EAAC1). Immunoblot analysis of human and rat brain total homogenates and isolated synaptosomes from frontal cortex revealed that the antibody immunoreacted with a protein band of apparent Mr approximately 70 kDa. Deglycosylation of immunoprecipitates obtained using the monoclonal antibody yielded a protein with a lower apparent Mr (approximately 65 kDa). These results are consistent with the molecular size of the human EAAC1 predicted from the cloned cDNA. Analysis of the transfected COS-1 cells by immunocytochemistry confirmed that the monoclonal antibody is specific for the neuron-specific glutamate transporter. Immunocytochemical studies of rat cerebral cortex, hippocampus, cerebellum, substantia nigra and spinal cord revealed intense labeling of neuronal somata, dendrites, fine-caliber fibers and puncta. Double-label immunofluorescence using antibody to glial fibrillary acidic protein as a marker for astrocytes demonstrated that astrocytes were not co-labeled for EAAC1. The localization of EAAC1 immunoreactivity in dendrites and particularly in cell somata suggests that this transporter may function in the regulation of other aspects of glutamate metabolism in addition to terminating the action of synaptically released glutamate at central synapses.

Adult↗

PubMed1996

Studies of the calmodulin-binding site of twitchin with synthetic peptides using fluorescence and CD spectroscopy.

The calcium-dependent interaction of two synthetic peptides derived from the putative calmodulin-binding site in the protein kinase autoinhibitory region of twitchin was studied by fluorescence and CD spectroscopy. The peptides interacted with dansylcalmodulin in the presence of Ca2+ as shown by a change in the fluorescence emission spectra. Fluorescence titration of dansylcalmodulin with the peptides was used to quantify this interaction. The peptides appeared to assume a helical conformation in a non-polar environment as seen by CD spectroscopy. The ellipticity of Ca2+ calmodulin was enhanced in the presence of peptides compared with that of Ca2+ calmodulin and peptides alone, indicating that the peptides had formed a complex with calmodulin. These results support the assignment of the twitchin calmodulin-binding site.

Amino Acid Sequence↗

PubMed1995

Phosphorylation of myosin regulatory light chains by the molluscan twitchin kinase.

The unusually large (approximately 600 to > 3000 kDa) myosin-associated proteins of the titin/twitchin superfamily are considered to be important cytoskeletal rulers for thick filament assembly in muscle. This function is maintained by approximately 60-240 modular fibronectin-type-III and immunoglobulin-C2 repeats in these proteins which further contain a protein serine/threonine kinase domain of unknown function. In this study, the bacterially expressed kinase domain of Aplysia twitchin was used in order to identify a potential physiological substrate. Addition of the recombinant kinase to Aplysia actomyosin preparations resulted in the specific phosphorylation of the 19-kDa myosin regulatory light chains. The twitchin kinase phosphorylated purified light chains on Thr15 in a region which shared a high degree of similarity with the phosphorylation site for vertebrate smooth muscle myosin light chain kinase. Peptide analogs of the twitchin substrate sequence and the similar sequence in vertebrate smooth muscle myosin light chains were phosphorylated with good kinetic properties. These data reveal the first potential substrate for any of the giant protein kinases and support a dual role of twitchin in molluscan muscle as a cytoskeletal protein as well as a myosin light chain kinase.

Amino Acid Sequence↗

PubMed1994

Autophosphorylation of molluscan twitchin and interaction of its kinase domain with calcium/calmodulin.

An approximately 750-kDa member of the family of giant titin/twitchin-like myosin-associated proteins was highly purified from muscle of the marine mollusc Aplysia californica. Purified twitchin was able to autophosphorylate on threonine, which demonstrates its protein serine/threonine kinase activity. cDNA sequence analysis of the cloned kinase domain of molluscan twitchin revealed that it is most closely related with the kinase domains of Caenorhabditis elegans twitchin (62% identity) and vertebrate myosin light chain kinases (45% average identity). Analysis of the cDNA sequence further suggested the presence of a potential calmodulin-binding site in a putative autoinhibitory region. The functional activity of this site was demonstrated by the calcium-dependent binding of purified twitchin to immobilized calmodulin and the fact that this interaction could be competed with synthetic peptides deduced from the cDNA sequence. Furthermore, biotinylated calmodulin bound to immobilized twitchin in gel-overlay assays with nanomolar affinity (EC50 approximately equal to 70 nM). The potential regulation of twitchin by calcium/calmodulin indicates that titin-like molecules may serve dynamic functions during contraction-relaxation cycles in muscle in addition to their functions as cytoskeletal proteins.

