Biomedical subjects

J Hoebeke

Publications and source records attributed to J Hoebeke.

At least 55 records · Page 3Linked to original sources

PubMed1998

A partially modified retro-inverso pseudopeptide modulates the cytokine profile of CTL specific for an influenza virus epitope.

There is considerable evidence that peptides corresponding to MHC class I-restricted epitopes can be used as immunogens or immunomodulators. Pseudopeptides containing isosteric replacements of the amide bond provide more stable analogues, which may even have enhanced biologic activity. But there have been very few studies on the use of pseudopeptides to initiate or modulate the cellular immune response. This study describes the immunogenicity of a partially modified retro-inverso pseudopeptide of an influenza virus epitope and shows that this pseudopeptide modulates the cytokine profile expressed by CD8+ CTL generated from primed precursors. Moreover, the pseudopeptide is much more efficient at low concentration than the wild-type epitope to stimulate IFN-gamma secretion by CD8+ T effector cells. These results are analyzed with reference to changes in the conformation of the MHC molecule/peptide complex deduced from molecular modeling. The findings support the idea that partially modified retro-inverso analogues can be used as altered peptide ligands to enhance the stimulation of natural epitope-specific CTL and to modify their functional properties. Hence, pseudopeptide ligands might be promising tools for use in immunotherapy.

Cell Line↗

PubMed1998

An agonist-like monoclonal antibody against the human beta2-adrenoceptor.

Monoclonal antibodies were produced against a peptide corresponding to the second extracellular loop of the human beta2-adrenoceptor. One of these monoclonals, inducing an agonist-like effect in neonatal rat cardiomyocytes, was used to define the structural and physiological basis of this activity. The epitope recognized by the antibody corresponds to the sequence Trp-Tyr-Arg-Ala-Thr-His-Gln-Glu as determined by peptide scanning. Analysis by alanine modification of the peptide epitope showed the importance of the Trp, and Glu residues in antibody recognition The apparent affinity of the antibody assessed either by surface plasmon resonance or by functional titration on its agonist-like activity showed a similar value (10(8) M(-1)). The antibody recognized the receptor in its native form as shown by immunofluorescence experiments on A431 cells but not in its denatured form as shown by its absence of staining in immunoblots. The positive chronotropic effect in vitro was specifically blocked by both the antigenic peptide and the specific beta2-antagonist (+/-)-1-[2,3-(Dihydro-7-methyl1H-inden-4-yl)oxy]-3-[(1-methy lethyl)amino]-2-butanol hydrochloride (ICI1118,551). This activity was mediated through activation of Ca2+ L-type channels as assessed in guinea pig cardiomyocytes. These results suggest that the epitope is located in an extracellular alpha-helix, whose recognition by the antibody could stabilize the receptor in its 'active' conformation.

Adrenergic beta-2 Receptor Agonists↗

PubMed1998

Immunochemical and functional characterization of an agonist-like monoclonal antibody against the M2 acetylcholine receptor.

Monoclonal antibodies were raised against a peptide corresponding to the second extracellular loop of the M2 acetylcholine receptor. One of the monoclonal antibodies, B8E5, was selected for further characterization on the basis of its high yield, its isotype (IgG2a), its dissociation kinetics and its agonist-like activity. The epitope recognized by B8E5 corresponded to the N-terminal part of the second extracellular loop of the receptor (V-R-T-V-E-) as determined by competition immunoassays and epitope scanning. The KA of B8E5 for the target peptide was assessed by surface plasmon resonance (SPR) to be 6.5x10(7) M(-1) by equilibrium and 3.7x10(7) M(-1) by kinetic analysis. B8E5 recognized the M2 acetylcholine receptor on rat cardiac tissue. It only recognized the non-reduced receptor in immunoblots. The antibody had no effect on antagonist binding but decreased the affinity for the agonist carbachol. B8E5 decreased the beating frequency of neonatal rat cardiomyocytes. The effect was specific since it was blocked by the target peptide and the antagonist atropine. The EC50 of the antibody corresponded to the KA measured by surface plasmon resonance. The physiological effect of the antibody did not lead to desensitization. The Fab fragments had no physiological effect; subsequent addition of anti-mouse IgG however restored the physiological effect. These results confirm that the N-terminus of the second extracellular loop is a functional target for antibodies against the M2 acetylcholine receptor. They suggest that the functional epitope is only accessible in the non-reduced receptor. The antibodies act through a functional dimerization of the receptor.