Amino Acid Sequence↗

PubMed1994

Circular dichroism (CD) studies on biological activity of mast cell degranulating (MCD) peptide analogs.

Analogs of MCD peptide were synthesized by solid-phase methods. Positive charges were deleted at the N-and/or C-terminus, including the helical portion of the molecule. Four peptides were prepared by removing residues 16-18 (Arg-Lys-Ile), 1-2 (Lys), 1-2 and 16-18 and by acetylation of the amino end (Ile). Analogs were tested on mast cells for histamine-releasing activity. Although the helicity of these derivatives, determined by circular dichroism (CD), was not significantly different from the native MCD peptide, two analogs with C-terminal deletions showed a 5- to 10-fold decrease in activity. These findings suggest that the C-terminus is more important than the N-terminus in determining bioactivity of MCD peptide.

Amino Acid Sequence↗

PubMed1992

The Q205LGo-alpha subunit expressed in NIH-3T3 cells induces transformation.

The growth functions of the heterotrimeric G protein G(o) was studied by expression in heterologous systems. The alpha-subunit of G(o) was mutated to convert Gln-205 to Leu (Q205L). Mutation of this conserved glutamine residue in G protein alpha-subunits is thought to persistently activate G proteins by inhibiting their GTPase activity. The wild type and mutant G(o)-alpha subunits were expressed in NIH-3T3 fibroblasts. These cells do not contain any measurable amounts of G(o)-alpha mRNA or protein. Transfection of wild type or Q205LG(o)-alpha subunit cDNA under the control of a dexamethasone-inducible promoter results in dexamethasone-dependent transcription of the mRNA and expression of the protein. The Q205LG(o)-alpha, but not wild type G(o)-alpha, stimulates mitogenesis in NIH-3T3 fibroblasts without significantly stimulating phospholipase C activity. Continuous expression of mutant G(o)-alpha induces focus formation, whereas transfections with vector alone or vector containing the native G(o)-alpha cDNA were without significant transforming effect in NIH-3T3 cells. Q205L G(o)-alpha did not induce focus formation in RAT-1 fibroblasts. Q205LG(o)-alpha-transformed NIH-3T3 cells are capable of anchorage-independent growth, as assessed by colony formation in soft agar. Q205LG(o)-alpha transformed cells induced tumors when injected into Nu/Nu mice. These results indicate that mutant G(o)-alpha subunits whose GTPase activity is presumably inhibited can induce the neoplastic transformation of NIH-3T3 cells in a phospholipase C-independent manner.

3T3 Cells↗

PubMed1992

Mast cell degranulating (MCD) peptide analogs with reduced ring structure.

Mast cell degranulating (MCD) peptide, a component of bee venom, is a 22 amino acid peptide with two disulfide bridges. In this first structure-activity study of MCD peptide, three analogs were synthesized and tested: two analogs shortened by omitting sequences 6-10 and 8-13, respectively, and one analog lacking the disulfide bridge between cysteine residues 5 and 19. These analogs were synthesized by solid-phase methods and were compared to MCD peptide in two assays for inflammation: histamine release from mast cells and superoxide anion release from neutrophils. All three analogs produced histamine release, although with only about one fifth of the activity of MCD peptide. Superoxide anion-releasing activity, however, did not parallel histamine release. MCD peptide did not release superoxide anion, while the 6-10 and 8-13 deletion analogs were strong and weak stimulants, respectively, of this anion. CD spectra showed that the secondary structures of the three analogs were very similar to that of MCD peptide, so that a change in secondary structure cannot completely explain the changes in releasing activities. Charge differences between the two deletion analogs and MCD peptide may explain some of the differences in activity. This is the first demonstration that the various activities of MCD peptide can be separated, and provides a lead through which the purported antiinflammatory activity of MCD peptide may possibly be explored in the future.