Amino Acid Sequence↗

PubMed1998

Functionally active cardiac antibodies in chronic Chagas' disease are specifically blocked by Trypanosoma cruzi antigens.

Antibodies of chronic chagasic patients have been shown to interfere with electric and mechanical activities of cardiac embryonic myocytes in culture and with whole mammalian hearts. A mechanism proposed for this effect involves interaction of the antibodies with G-protein-linked membrane receptors, thus leading to activation of beta adrenergic and muscarinic receptors; more specifically, IgG of chagasic patients would interact with the negatively charged regions of the second extracellular loop of these receptors. We performed competition experiments to test this hypothesis. We evaluated the effect of sera/IgG from patients previously known to depress electrogenesis and/or atrioventricular conduction in isolated rabbit hearts after incubation with live and lysed parasites, the peptide corresponding to the second extracellular loop (O2) of the M2 receptor, and different peptides derived from two ribosomal proteins of T. cruzi: P0 and P2beta. Our results indicate that 1) the antigenic factor inducing the functionally active IgGs in the chagasic patients is probably an intracellular T. cruzi antigen; 2) IgG/serum is interacting with the O2 region of the M2 receptor in the rabbit heart; and 3) the negative charges present in the ribosomal proteins of T. cruzi are important in mediating the interaction between the patients' serum/IgG and the receptor.

Amino Acid Sequence↗

PubMed1998

Immunoreactivity of sera to a peptide derived from the serotonin 5-HT1A receptor in a group of children with developmental disorders: possible role in non-autistic epilepsy.

The presence of autoantibodies against the serotoninergic 5-HT1A receptor has been reported in serum from an autistic child using radioligand binding studies. It is now well established that, in cardiovascular diseases with an autoimmune component, patients present in their sera autoantibodies directed against the second extracellular loop of some G-protein coupled membrane receptors. We thus investigated by an enzyme-immunoassay method the presence of anti-5-HT1A receptor antibodies in sera of children with developmental disorders using synthetic peptides corresponding to the first and the second extracellular loops of this receptor. The population of children with developmental disorders was divided in autistic children with or without EEG abnormalities, and in non-autistic children with or without EEG abnormalities. We found that 6 out of 10 sera of non-autistic children with an abnormal EEG recognized the second extracellular loop of the 5-HT1A receptor. This is significantly higher than the other groups of children with developmental disorders or a healthy control group. These observations support the existence of an autoimmune component in epilepsy.

Adolescent↗

PubMed1998

Immunohistochemical localization of angiotensin II receptors (AT1) in the heart with anti-peptide antibodies showing a positive chronotropic effect.

Antibodies were produced against a synthetic peptide corresponding to amino acids (165-191) of the second extracellular loop of the human angiotensin II receptor subtype 1 (AT1) in rabbits. The purified antibodies had an apparent affinity of about 1 nM and were monospecific for the AT1-receptor peptide. Chemical modification of the carboxyl groups (glu at positions 173 and 185) and the sulfhydryl group (cys at position 180) of the AT1-receptor peptide did not alter the relative affinity of the coated AT1-receptor peptide to antibodies. The antibodies specifically stained CHO cells expressing the rat AT1a receptor. Immunoblots on rat kidney revealed that the antibody recognized a protein band of 59 +/- 3 kDa in a dose-dependent manner and this band was no longer detected after preincubating the antibodies with AT1-receptor peptide. Using electron microscopic and immunofluorescence immunocytochemistry techniques, angiotensin II receptors were detected in (1) the sarcolemma, T-tubules and nuclei of rat cardiomyocytes, (2) the transluminal side of endothelial cells and (3) fibroblast cells. These localizations are specific, as the immunostaining did not appear when preimmune rabbit serum was used and was blocked after preincubating antibodies with antigenic peptide. Functionally, these antibodies did not affect the ligand binding properties of the receptors but displayed agonist-like activity as shown by dose-dependent increases in beating frequency in cultured neonatal cardiomyocytes. These results suggest that the antibodies against the second extracellular loop of human AT1 receptors were able to specifically recognize AT1 receptors. In addition, they extend the observation that the second extracellular loop of the G-protein coupled membrane receptors is a specific target for antibodies with agonist-like activity.

Amino Acid Sequence↗

PubMed1997

Antibodies to ribosomal P proteins of Trypanosoma cruzi in Chagas disease possess functional autoreactivity with heart tissue and differ from anti-P autoantibodies in lupus.

Anti-P antibodies present in sera from patients with chronic Chagas heart disease (cChHD) recognize peptide R13, EEEDDDMGFGLFD, which encompasses the C-terminal region of the Trypanosoma cruzi ribosomal P1 and P2 proteins. This peptide shares homology with the C-terminal region (peptide H13 EESDDDMGFGLFD) of the human ribosomal P proteins, which is in turn the target of anti-P autoantibodies in systemic lupus erythematosus (SLE), and with the acidic epitope, AESDE, of the second extracellular loop of the beta1-adrenergic receptor. Anti-P antibodies from chagasic patients showed a marked preference for recombinant parasite ribosomal P proteins and peptides, whereas anti-P autoantibodies from SLE reacted with human and parasite ribosomal P proteins and peptides to the same extent. A semi-quantitative estimation of the binding of cChHD anti-P antibodies to R13 and H13 using biosensor technology indicated that the average affinity constant was about 5 times higher for R13 than for H13. Competitive enzyme immunoassays demonstrated that cChHD anti-P antibodies bind to the acidic portions of peptide H13, as well as to peptide H26R, encompassing the second extracellular loop of the beta1 adrenoreceptor. Anti-P antibodies isolated from cChHD patients exert a positive chronotropic effect in vitro on cardiomyocytes from neonatal rats, which resembles closely that of anti-beta1 receptor antibodies isolated from the same patient. In contrast, SLE anti-P autoantibodies have no functional effect. Our results suggest that the adrenergic-stimulating activity of anti-P antibodies may be implicated in the induction of functional myocardial impairments observed in cChHD.

Amino Acid Sequence↗

PubMed1997

Anti-M2 muscarinic receptor antibodies inhibit beta-adrenoceptor-mediated inotropic response in rat myocardium.

The modulation of the inotropic effect by affinity-purified antibodies against a synthetic peptide corresponding to the second extracellular loop of the human muscarinic M2 receptors was studied in adult rat ventricular myocardium. These anti-muscarinic M2 receptor antibodies shifted the dose-response relationship of the beta-adrenoceptor agonist isoproterenol to higher concentrations whereas preimmune rabbit immunoglobulin G (IgG) or antibodies against the N-terminus of the beta 1-adrenoceptor had no effect. This effect of anti-muscarinic M2 receptor antibodies was fully blocked after preincubation with the antigenic peptide. No significant change of maximal inotropic response to isoproterenol was observed in the presence of anti-muscarinic M2 receptor antibodies. The anti-muscarinic M2 receptor antibodies did apparently not hamper the access of the muscarinic receptor agonist carbachol. The muscarinic receptor antagonist atropine attenuated the effect of the anti-muscarinic M2 receptor antibodies. The present study demonstrates for the first time in intact adult ventricular myocardium a specific stimulatory muscarinic activity of antibodies raised against a part of the muscarinic M2 receptor protein.

Adrenergic beta-Agonists↗

PubMed1997

Peptides derived from cardiovascular G-protein-coupled receptors induce morphological cardiomyopathic changes in immunized rabbits.

An experimental model of early-stage cardiomyopathy was created by immunizing rabbits for 1 year with synthetic peptides corresponding to the sequence of the second extracellular loop of either beta-adrenoceptors or M2-muscarinic receptors. Thirty male rabbits were used and divided into three groups: a control group (n = 10), a group immunized with the peptide corresponding to the beta-adrenoceptor (beta 1 group) (n = 10) and a group immunized with the peptide corresponding to the M2-muscarinic receptor (M2 group) (n = 10). If the sera from both groups of immunized rabbits high-titres of anti-peptide antibodies were found throughout the study period but not in the sera from control rabbits or in the preimmune sera of immunized rabbits. No significant cross-reaction with peptides other than those used for immunization was found. The myocardial receptor density of both immunized groups displayed a strong trend toward receptor up-regulation. This was significant in the beta 1 group but not in the M2 group. Both groups of immunized rabbits displayed significantly enlarged ventricles and thinner walls, as compared with the control group. However, in contrast to the beta 1 group, which showed enlarged cavities in both left and right ventricles, the M2 group was mainly affected in the right ventricles. Moreover, morphological examinations of the hearts of rabbits from both immunized groups demonstrated focal myofibrillar lysis, loss of myofilament, mitochondrial swelling and condensation, sarcoplasmic vacuolation, deposition of dense granules in the sarcoplasm and the myofibrils. One of the sex control rabbit hearts which were examined showed mild degenerative changes in the myocardium and scant mononuclear cell infiltration. However, when all the control rabbit hearts were examined by electron microscopy, no significant alterations were found. These results suggest that immunization by peptides, corresponding to the target sequences for anti-receptor autoantibodies in idiopathic dilated cardiomyopathy, induces morphological changes in the heart similar to those found in the human disease.