Amino Acid Sequence↗

PubMed1990

Expression of murine Fc receptors for IgG.

There are two distinct genes that encode murine low affinity Fc gamma RII, murine Fc gamma RII alpha, and murine Fc gamma RII beta, which are transcribed in specific cell lineages. Fc gamma RII alpha transcripts are present in macrophages, NK cells, and mesangial cells; Fc gamma RII beta transcripts are expressed in Fc gamma R-bearing B cells, T cells, and macrophages. We have devised a sandwich ELISA to quantify the expression of Fc gamma RII alpha protein. The ELISA is specific for Fc gamma RII alpha, and does not detect the closely related Fc gamma RII beta protein. Upon stimulation with IFN-gamma the Fc gamma RII beta- macrophage cell line J774a expressed a twelvefold enhanced level of Fc gamma RII alpha protein. Peritoneal macrophages synthesized varying amounts of Fc gamma RII alpha. High levels of Fc gamma RII alpha were observed in resident and thioglycollate-elicited peritoneal macrophages, but no Fc gamma RII alpha was detected in Bacillus Calmette Guérin-elicited macrophages. J774a cells stimulated with rIL-6 bound approximately twice as much anti-Fc gamma RII mAb 2.4G2 IgG as did unstimulated controls. However, the Fc gamma RII alpha-specific ELISA showed no change in the amount of Fc gamma RII alpha expressed. A probe encompassing the extracellular coding sequence of Fc gamma RII beta hybridized to two distinct transcripts that were elevated in rIL-6-stimulated J774a cells. One of these transcripts had the same mobility in electrophoresis as Fc gamma RII alpha mRNA and hybridized to an Fc gamma RII alpha-specific probe, whereas the other transcript was larger and did not hybridize to probes specific for either Fc gamma RII alpha or Fc gamma RII beta. Moreover, we confirmed, with an Fc gamma RII beta-specific probe, that J774a cells do not make Fc gamma RII beta mRNA. Thus, the larger transcript appears to encode a novel Fc gamma RII. We suggest that the increased level of binding of the anti-Fc gamma RII mAb 2.4G2 to rIL-6-induced cells represents translation of a Fc gamma R distinct from Fc gamma RII alpha or Fc gamma RII beta.

Animals↗

PubMed1990

The conformation of deamino-oxytocin: X-ray analysis of the 'dry' and 'wet' forms.

Two crystal structures of (1 beta-mercaptopropionic acid) deamino-oxytocin are reported. The 'dry form' in space group C2 has cell dimensions a = 27.08 +/- 0.03, b = 9.06 +/- 0.01, c = 22.98 +/- 0.02 A, beta = 102.06 +/- 0.03 with one deamino-oxytocin and six water molecules per asymmetric unit. The 'wet form' in space group P2(1) has cell dimensions a = 27.27 +/- 0.02, b = 9.04 +/- 0.01, c = 23.04 +/- 0.02 A, beta = 102.24 +/- 0.02, with two deamino-oxytocin and 13 water molecules per asymmetric unit. A local twofold parallel to the monoclinic axis gives a pseudo C2 packing. Initial phases of the 'dry form' were calculated by the heavy-atom method from the isomorphous and anomalous difference Pattersons and anomalous difference Fouier synthesis. The structure was refined by using restrained least-squares at 1.2 A resolution to a crystallographic R = 0.10. The molecular replacement method yielded the P2(1) structure that was refined with geometric restraints to R less than 0.09, by using all data to 1.09 A resolution. Deamino-oxytocin consists of a cyclic tocin ring formed by six amino acids, closed by a disulphide bridge, S1-S6, and held by two trans-annular hydrogen bonds N2-O5 and N5-O2 with a type II turn at residues 3 and 4. A flexible tripeptide tail has a loosely hydrogen-bonded type I beta-turn between N9 and O6. The sulphur of cysteine at position 1 is disordered in all the molecules leading to alternative hands of disulphide. The conformational flexibility of Ile 3, Asn 5, Pro 7 side chains and the disulphide bridge is consistent with previous models of oxytocin in which flexibility is necessary for biological activity.

Amino Acid Sequence↗