Amino Acid Sequence↗

PubMed1997

Evaluation of alkaline phosphatase immunoassay and comparison with other diagnostic methods in areas of low transmission of schistosomiasis.

The alkaline phosphatase immunoassay (APIA) is an antibody detection technique which permits the diagnosis of schistosomiasis using a butanolic extract preparation from adult worms. APIA has demonstrated high sensitivity and specificity in previous reports with well characterized human sera. Its potential as a diagnostic tool for epidemiological surveillance was assessed in comparison with three other diagnostic tests: stool examination, ELISA with soluble egg antigen (SEA) and the circumoval precipitin test (COPT). APIA was 100% specific in an area without Schistosoma mansoni transmission and had 89% sensitivity in an endemic area where 69% of the infected subjects excreted less than 100 eggs g of faeces. It was found to be less sensitive in children under 5 years of age who were positive by the COPT test. APIA can be applied as an initial screening test, based on its high sensitivity, specificity, absence of cross-reactivity with intestinal parasites and the fact that it is a technique suitable for use in epidemiological surveillance.

Adolescent↗

PubMed1997

Differences in immunological response to a T. gondii protein (SAG1) derived peptide between two strains of mice: effect on protection in T. gondii infection.

This study presents the analysis of the immunogenicity, antigenicity and protective effects of a peptide derived from the major surface antigen of Toxoplasma gondii, SAG1. This synthetic peptide carrying three predicted H-2k restricted T cell epitopes was used to immunize mice. The protective effect of the peptide was evaluated in CBA/J and C57BL/6 mice using the decrease in brain cyst load as evidence of protection. Immunization of C57BL/6 mice yielded high antibody titres but had no protective effect after oral challenge. Immunized CBA/J, mice which responded with a lower titre, showed a 35% reduction in cyst burden after oral challenge. Both strains yielded antibodies which recognized the cognate SAG1 protein on immunoblot assay. Using the BIAcore, system, it was shown that at lower titres the CBA/J mouse sera recognized the native SAG1 protein more effectively than the C57BL/6 mouse sera, yielding much higher anti-peptide titres. Lymphoproliferation assays using the peptide experimentally confirmed the predicted T-cell epitopes and showed that they were also recognized by cells of T. gondii infected mice. The anti-peptide subclass analysis suggested a Th1 orientation in CBA/J mice, whereas a Th2 orientation was observed in C57BL/6 mice. Finally, fine analysis of sequences recognized under MHC class I indicated the existence of a T-cell epitope in the H-2k haplotype (CBA/J mice) but not in the H-2b haplotype (C57BL/6 mice). This study provides a structural basis to the understanding of the vaccination response to one of the T. gondii antigens in different strains of mice.

Animals↗

PubMed1996

Anti-peptide antibodies sensitive to the 'active' state of the beta2-adrenergic receptor.

Antibodies directed against a peptide corresponding to the second loop of the human beta2-adrenergic receptor were induced in rabbits by immunisation with the free peptide in complete Freund's adjuvant. The resulting antibodies were affinity-purified and shown to be monospecific for the target receptor. They were able to stimulate the L-type Ca2+ channels in whole-cell patch-clamp experiments on isolated adult guinea-pig cardiomyocytes. This effect was similar to that obtained by the specific beta2-adrenergic agonist zinterol. The antibody effects could be blocked with the specific beta2-adrenergic inverse agonist ICI118,551 but not with the neutral antagonist alprenolol. These results suggest that the antibodies recognise the active conformer of the beta2-adrenergic receptor.

Amino Acid Sequence↗

PubMed1996

Cystatins up-regulate nitric oxide release from interferon-gamma-activated mouse peritoneal macrophages.

Up-regulation of nitric oxide (NO) production by activated murine macrophages was observed during infection by Trypanosoma cruzi, the etiological agent of Chagas' disease. Cell infection by T. cruzi depends at least in part on cruzipain, a membrane-associated papain-related proteinase which is sensitive to inhibition by synthetic inhibitors of cysteine proteinases. Using the natural cysteine proteinase inhibitor chicken cystatin, a representative member of cystatin family 2, to investigate the effect of cruzipain on macrophage infection and NO release, we found that the inhibitor alone up-regulated NO release from interferon-gamma-activated macrophages. A 12-fold increase in NO production was observed in the presence of 1 microM chicken cystatin. This overproduction was concentration-dependent and could be detected at concentrations as low as 10 nM and remained in the presence of polymyxin B. Representative members of the other cystatin families, i.e. stefin B (family 1), T-kininogen, and its inhibitory domains (family 3), were also able to enhance NO production from interferon-gamma-activated macrophages. Neither E64, an irreversible inhibitor of cysteine proteinases, nor inhibitors of aspartyl and serine proteinases (aprotinin, pepstatin, and soybean trypsin inhibitor) enhanced NO production. Upon complexation with saturating amounts of reduced-alkylated papain, cystatins still remained active in increasing NO production, suggesting that the cystatin inhibitory site was not involved in the mechanism. The results demonstrate that members of all 3 cystatin families share another common property unrelated to their function of cysteine proteinase inhibitors, i.e. up-regulation of NO production, which biological significance remains to be elucidated.

Animals↗

PubMed1996

Structural and functional analysis of the B cell epitopes recognized by anti-receptor autoantibodies in patients with Chagas' disease.

IgG fractions of patients were screened for autoantibodies against the beta1- and beta2-adrenoceptors and the M2 acetylcholine receptor by enzyme immunoassays and surface plasmon resonance (SPR) using peptides corresponding to the second extracellular loop of these receptors. A high prevalence of anti-M2 acetylcholine receptor and, in decreasing order, of anti-beta1- and anti-beta2-adrenoceptor autoantibodies was shown. The enzyme immunoassays and the SPR studies on the anti-beta1 adrenoceptor and the M2 acetylcholine receptor autoantibodies were dependent on the ionic strength of the interaction buffer, suggesting the importance of electrostatic interactions in Ab recognition. IgG fractions showed chronotropic effects on neonatal rat cardiomyocytes in vitro. The positive chronotropic effect was enhanced in the presence of 1 microM of atropine, demonstrating a muscarinic stimulation by the IgG fractions in the presence of a beta-adrenergic stimulation, which was blocked by the use of 1 microM of the beta1-selective antagonist bisoprolol. The beta2-selective antagonist ICI 118,551 only partially inhibited the positive chronotropic effect induced by the IgG fractions, confirming the minor functional importance of autoantibodies against the beta2-adrenoceptor. Affinity-purified Abs confirmed that Abs against the beta1-adrenoceptors and the M2 muscarinic receptors exist together with an Ab population recognizing a cross-reactive epitope on both receptors. This epitope could be identified as a polyanionic stretch present in the second extracellular loop of both the beta1-adrenoceptor and the M2 acetylcholine receptor. This stretch corresponds to the previously determined cross-reactive epitope between the P0 ribosomal protein of Trypanosoma cruzi and the beta1-adrenoceptor.

Adrenergic beta-Antagonists↗

PubMed1996

Alternative splicing determines the binding of platelet-derived growth factor (PDGF-AA) to glycosaminoglycans.

We have shown previously that the platelet-derived growth factor (PDGF) and a synthetic oligopeptide, corresponding to the basic carboxyl-terminal amino acid extension of the long PDGF-A isoform, bind to heparin. Here, we have expressed the long (rA125) and the short (rA109) variants of PDGF A-chains in Escherichia coli and produced the functional homodimers. Surface plasmon resonance analyses showed that while the dimeric rA125 bound with high affinity to low molecular weight heparin, the rA109, lacking the basic extension, did not. This strongly indicated that high affinity binding is due to the carboxyl-terminal extension. Investigations of kinetics and thermodynamics suggested an allosteric binding mechanism. Thus, dimeric rA125 contains two equivalent binding sites. Following low affinity binding of heparin to one binding site, the dimer undergoes a conformational change, increasing the affinity for heparin about 40 times. This positive cooperativity requires the basic amino acid extension in both monomers of the dimeric PDGF molecule. Thermodynamics of the reaction, showing an entropy-driven endothermic process, suggest the involvement of hydrophobic interactions in this rearrangement. Three amino acids in the basic carboxyl-terminal extension were essential for the interaction: the basic residues Arg111 and Lys116, and the polar Thr125. We also found that other glycosaminoglycan species, corresponding to those produced by human arterial smooth muscle cells, bound to dimeric rA125 and that heparan sulfate showed the highest affinity.

Alternative Splicing↗

PubMed1996

Immunochemical characterization of antibody-coated nanoparticles.

A new method using surface plasmon resonance (SPR) through the BIAcore was used to demonstrate the specific interaction between an anti-CD4 monoclonal antibody (IOT4a), adsorbed on poly(methylidene malonate 2.1.2) (PMM 2.1.2) nanoparticles, and the CD4 molecule. The results obtained were compared with the interaction of the same immunonanoparticles with rabbit anti-mouse Fc antibodies. The molar ratio (Fc)/(Fab) was 1, suggesting that the same number of epitopes on the Fc and the Fab fragments were accessible after IOT4a adsorption onto nanoparticles. Comparing the observed association rates of free antibody and antibody adsorbed on nanoparticles, the number of molecules of IOT4a antibody on PMM 2.1.2 nanoparticles was estimated as between 2.6 and 3 per nanoparticle. The properties of the antibody-coated nanoparticles are compatible with their use as antibody-targeted pharmacophores.

Adsorption↗

PubMed1996

B cell epitopes of HIV type 1 p24 capsid protein: a reassessment.

The objective of the present study was to identify p24 antigenic domains recognized during natural human immunodeficiency virus type 1 (HIV-1) infection, the determination of the major epitopes of p24 having significant applications for both the improvement of diagnostic approaches and the development of vaccines. Reactivity of 20 HIV-1-infected patients and 8 HIV-1-negative patients was analyzed using an enzyme-linked immunosorbent assay (ELISA) developed with 45 overlapping synthetic pentadecapeptides, spanning amino acids 133 to 363 of HIV-1 p55gag precursor. Two peptides covering aa 178-192 and 288-302 of p55 were recognized by 40 and 45% of HIV-1 antibody-positive human samples, respectively. A peptide covering aa 272-322 of p55 was synthesized and recognized by most human sera in indirect ELISA. However, inhibition assays indicated that this sequence does not contain all of the immunodominant domains of p24 since it was not sufficient to block binding of human sera to whole p24. A three-dimensional model of p24 derived from the Mengovirus VP2 suggests that the two distant sequences recognized by human sera containing antibodies to HIV-1 could possibly be a part of a conformational epitope built up by two loops corresponding to aa 183-186 and 289-292.

Amino Acid Sequence↗

PubMed1996

A synthetic peptide corresponding to the second extracellular loop of the human M2 acetylcholine receptor induces pharmacological and morphological changes in cardiomyocytes by active immunization after 6 months in rabbits.

We have previously shown the presence of functional autoantibodies against the second extracellular loop of the human M2 acetylcholine receptor as an autoimmune epitope in 39% of patients with idiopathic dilated cardiomyopathy (DCM). To see if this autoimmune DCM can be experimentally reproduced, a synthetic peptide corresponding to the above autoimmune epitope was used as an autoantigen to immunize rabbits monthly for 6 months. Affinity-purified antibodies were able to display the negative chronotropic effects on neonatal rat cardiomyocytes in culture. The density of the M2 acetylcholine receptors was increased in the immunized rabbits compared to controls but the receptors showed only low-affinity binding sites for the agonist carbachol. Ultrastructural changes such as moderate sarcolemma alterations, mitochondrial swelling, disruption of cristae, and formation of myelin-like structures in the myocardial cells of rabbits immunized for 6 months suggest an immune-induced cardiotoxicity. With only a limited infiltration of inflammatory cells, these results point toward a pathophysiological role of anti-M2 acetylcholine receptor autoantibodies.

Amino Acid Sequence↗